CN111527211B - 生产霉孢菌素样氨基酸的微生物及利用其生产霉孢菌素样氨基酸的方法 - Google Patents

生产霉孢菌素样氨基酸的微生物及利用其生产霉孢菌素样氨基酸的方法 Download PDF

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CN111527211B
CN111527211B CN201880066300.6A CN201880066300A CN111527211B CN 111527211 B CN111527211 B CN 111527211B CN 201880066300 A CN201880066300 A CN 201880066300A CN 111527211 B CN111527211 B CN 111527211B
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金率
李圭成
李周姬
石宗哲
张在佑
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CJ CheilJedang Corp
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Abstract

本申请涉及生产霉孢菌素样氨基酸的微生物，以及使用相同微生物生产霉孢菌素样氨基酸的方法。本申请的微生物具有提高的的生产霉孢菌素样氨基酸的能力，可以有效地用于霉孢菌素样氨基酸的生产。

Description

生产霉孢菌素样氨基酸的微生物及利用其生产霉孢菌素样氨 基酸的方法
技术领域
本公开涉及生产霉孢菌素样氨基酸的微生物，以及涉及利用该微生物生产霉孢菌素样氨基酸的方法。
背景技术
从太阳发射的紫外线辐射由UV-A(范围从大约320nm至400nm的紫外线A)、UV-B(范围从大约290nm至320nm的紫外线B)和UV-C(范围从大约100nm至280nm的紫外线C)组成。在太阳光线中，大约6％的UV-A和UV-B到达地球表面，另一方面，因为UV-C在地球的臭氧层和大气中被吸收并散射，它并未到达地球表面。
尽管这些UV光线可能会提供有益的方面，如体内维生素D的合成、皮肤疾病的治疗、杀菌效果等，但它们也会带来有害的方面，如晒伤、皮肤癌、衰老、光敏性皮肤病和诱变。众所周知，UV-A甚至穿透到真皮层，主要引起色素形成和皮肤老化，并参与光敏性皮肤病的发生(development)，而UV-B则涉及通过用高能光线穿透表皮和真皮层的上部而引起晒伤、色素形成和皮肤癌。
为了防止由太阳光线引起的这些副作用，已经尝试了阻挡日光。为此，防晒剂的类型可分为化学防晒剂和物理防晒剂。化学防晒剂主要通过吸收阻挡日光的穿透，而物理防晒剂通过日光的反射和散射阻挡日光。
化学防晒剂含有一种或多种吸收UV的成分，如主要吸收UV-B的PABA、PABA酯(戊基二甲基PABA、辛基二甲基PABA)、肉桂酸酯(西诺沙酯(cinoxate))、水杨酸酯(水杨酸高孟酯(Homomenthyl salicylate))、樟脑等，以及主要吸收UV-A的二苯甲酮(氧苯酮、二羟苯宗、舒利苯酮(sulisobenzone))、二苯甲酰甲烷、邻氨基苯甲酸等。尽管这些化学防晒剂可通过吸收UV光线来提供UV保护作用，但其中一些可引起皮肤或眼睛中的刺激。特别地，已知PABA、PABA酯、二苯甲酮、肉桂酸酯等引起接触性皮炎。另外，已经报道了与皮肤中发生超敏反应有关的一些其它的问题，且因此，在一些国家中化学防晒剂的使用及它们使用的量被限制。
含有自然界中存在的成分的物理防晒剂可通过反射和散射穿透皮肤的UV光线来保护皮肤。例如，物理防晒剂(如二氧化钛、滑石(硅酸镁)、氧化镁、氧化锌、高岭土等)可提供对UV-A和UV-B两者的UV保护作用。进一步，它们具有的优点在于它们不具有诸如接触性皮炎的副作用，并且不容易被水洗掉。相反，它们也具有的缺点在于，在实施其期望的制剂时，它可能难以维持物理防晒剂的有效量，并且当被施加到皮肤时它们留下白色的脱落物(cast)。
霉孢菌素样氨基酸(MAA)是在自然生物中发现的物质，并已知可有效吸收UVA(320nm至400nm)和UVB(290mm至320mm)。根据作为前体的氨基酸、环己烯酮或环己烯亚胺(cyclohexenimine)环的类型，自然界中有35种或更多种MAA，(Mar.Biol.,1991,108:157-166；Planta Med.,2015,81:813-820)。近来，在微观藻类中发现了各种糖基化的MAA，已报道它们起着卓越的抗氧化剂的作用(Journal of Photochemistry and Photobiology,2015,142:154-168)。另外，已知MAA不仅提供UV保护，而且还显示抗氧化、抗渗透和抗热应力(Comp.Biochem.Physiol.C Toxicol.Pharmacol.,2007,146:60-78；J.Photochem.Photobiol.B.,2007,89:29-35)。
然而，微观藻类中生产的MAA的量很少，在几μg的水平，并且通过培养微观藻类来分离、提取和纯化MAA的条件复杂，且因此，难以大规模生产MAA。
现有技术文献
非专利文献
(非专利文献1)Comp.Biochem.Physiol.B 1995,112:105-114。
(非专利文献2)FEMS Microbiol Lett.2007,269:1-10。
(非专利文献3)Ann.Rev.Physiol.2002,64:223-262。
(非专利文献4)Mar.Biol.1991,108:157-166。
(非专利文献5)Journal of Photochemistry and Photobiology B:Biology.2015,142:154-168
(非专利文献6)Biol.Rev.1999,74:311-345。
(非专利文献7)Mol.Biol.Evol.2006,23:1437-1443。
(非专利文献8)Science,2010,329:1653-1656。
(非专利文献9)Genomics 2010,95:120-128。
(非专利文献10)Geomicrobiol.J.1997.14:231-241。
(非专利文献11)Comp.Biochem.Physiol.C Toxicol.Pharmacol.2007.146:60-78。
(非专利文献12)Can.J.Bot.2003.81:131-138。
(非专利文献13)J.Photochem.Photobiol.B.2007,89:29-35。
(非专利文献14)J.Bacteriol.2011.193(21):5923-5928。
(非专利文献15)Planta Med.2015.81:813-820
(非专利文献16)ACS Appl.Mater.Interfaces.2015.7:16558-16564
(非专利文献17)Appl Environ Microbiol.2016,82(20):6167-6173
(非专利文献18)ChemBioChem.2015,16:320-327
(非专利文献19)Methods Mol Biol.2013,1073:43-7
(非专利文献20)Enzyme Microb Technol.,2016,Jan,82:96-104
(非专利文献21)Nature Review,2011,9:791-802
发明内容
技术问题
本发明人做出了大量的努力以增加微生物中MAA的生产。结果，通过对生产MAA的微生物中的3-脱氢奎尼酸脱水酶活性的失活的各种研究确认了MAA的生产被增加，从而完成了本公开。
技术方案
本公开的一个目的是提供生产霉孢菌素样氨基酸的微生物，其中与非修饰的微生物相比，3-脱氢奎尼酸脱水酶的活性被失活。
本公开的另一个目的是提供生产霉孢菌素样氨基酸的方法，其包括：
培养微生物；以及
从培养的微生物或培养基回收霉孢菌素样氨基酸。
有益效果
本公开的微生物显示提高的生产霉孢菌素样氨基酸的能力，且因此可以有效地用于霉孢菌素样氨基酸的生产。
具体实施方式
可以如下解释本公开的具体细节。
同时，本公开中公开的说明和实施方式可以分别应用于其它说明和实施方式。即，本文公开的各种要素的所有组合均属于本公开的范围。另外，本公开的范围不应被下文描述的具体描述限制。另外，本领域普通技术人员可仅使用常规实验就能够识别或确认本申请中描述的本发明的特定方面的许多等同物。此外，还意图将这些等同物包括在本公开中。
为了实现上述目的，本公开的一方面提供了生产霉孢菌素样氨基酸的微生物，其中与非修饰的微生物相比，3-脱氢奎尼酸脱水酶的活性被失活。
如本文所用，术语“3-脱氢奎尼酸脱水酶”是指催化由以下反应方案代表的可逆反应的酶，并且可以具体地将3-脱氢奎尼酸转化为3-脱氢莽草酸，但不限于此。
[反应方案]
如本文所用，术语“失活”是指微生物本来具有的酶蛋白的活性与其内源性活性或修饰前的活性相比被减弱的情况；蛋白质不以任何方式表达的情况；或蛋白质表达但表现无活性的情况。失活是这样的概念，其包括由于编码酶的多核苷酸中的修饰等，与微生物本来具有的酶的活性相比，酶本身的活性被减弱或消除的情况；由于编码酶的基因的表达的抑制或翻译的抑制等，与野生型微生物相比，细胞内的总酶活性水平被减弱或被消除的情况；编码酶的基因的部分或全部被缺失的情况；以及其组合，但不限于此。术语“非修饰的微生物”是指当通过由用于比较的微生物具有的特定蛋白质中由于自然或者人为因素遗传修饰而使微生物的性状被修饰时，在其修饰之前具有的由亲本菌株本来具有的特定蛋白质的活性的微生物。如本文所用，“非修饰的微生物”可与“具有内源性活性的微生物”互换使用。
酶活性的失活可以通过本领域公知的各种方法实现。方法的实例包括：1)缺失染色体上编码酶的基因的部分或全部的方法；2)修饰表达调节序列使得染色体上编码蛋白质的基因的表达被减弱的方法；3)修饰染色体上编码蛋白质的基因序列使得蛋白质的活性被移除或减弱的方法；4)引入与染色体上编码蛋白质的基因的转录物互补结合的反义寡核苷酸的方法(例如，反义RNA)；5)在Shine-Dalgarno序列的上游人工添加与在染色体上编码蛋白质的基因的Shine-Dalgarno序列互补的序列以形成二级结构，从而使核糖体的附着不可能的方法；以及6)在编码蛋白质的多核苷酸序列的开放阅读框(ORF)的3'端添加待逆转录的启动子的逆转录工程(RTE)方法，或其组合，但不特别限于此。
缺失染色体上编码蛋白质的基因的部分或全部的方法可以通过将染色体插入到微生物中的载体，用具有部分缺失的核酸序列的多核苷酸或标记基因置换染色体内编码内源性目标蛋白的多核苷酸来进行。作为缺失多核苷酸的部分或全部的方法的实例，可以使用通过同源重组缺失多核苷酸的方法，但不限于此。
修饰表达调节序列的方法可以通过缺失、插入、保守或非保守取代、或其组合来诱导表达调节序列中的修饰，以进一步减弱表达调节序列的活性来进行；或通过用具有更弱活性的核酸序列置换序列来进行。表达调节序列可包括启动子、操纵子序列、编码核糖体结合域的序列、以及调节转录和翻译的终止的序列，但不限于此。
修饰染色体上的基因序列的方法可以通过缺失、插入、保守或非保守取代、或其组合来诱导基因序列中的修饰，以进一步减弱酶的活性来进行；或通过用修饰以具有更弱活性的基因序列、或修饰以完全没有活性的基因序列置换序列来进行，但不限于此。
在多核苷酸是指在能够执行功能的多核苷酸的集合(assemblage)的情况下，可以将多核苷酸描述为基因。在本公开中，多核苷酸和基因可以互换使用，并且多核苷酸序列和核苷酸序列可以互换使用。
如本文所用，尽管术语“部分”可根据多核苷酸的种类而变化，但可具体地指1至300个核苷酸、更具体地1至100个核苷酸、和甚至更具体地1至50个核苷酸，但不特别地限于此。
在本公开的微生物中，与非修饰的微生物相比，选自2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶I/II、和3-脱氢奎尼酸合酶中的至少一种(具体地一种或更多、两种或更多种、三种或更多种、或所有酶)的活性可以被增强。
2-脱氢-3-脱氧磷酸庚糖酸醛缩酶是指催化由以下反应方案代表的可逆反应的酶，并且可以具体地合成3-脱氧-阿拉伯-庚酮糖7-磷酸(3-deoxy-arabino-heptulosonate7-phosphate)，但不限于此。
[反应方案]
磷酸烯醇丙酮酸合成酶是指催化由以下反应方案代表的可逆反应的酶，并且可以具体地合成磷酸烯醇丙酮酸，但不限于此。
[反应方案]
转酮醇酶I/II是指催化由以下反应方案代表的可逆反应的酶。
[反应方案]
景天庚酮糖7-磷酸+D-甘油醛3-磷酸＝D-核糖5-磷酸+D-木酮糖5-磷酸
3-脱氢奎尼酸合酶是指催化由以下反应方案代表的可逆反应的酶，并且可以具体地合成3-脱氢奎尼酸(3-DHQ)，但不限于此。
[反应方案]
如本文所用，术语“活性的增强”意思是与微生物具有的内源性活性或修饰前的活性相比，酶蛋白的活性被引入、或活性被增强。活性的“引入”意思是微生物本来不具有的特定多肽的活性被天然或人工表达。例如，活性的增强可以包括通过引入2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶I/II、和/或3-脱氢奎尼酸合酶的增强；或2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶I/II、和/或3-脱氢奎尼酸合酶的内源性活性的增强。具体地，本公开中活性的增强可以通过以下方法进行：
1)增加编码酶的多核苷酸的拷贝数的方法；
2)修饰表达调节序列使得多核苷酸的表达增加的方法；
3)修饰染色体上的多核苷酸序列使得酶活性增强的方法；以及
4)通过上述方法的组合进行修饰以增强活性的方法，但不限于此。
上述方法1)中的多核苷酸的拷贝数的增加可以以其中将多核苷酸可操作地连接至载体的形式进行，或者通过插入到宿主细胞的染色体中来进行，但不特别限于此。另外，在增加拷贝数的方面，可以通过将展示酶活性的外源多核苷酸或其中外源多核苷酸的密码子已被优化的修饰多核苷酸引入到宿主细胞中来进行。只要外源多核苷酸展示与酶的活性相同或相似的活性，就可以不受其来源或序列的限制而使用。可以由本领域技术人员通过选择本领域已知的合适的转化方法来进行引入，并且随着引入的多核苷酸在宿主细胞中表达，可以生产酶，从而增加其活性。
接下来，方法2)中的表达调节序列的修饰使得多核苷酸的表达增加，可以通过核酸序列的缺失、插入或非保守或保守取代、或其组合来诱导序列中的修饰，以进一步增强表达调节序列的活性，或通过用具有更强活性的核酸序列置换来进行，但不特别限于此。另外，表达调节序列可以包括启动子、操纵子序列、编码核糖体结合域的序列、调节转录和翻译的终止的序列等，但不特别限于此。
具体地，代替原始启动子的强异源启动子可能被连接到多核苷酸表达单元的上游区域。强启动子的实例包括CJ7启动子、lysCP1启动子、EF-Tu启动子、groEL启动子、aceA或aceB启动子等。更具体地，可以通过将多核苷酸与lysCP1启动子(W02009/096689)或CJ7启动子(W02006/065095)可操作地连接来增强编码酶的多核苷酸的表达率，但不限于此。
进一步，方法3)中的染色体上的多核苷酸序列的修饰可以通过核酸序列的缺失、插入、或非保守或保守取代、或其组合来诱导表达调节序列中的修饰，以进一步增强多核苷酸序列的活性，或通过用修饰的多核苷酸序列置换多核苷酸序列以具有更强活性来进行，但不特别限于此。
最后，方法4)中通过方法1)至方法3)的组合来增强活性的修饰方法可以通过组合应用以下方法中的至少一个来进行：增加编码蛋白质的多核苷酸的拷贝数；修饰表达调节序列使得多核苷酸的表达增加；修饰染色体上的多核苷酸序列，以及修饰展示酶的活性的外源多核苷酸或其密码子优化的修饰的多核苷酸。
如本文所用，术语“载体”是指为能够在适当的宿主中表达目标蛋白，可操作地被连接至合适的调节序列的，含有编码目标蛋白的多核苷酸的核苷酸序列的DNA构建体。调节序列包括能够起始转录的启动子、用于控制转录的任何操纵子序列、编码适当的mRNA核糖体结合结构域的序列、和调节转录和翻译的终止的序列。在转化到合适的宿主细胞中之后，载体可以与宿主基因组无关地复制或起作用，和可以被整合入宿主基因组本身。
只要本公开中使用的载体可在宿主细胞中复制，其不受特别地限制，并且可以使用本领域中已知的任何载体。常规使用的载体的实例可包括天然或重组质粒、粘粒、病毒和噬菌体。例如，作为噬菌体载体或粘粒载体，可以使用pWE15、M13、MBL3、MBL4、IXII、ASHII、APII、t10、t11、Charon4A、Charon21A等，且作为质粒载体，可以使用基于pBR、基于pUC、基于pBluescriptII、基于pGEM、基于pTZ、基于pCL、基于pET等的载体。具体地，可以使用载体pDZ、pACYC177、pACYC184、pCL、pECCG117、pUC19、pBR322、pMW118、pCC1BAC等，但载体不限于此。
可以在本公开中使用的载体不受特别地限制，并且可以使用已知的表达载体。另外，可通过用于染色体插入的载体将编码目标蛋白的多核苷酸插入到染色体中。可以使用本领域已知的任何方法，例如通过同源重组，将多核苷酸插入到染色体中，但不限于此。可以进一步包括用于确认插入到染色体中的选择标记。选择标记用于选择用载体转化的细胞(即，为了确认目标核酸分子是否已被插入)，以及可以使用提供可选表型(如药物抗性、营养需求、对细胞毒剂的抗性、或表面蛋白的表达)的标记。在用选择剂处理的环境下，只有表达选择标记的细胞能存活或表达其它表型性状，并且因此转化的细胞能容易地被选择。
如本文所用，术语“转化”是指将包括编码目标多肽的多核苷酸的载体引入到宿主细胞中，从而使多核苷酸编码的蛋白质能够在宿主细胞中表达的过程。只要转化的多核苷酸可在宿主细胞中表达，那么无论它是被插入到宿主细胞的染色体中并位于其中、或位于染色体外都无关紧要，且这两种情况都可能被包括在内。另外，多核苷酸包括编码目标多肽的DNA和RNA。只要多核苷酸可被引入到宿主细胞中并在其中表达，它可以以任何形式被引入。例如，多核苷酸可以以表达盒的形式被引入到宿主细胞中，表达盒是包括自主表达所必要的所有元件的基因构建体。表达盒可常规地包括可操作地连接到多核苷酸的启动子、终止子、核糖体结合域和终止密码子。表达盒可以是能够自复制的表达载体的形式。另外，多核苷酸可以原样被引入到宿主细胞中并可操作地被连接到其在宿主细胞中表达所必要的序列，但不限于此。转化载体的方法包括将核酸引入到细胞中的任何方法，并且可以通过根据宿主细胞选择本领域已知的合适的标准技术来进行。例如，可以经由电穿孔、磷酸钙(CaPO4)沉淀、氯化钙(CaCl2)沉淀、显微注射、聚乙二醇(PEG)技术、DEAE-葡聚糖技术、阳离子脂质体技术、乙酸锂-DMSO技术等进行转化，但方法不限于此。
进一步，如上所用，术语“可操作地连接”是指起始和介导编码本公开的目标蛋白的多核苷酸转录的上述多核苷酸序列与启动子序列之间的功能性连接。可操作连接可使用本领域已知的遗传重组技术制备，并且位点特异性DNA切割和连接可使用本领域已知的酶(如裂解酶、连接酶等)进行，但不限于此。
3-脱氢奎尼酸脱水酶、2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶、和3-脱氢奎尼酸合酶的遗传信息可从已知数据库获得，且其实例包括美国国家生物技术信息中心(National Center for Biotechnology Information(NCBI))的GenBank等，但是已知数据库不限于此。
因为根据微生物种类或微生物展示活性的蛋白质的氨基酸序列中可能存在差异，所以3-脱氢奎尼酸脱水酶、2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶、和3-脱氢奎尼酸合酶可能不受它们的来源或序列限制。
具体地，3-脱氢奎尼酸脱水酶可以是包括SEQ ID NO:72或80的氨基酸序列的蛋白质，并且2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶、和3-脱氢奎尼酸合酶可以是分别包括SEQ ID NO:74、76、78和84的氨基酸序列的蛋白质，但不限于此。在本公开中，“包括氨基酸序列的蛋白质”可以与表述“具有氨基酸序列的蛋白质”或“由氨基酸序列组成的蛋白质”互换使用。
另外，在本公开中，只要蛋白质具有与每种酶的生物活性相同或相应的生物活性，酶可以不仅包括具有上述SEQ ID NO:的氨基酸序列的蛋白质，而且包括与上述氨基酸序列具有80％或更多、具体地90％或更多、更具体地95％或更多、甚至更具体地99％或更多的同源性的蛋白质。
进一步，明显的是，作为与上述序列具有同源性的氨基酸序列，只要氨基酸序列与具有上述SEQ ID NO:的氨基酸序列的酶蛋白的生物活性具有实质上相同或相应的生物活性，则缺失、修饰、取代或添加了部分序列的氨基酸序列也落入本公开的范围内。
本公开的编码3-脱氢奎尼酸脱水酶、2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶、和3-脱氢奎尼酸合酶的多核苷酸只要具有与每种酶的生物活性相同或相应的生物活性，可包括具有上述SEQ ID NO:的氨基酸序列的多核苷酸、或编码与氨基酸序列具有80％或更多、具体地90％或更多、更具体地95％或更多、甚至更具体地99％或更多的同源性的蛋白质的多核苷酸。
另外，至于编码2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶、和3-脱氢奎尼酸合酶的多核苷酸，考虑到由于密码子简并性，生物表达蛋白质优选的密码子，可以在不改变多肽的氨基酸序列的范围内对编码的区域实施各种修饰。因此，多核苷酸可不受限制地包括编码每种酶蛋白的任何多核苷酸序列。
进一步，可以不受限制地包括可从已知的基因序列——例如，在严格条件下与核苷酸序列的全部或部分互补的序列杂交以编码具有3-脱氢奎尼酸脱水酶、2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶、和3-脱氢奎尼酸合酶活性的蛋白质的任何序列——制备的探针。
如本文所用，术语“同源性(homology)”是指与给定氨基酸序列或核苷酸序列的一致程度，并且可以表示为百分比。在本说明书中，具有与给定氨基酸序列或核苷酸序列的活性相同或相似的活性的同源序列，可以用术语“％同源性”表明。例如，可以使用用于计算诸如得分、同一性和相似性的参数的标准软件(具体地，BLAST 2.0)，或通过在限定的严格条件下经由杂交实验比较序列，来确认同源性，并且限定的适当的杂交条件在本领域技术内并且可以通过本领域技术人员众所周知的方法来确定(例如，J.Sambrook等人,MolecularCloning,A Laboratory Manual,2nd Edition,Cold Spring Harbor Laboratory press,Cold Spring Harbor,New York,1989；F.M.Ausubel等人,Current Protocols inMolecular Biology,John Wiley&Sons,Inc.,New York)。术语“严格条件”是指允许多核苷酸之间特异性杂交的条件。这些条件被具体地公开于文献(例如，J.Sambrook等人)中。
如本文所用，术语“霉孢菌素样氨基酸(MAA)”是指吸收紫外光线的环状化合物。在本公开中，只要霉孢菌素样氨基酸可吸收UV光线，可以不受限制地包括任何霉孢菌素样氨基酸，但是其具体地可以是含有环己烯酮或环己烯亚胺的中心环的化合物；或其中各种物质(如氨基酸等)被结合到中心环的化合物。更具体地，它可以是霉孢菌素-2-甘氨酸、palythinol、palythenic acid、deoxygadusol、霉孢菌素-甲胺-苏氨酸、霉孢菌素-甘氨酸-缬氨酸、palythine、asterina-330、shinorine、porphyra-334、euhalothece-362、霉孢菌素-甘氨酸、霉孢菌素-鸟氨酸、霉孢菌素-赖氨酸、霉孢菌素-谷氨酸-甘氨酸、霉孢菌素-甲胺-丝氨酸、霉孢菌素-牛磺酸、palythene、palythine-丝氨酸、palythine-丝氨酸-硫酸盐、palythinol、usujirene或其组合。
在本公开中，霉孢菌素样氨基酸可与MAA和MAAs互换使用。
如本文所用，术语“生产霉孢菌素样氨基酸的微生物”是指包括参与霉孢菌素样氨基酸的生物合成的酶的基因或基因的簇的微生物。另外，如本文所用，术语“霉孢菌素样氨基酸生物合成基因”是指编码参与霉孢菌素样氨基酸的生物合成中的酶的基因，并且还包括基因的簇。只要包括基因的微生物可以生产霉孢菌素样氨基酸，霉孢菌素样氨基酸生物合成基因包括微生物的外源和/或内源基因。外源基因可以是同源和/或异源的。
只要包括霉孢菌素样氨基酸生物合成基因的微生物产生参与霉孢菌素样氨基酸生物合成中的酶并因此产生霉孢菌素样氨基酸，则霉孢菌素样氨基酸生物合成基因可以不受衍生自该基因的微生物的种类限制。具体地，它可以是蓝细菌(cyanobacteria)的物种：多变鱼腥藻(Anabaena variabilis)、点状念珠藻(Nostoc punctiforme)、产泡节球藻(Nodularia spumigena)、蓝丝菌属(Cyanothece sp.)PCC 7424、鞘丝藻属(Lyngbya sp.)PCC 8106、铜绿微囊藻(Microcystis aeruginosa)、原型微鞘藻(Microcoleuschthonoplastes)、蓝丝菌属(Cyanothece sp.)ATCC 51142、瓦氏鳄球藻(Crocosphaerawatsonii)、蓝丝菌属(Cyanothece sp.)CCY 0110、静水柱孢藻属(Cylindrospermumstagnale sp,)PCC 7417、嗜盐隐杆藻(Aphanothece halophytica)或红海束毛藻(Trichodesmium erythraeum)；或真菌的物种：稻瘟病菌(Magnaporthe oryzae)、小麦黄斑叶枯病菌(Pyrenophora tritici-repentis)、棒曲霉菌(Aspergillus clavatus)、赤球丛赤壳(Nectria haematococca)、构巢曲霉(Aspergillus nidulans)、玉蜀黍赤霉(Gibberella zeae)、苜蓿黄萎病菌(Verticillium albo-atrum)、灰葡萄孢霉(Botryotinia fuckeliana)、或小麦颖枯病菌(Phaeosphaeria nodorum)；或海葵(Nematostella vectensis)、三角异囊藻(Heterocapsa triquetra)、尖尾藻(Oxyrrhismarina)、微小卡罗藻(Karlodinium micrum)、奇迹束丝放线菌(Actinosynnema mirum)等，但不限于此。
根据一个实施方式，本公开的生产霉孢菌素样氨基酸的微生物可以包括霉孢菌素样氨基酸生物合成基因。
具体地，只要微生物可生产霉孢菌素样氨基酸，霉孢菌素样氨基酸生物合成基因可以不被酶或衍生的微生物的名称限制，并且可以具体地包括选自2-脱甲基(demetyl)4-deoxygadusol合酶、O-甲基转移酶和C-N连接酶的至少一种、具体地一种或更多种、两种或更多种、或三种或更多种、或所有的酶蛋白；或编码具有与其相同和/或相似活性的酶蛋白的基因。
例如，2-脱甲基4-deoxygadusol合酶是将景天庚酮糖-7-磷酸转化为2-脱甲基-4-deoxygadusol的酶。O-甲基转移酶是将2-脱甲基-4-deoxygadusol转化为4-deoxygadusol的酶，并且4-deoxygadusol的糖基化(glycylation)由C-N连接酶催化。
另外，生产霉孢菌素样氨基酸的微生物可以包括具有将另外的氨基酸残基附接到霉孢菌素样氨基酸的活性的酶的基因，或者基因的簇。只要生产霉孢菌素样氨基酸的微生物可以生产附接有两个或更多个氨基酸残基的霉孢菌素样氨基酸，基因或基因的簇可以不受酶或衍生的微生物的名称限制，并且可以具体地包括选自非核糖体肽合成酶(NRPS)、非核糖体肽合成酶样酶(non-ribosomal peptide synthetase-like enzyme)(NRPS样酶(NRPS-like enzyme))、和D-Ala D-Ala连接酶(DDL)的至少一种、具体地一种或更多种、两种或更多种、三种或更多种、或所有的酶蛋白；或编码具有与其相同和/或相似活性的酶蛋白的基因。一些霉孢菌素样氨基酸可在霉孢菌素-甘氨酸中包括第二个氨基酸残基。选自非核糖体肽合成酶、非核糖体肽合成酶样酶、和D-Ala D-Ala连接酶的至少一种酶可以将第二个氨基酸残基附接至霉孢菌素-甘氨酸。
根据一个实施方式，只要酶具有将第二个氨基酸附接至霉孢菌素-甘氨酸的活性，如非核糖体肽合成酶、非核糖体肽合成酶样酶和D-Ala D-Ala连接酶，生产霉孢菌素样氨基酸的微生物可以包括任何酶，而不限于酶的名称或衍生的微生物的物种。
在一个实施方式中，在多变鱼腥藻中发现的非核糖体肽合成酶样酶(Ava_3855)或在点状念珠藻中发现的D-Ala D-Ala连接酶(NpF5597)可以通过将丝氨酸残基附接到霉孢菌素-甘氨酸生产shinorine。在另一个实施方式中，霉孢菌素-2-甘氨酸可以通过经由在嗜盐隐杆藻中发现的D-Ala D-Ala连接酶同系物(Ap_3855)的第二个甘氨酸残基的附接而形成。相似地，在奇迹束丝放线菌中，丝氨酸或丙氨酸可通过D-Ala D-Ala连接酶附接以形成shinorine或霉孢菌素-甘氨酸-丙氨酸。根据本公开的一个实施方式的微生物可以包括由选自上述酶或具有与其相同和/或相似活性的酶的适合于生产期望的霉孢菌素样氨基酸的酶。
可以在本公开中使用的2-脱甲基4-deoxygadusol合酶、O-甲基转移酶和C-N连接酶、非核糖体肽合成酶、非核糖体肽合成酶样酶和/或D-Ala D-Ala连接酶可不受衍生的微生物的物种限制，并且只要已知其具有相同和/或相似的功能和活性，可以不受限制地包括任何酶。另外，这些酶之间的同源性的数值范围可以不被限制。例如，静水柱孢藻PCC 7417的MylA、MylB、MylD、MylE和MylC与衍生自多变鱼腥藻和点状念珠藻的2-脱甲基4-deoxygadusol合酶、O-甲基转移酶和C-N连接酶和D-Ala D-Ala连接酶同源，并且这些同系物之间的相似性是约61％至88％(Appl Environ Microbiol,2016,82(20),6167-6173；JBacteriol,2011,193(21),5923-5928)。即，只要已知其展示相同和/或相似的功能和效果，可以用于本公开的酶可以不特别地受衍生的微生物的物种或序列同源性限制。进一步，将现有技术中公开的非专利文献通过引用以其整体并入本文。
另外，霉孢菌素样氨基酸生物合成基因可以是编码包括SEQ ID NO:2、4、86、88、90、92、94、96、98、100、102或104的氨基酸序列的蛋白质的多核苷酸，但不限于此。
另外，霉孢菌素样氨基酸生物合成基因可以包括编码包括与SEQ ID NO:2、4、86、88、90、92、94、96、98、100、102或104的氨基酸序列具有50％、60％或70％或更多，具体地80％或更多，更具体地90％或更多、甚至更具体地95％、和甚至更具体地99％的同源性的氨基酸序列的蛋白质的核苷酸序列，以及只要微生物生产霉孢菌素样氨基酸，就可以不受限制地包括编码具有不同于(deviating from)上述的同源性的蛋白质的任何核苷酸序列。具体地，霉孢菌素样氨基酸生物合成基因可以包括但不限于SEQ ID NO:1、3、85、84、89、91、93、95、97、99、101或103的核苷酸序列。
另外，明显地，作为与上述氨基酸序列具有同源性的氨基酸序列，只要氨基酸序列具有与上述具有SEQ ID NO:的氨基酸序列的蛋白质实质上相同或相应的生物活性，具有部分序列的缺失、修饰、取代或添加的氨基酸序列也落入本公开的范围内。
另外，考虑到由于密码子简并性，表达蛋白质的生物优选的密码子，可以在不改变多肽的氨基酸序列的范围内对编码的区域执行各种修饰。因此，霉孢菌素样氨基酸生物合成基因可不受限制地包括编码参与霉孢菌素样氨基酸合成中的蛋白质的任何核苷酸序列。
另外，可以不受限制地包括可从已知的基因序列——例如，在严格条件下与核苷酸序列的全部或部分互补的序列杂交以编码参与霉孢菌素样氨基生物合成中的蛋白质的任何序列——制备的探针。
根据实施方式，生产霉孢菌素样氨基酸的微生物可以包括衍生自不同来源的霉孢菌素样氨基酸生物合成基因。
在本公开中，本文的蛋白质的失活、蛋白质的活性的增强、和/或基因的引入可以同时、顺序地或以相反的顺序进行。
如本文所用，术语“生产霉孢菌素样氨基酸的微生物”具有内源性和/或外源引入的霉孢菌素样氨基酸生物合成基因，并因此可以生产霉孢菌素样氨基酸，并且另外，它可以是其中通过失活内源性3-脱氢奎尼酸脱水酶活性来增加生产霉孢菌素样氨基酸的能力的微生物。霉孢菌素样氨基酸生物合成基因的引入和3-脱氢奎尼酸脱水酶的失活可以同时、顺序地或以相反的顺序进行。
另外，本公开的微生物可以是本来具有霉孢菌素样氨基酸生物合成基因的天然微生物；和已经引入有异源和/或同源霉孢菌素样氨基酸生物合成基因的微生物，但不限于此。
另外，本公开的微生物可以是具有由内源和/或引入的霉孢菌素样氨基酸生物合成相关基因编码的酶的增强活性的微生物，但不限于此。
另外，只要本公开的微生物具有修饰前的3-脱氢奎尼酸脱水酶的活性，本公开的微生物可以没有限制，并且具体地，它可以是棒杆菌属(genus Corynebacterium)的微生物、埃希氏菌属(genus Escherichia)的微生物、或酵母。
棒杆菌属的微生物可以具体地是谷氨酸棒杆菌(Corynebacterium glutamicum)、产氨棒杆菌(Corynebacterium ammoniagenes)、乳发酵短杆菌(Brevibacteriumlactofermentum)、黄色短杆菌(Brevibacterium flavum)、热氨基棒杆菌(Corynebacterium thermoaminogenes)、有效棒杆菌(Corynebacterium efficiens)等，并且更具体地，它可以是谷氨酸棒杆菌，但不限于此。
埃希氏菌属的微生物可以具体地是艾伯替埃希氏菌(Escherichia albertii)、大肠杆菌(Escherichia coli)、弗氏埃希氏菌(Escherichia fergusonii)、赫氏埃希氏菌(Escherichia hermannii)、伤口埃希氏菌(Escherichia vulneris)等，并且更具体地，它可以是大肠杆菌，但不限于此。
酵母可以具体地是属于子囊菌门(Ascomycota)的酵母亚门(Saccharomycotina)、外囊菌亚门(Taphrinomycotina)、或担子菌门(Basidiomycota)的伞菌亚门(Agaricomycotina)、柄锈菌亚门(Pucciniomycotina)等的微生物，并且更具体地，它可以是酵母属(genus Saccharomyces)的微生物、裂殖酵母属(genus Schizosaccharomyces)的微生物、发夫酵母属(genus of Phaffia)的微生物、克鲁维酵母属(genus ofKluyveromyces)的微生物、毕赤酵母属(genus of Pichia)的微生物、或假丝属(genus ofCandida)的微生物、并且甚至更具体地，它可以是酿酒酵母(Saccharomyces cerevisiae)，但不限于此。
在本公开中，生产霉孢菌素样氨基酸的酵母可以引入有编码3-脱氢奎尼酸合酶的基因或可以具有增强的3-脱氢奎尼酸合酶活性。例如，如果为了使酵母中的3-脱氢奎尼酸脱水酶活性失活而缺失部分或全部ARO1，则3-脱氢奎尼酸合酶的功能丧失，并且因此其可能难以合成3-DHQ。因此，当缺失酵母中部分或全部ARO1基因时，可以引入编码3-脱氢奎尼酸合酶的基因(例如，aroB基因)，但不限于此。
本公开的另一方面提供了生产霉孢菌素样氨基酸的方法，其包括：
培养本公开的微生物；以及
从培养的微生物或培养基中回收霉孢菌素样氨基酸。
“微生物”和“霉孢菌素样氨基酸”如上所述。
如本文所用，术语“培养”是指在适当调节的环境中使微生物生长。本公开的培养过程可以根据本领域已知的适当的培养基和培养条件来实现。根据待选择的微生物，可以由本领域技术人员容易地调节培养过程以使用。培养微生物的步骤可以通过已知的分批培养法、连续培养法、补料分批培养法等进行，但不特别限于此。对用于培养本公开的微生物的培养基和其它培养条件没有特别的限制，而可以使用用于微生物常规培养的任何培养基。具体地，本公开的微生物可以在需氧条件下在含有合适的碳源、氮源、磷源、无机化合物、氨基酸和/或维生素等的常规培养基中培养，同时调节温度、pH等。具体地，可以使用碱性化合物(例如，氢氧化钠、氢氧化钾或氨)或酸性化合物(例如，磷酸或硫酸)将培养物的pH调节至合适的pH(例如，pH 5至9、具体地pH 6至8、和最具体地pH 6.8)，但不限于此。另外，可以将氧气或含氧气体注入到培养物中以维持培养物的需氧状态；或可以注入氮气、氢气或二氧化碳气体、或不注入气体以维持厌氧或微需氧状态，但不限于此。另外，培养温度可以被维持在20至45℃下、具体地在25至40℃下，并且培养可以进行约10至160小时，但培养不限于此。另外，在使用消泡剂(如脂肪酸聚乙二醇酯等)的培养期间可防止泡沫形成，但不限于此。
另外，作为用于待使用培养基的碳源，可以单独或组合使用糖和碳水化合物(例如，葡萄糖、蔗糖、乳糖、果糖、麦芽糖、糖蜜、淀粉和纤维素)、油和脂肪(例如，大豆油、葵花籽油、花生油和椰子油)、脂肪酸(例如，棕榈酸、硬脂酸和亚油酸)、醇(例如，甘油和乙醇)、有机酸(例如，乙酸)等，但不限于此。作为氮源，可以单独或组合使用含氮有机化合物(例如，蛋白胨、酵母提取物、肉汁、麦芽提取物、玉米浆、大豆粉和尿素)或无机化合物(例如，硫酸铵、氯化铵、磷酸铵、碳酸铵和硝酸铵)等，但不限于此。作为磷源，可以单独使用或组合使用磷酸二氢钾、磷酸氢二钾、其相应的含钠盐等，但不限于此。进一步，培养基中可含有必需的促生长物质，如其它金属盐(例如，硫酸镁或硫酸铁)、氨基酸、维生素等。
培养物生产的MAA可能被分泌到培养基中，或者可保留在细胞中。
如本文所用，术语“培养基”是指在培养本公开的微生物后获得的产物。培养基是包括含有微生物的形式和其中通过离心、过滤等从含有微生物的培养液移除微生物的形式两者的概念。
在回收在本公开的培养步骤中生产的MAAs的步骤中，可以使用本领域已知的适当方法从培养液中收集期望的MAAs。例如，可以使用离心、过滤、阴离子交换色谱、结晶、HPLC等，并且可以使用本领域已知的适当方法从培养的微生物或培养基中回收期望的MAAs。另外，回收MAAs的步骤可以进一步包括分离工艺和/或纯化步骤。
实施例
将通过实施例的方式更详细地描述本公开。然而，给出这些实施例仅出于说明性目的，并且本公开的范围不意图限于这些实施例或不被这些实施例限制。
＜衍生自大肠杆菌(E.coli)的MAAs生产重组微生物的构建及使用其的MAAs的生产>
实施例1：衍生自微观藻类的过表达shinorine生物合成基因的载体的构建
基于多变鱼腥藻(A.variabilis)的shinorine生物合成基因簇由四个基因组成，即2-脱甲基4-deoxygadusol合酶、O-甲基转移酶、C-N连接酶和非核糖体肽合成酶，并且点状念珠藻(蓝细菌属的物种)也可以使用该基因产生shinorine。使用多变鱼腥藻ATCC29413和点状念珠藻(N.punctiforme)ATCC29133的基因组DNA鉴定了shinorine生物合成基因簇。使用两种类型的载体pECCG 117_Ptrc_GFP_终止子和pECCG 117_Pcj1_GFP_终止子构建了四种载体，每种载体分别含有衍生自多变鱼腥藻ATCC29413和点状念珠藻ATCC29133的shinorine生物合成基因(Ava_ABCD和Npr_ABCD)。下表1中总结了四种shinorine生物合成基因表达载体的名称、以及用于构建载体的模板和引物。
[表1]
在使用模板和引物获得基因片段后，利用HD克隆试剂盒(ClontechLaboratories,Inc.)将每个基因片段连接至用EcoRV/XbaI限制性酶处理的pECCG117_Ptrc_GFP_终止子和pECCG 117_Pcj1_GFP_终止子载体。这些表达载体分别命名为pECCG117_Ptrc_Ava_ABCD、pECCG117_Pcj1_Ava_ABCD、pECCG117_Ptrc_Npr_ABCD、和pECCG117_Pcj1_Npr_ABCD，并通过测序确认了每种表达载体。Ava_ABCD和Npr_ABCD的核苷酸序列和氨基酸序列分别被指定为SEQ ID NO:1、SEQ ID NO:2、SEQID NO:3和SEQ ID NO:4。
实施例2：引入有shinorine生物合成基因表达载体的菌株的shinorine生产能力 的评价
为了确认大肠杆菌中的MAA生产能力，将实施例1中制备的四种质粒引入到野生型大肠杆菌W3110菌株中，以制备具有增强的shinorine生物合成的菌株。将因此生产的菌株平皿接种在含有卡那霉素的LB固体培养基上，以及然后在37℃的培养箱中培养过夜。将一个铂环(platinum loop)的在LB固体培养基中过夜培养的每种菌株接种到25ml的效价培养基[培养基组成：40g/L的葡萄糖、0.3g/L的KH2PO4、0.6g/L的K2HPO4、15g/L的(NH4)2SO4、1g/L的MgSO4·7H2O、2.5g/L的NaCl、1.2g/L的柠檬酸钠、2.5g/L的酵母提取物、40g/L的碳酸钙：pH7.0]中，以及然后在37℃下的培养箱中以200rpm的速率培养48小时。结果示于下表2中。
[表2]
菌株的名称 OD(600nm) Shinorine浓度(mg/L)
W3110 22.3 -
W3110/pECCG117_Ptrc_Ava_ABCD 20.1 121
W3110/pECCG117_PCJ1_Ava_ABCD 19.8 382
W3110/pECCG117_Ptrc_Npr_ABCD 21.0 96
W3110/pECCG117_PCJ1_Npr_ABCD 20.2 332
如上表2中所示，当shinorine生物合成基因被引入W3110时，确认了shinorine生产是可能的。另外，确认了通过增加启动子(引入启动子PCJ1)的强度增强生物合成途径而增加了shinorine的生产。
实施例3：其中3-脱氢奎尼酸脱水酶被失活的菌株的构建
Ava-A——微观藻类中用于MAAs的生物合成的第一个基因——共享并使用莽草酸途径中的DHQ(3-脱氢奎尼酸)和磷酸戊糖途径中的SH-7P(景天庚酮糖7-磷酸)作为底物。为了制备其中3-脱氢奎尼酸脱水酶通过aroD基因的缺失而被失活的菌株，采用了利用λ红重组酶(lambda red recombinase)的同源重组方法。使用了pKD3的氯霉素抗性基因作为基因插入标记，并且利用SEQ ID NO:9(正向)和SEQ ID NO:10(反向)的引物，通过PCR构建了包括部分aroD基因和pKD3质粒的氯霉素抗性基因的aroD缺失盒(aroD-deleted cassette)。在制备其中待缺失aroD基因(SEQ ID NO:71和72)的菌株(野生型大肠杆菌W3110)之后，将含有λ红重组酶基因的pKD46质粒转化到菌株中，并且随后，利用阿拉伯糖诱导了基因的表达，以制备感受态细胞。通过电穿孔将aroD缺失盒引入到感受态细胞中，以及然后将细胞平皿接种在含30mg/L的氯霉素的LB固体培养基上。利用SEQ ID NO:11(正向)和SEQ ID NO:12(反向)的引物对由此获得的菌株进行PCR，并且通过观察1300bp的扩增片段，确认了aroD基因缺失。
实施例4：其中3-脱氢奎尼酸脱水酶被失活的菌株的shinorine生产能力的评价
将实施例1中制备的4种质粒中由PCJ1启动子控制表达的2种质粒引入到实施例3中制备的其中缺失了aroD基因的菌株(W3110ΔaroD/pECCG117_PCJ1_Ava_ABCD和W3110ΔaroD/pECCG117_PCJ1_Npr_ABCD)中，以及然后将菌株平皿接种在含有卡那霉素的LB固体培养基上。随后，将aroD缺失菌株和aroD未缺失菌株分别在37℃的培养箱中培养过夜，并将一个铂环的每种菌株接种到25mL的效价培养基[培养基组成：40g/L的葡萄糖、0.3g/L的KH2PO4、0.6g/L的K2HPO4、15g/L的(NH4)2SO4、1g/L的MgSO4·7H2O、2.5g/L的NaCl、1.2g/L的柠檬酸钠、2.5g/L的酵母提取物、40g/L的碳酸钙：pH7.0]中，以及然后在37℃下的培养箱中以200rpm的速率培养48小时。结果示于下表3中。
[表3]
菌株的名称 OD(600nm) Shinorine浓度(mg/L)
W3110/pECCG117_PCJ1_Ava_ABCD 20.3 352
W3110ΔaroD/pECCG117_PCJ1_Ava_ABCD 18.7 683
W3110/pECCG117_PCJ1_Npr_ABCD 18.9 331
W3110ΔaroD/pECCG117_PCJ1_Npr_ABCD 17.9 601
如表3中所示，与在aroD未缺失的菌株中生产的shinorine的浓度相比，在aroD缺失菌株中生产的shinorine的浓度分别增加了194％和182％。因此，W3110ΔaroD/pECCG117_PCJ1_Ava_ABCD菌株和W3110ΔaroD/pECCG117_PCJ1_Npr_ABCD菌株(它们是aroD缺失菌株)被命名为CB06-0017和CB06-0018，并于2017年6月26日根据布达佩斯条约保藏在韩国微生物保藏中心(KCCM)，保藏号分别为KCCM12044P和KCCM12045P。
实施例5：具有2-脱氢-3-脱氧磷酸庚糖酸醛缩酶/磷酸烯醇丙酮酸合成酶/转酮醇 酶I/II增强活性的菌株的构建
为了增加MAA生产微生物的MAA生产能力，增强了2-脱氢-3-脱氧磷酸庚糖酸醛缩酶/磷酸烯醇丙酮酸合成酶/转酮醇酶I/II的活性。具体地，进一步引入了源自大肠杆菌W3110的三个基因，即aroG(2-脱氢-3-脱氧磷酸庚糖酸醛缩酶；SEQ ID NO:73和74)、ppsA(磷酸烯醇丙酮酸合成酶；SEQ ID NO:75和76)和tktA(转酮醇酶I/II；SEQ ID NO:77和78)。构建了pSKH130-ΔfhuA-Pn-aroG-Pn-ppsA-Pn-tktA质粒，以增强aroG、ppsA和tktA基因。pSKH130-ΔfhuA-Pn-aroG-Pn-ppsA-Pn-tktA质粒的构建中所用的模板和引物示于下表4中。
[表4]
使用上述模板和引物，将aroG、ppsA和tktA基因片段通过PCR扩增，以及然后分别引入到用BamH1-Pst1限制酶切割的pSKH130-ΔfhuA载体中。测序被应用以确认载体的克隆与否和基因序列，以及然后通过电穿孔将载体转化到野生型大肠杆菌W3110和aroD缺失的大肠杆菌W3110ΔaroD中。通过一次重组(交叉(crossover))将转化的基因引入到染色体中，然后通过二次重组(交叉)将质粒区从染色体的切除。使用SEQ ID NO:19(正向)和SEQID NO:20(反向)的引物，在大肠杆菌转化菌株(其中完成了二次重组)中确认了aroG、ppsA和tktA基因的引入。
实施例6：具有2-脱氢-3-脱氧磷酸庚糖酸醛缩酶/磷酸烯醇丙酮酸合成酶/转酮醇 酶I/II增强活性的菌株的shinorine生产能力的评价
将实施例1中制备的4种质粒中由PCJ1启动子控制表达的2种质粒分别引入到实施例5中制备的引入有aroG、ppsA和tktA基因的菌株中，以及然后将菌株平皿接种在LB固体培养基上。随后，将菌株在37℃的培养箱中培养过夜，并且将一个铂环的每种菌株接种到实施例4的25mL的效价培养基中，以及然后在37℃下在培养箱中以200rpm的速率培养48小时。结果示于下表5中。
[表5]
如表5中所示，与对照组相比，在其中三个基因(aroG、ppsA和tktA)增强的菌株中生产的shinorine浓度增加了约300％。
实施例7：ava_ABCD染色体插入载体和菌株的构建
为了将shinorine生物合成基因引入到大肠杆菌中，制备了pSKH130ΔpinR::Ava-ABCD质粒。基于pECCG117_Ptrc_Ava_ABCD作为模板，利用SEQID NO:21(正向)和SEQ ID NO:22(反向)的引物对，对Ava_ABCD进行PCR。用HD克隆试剂盒(ClontechLaboratories,Inc.)将约7kb PCR片段连接到用BamHI和PstI限制酶处理的pSKH130ΔpinR载体，以制备pSKH130ΔpinR::Ava_ABCD。随后，分别利用SEQ ID NO:23和24、SEQ ID NO:25和26、以及SEQ ID NO:25和27的正向和反向引物对，对Ptrc和PCJ1启动子片段进行PCR，以控制Ava-ABCD的表达。然后，使用/>HD克隆试剂盒(Clontech Laboratories,Inc.)将片段连接到用ScaI限制酶处理的pSKH130ΔpinR::Ava_ABCD载体，以制备pSKH130ΔpinR::Ptrc-Ava-ABCD和pSKH130ΔpinR::PCJ1-Ava-ABCD。通过电穿孔将重组质粒转化到实施例5中制备的W3110ΔaroDΔfhuA::Pn-aroG-Pn-ppsA-Pn-tktA菌株中，并且通过一次重组(交叉)将菌株引入到染色体中，然后通过二次重组(交叉)从染色体切除除目的基因以外的载体区域。
通过使用SEQ ID NO:28(正向)和SEQ ID NO:29(反向)的引物的PCR，在其中完成了二次重组的大肠杆菌转化菌株中确认了Ava_ABCD基因的引入。
实施例8：ava_ABCD染色体插入菌株的shinorine生产能力的评价
将实施例7中制备的菌株平皿接种在LB固体培养基上，以及然后在37℃的培养箱中培养过夜。随后，将一个铂环的每种菌株接种到25ml的实施例4的效价培养基中，以及然后在37℃下的培养箱中以200rpm的速率培养48小时。结果示于下表6中。
[表6]
如表6中所示，当将Ava-ABCD引入染色体中时，确认生产了shinorine，并且其浓度根据启动子的强度而增加。然而，也确认了与通过质粒具有增强的shinorine生物合成的菌株相比，shinorine的生产被降低。当将pECCG117_PCJ1_Ava_ABCD质粒额外地引入到染色体上引入有Ava-ABCD的菌株中时，与染色体上仅引入有Ava-ABCD的菌株(基于CJ1启动子)和仅引入有质粒的菌株相比，shinorine生产分别增加了353％和152％。
实施例9：MAA基因过表达载体的构建及其MAA生产能力的评价
4-deoxygadusol和霉孢菌素-甘氨酸是在shinorine生物合成期间产生的中间体，且同时是具有防UV光线作用的霉孢菌素样氨基酸。为了确认是否可以在大肠杆菌的AroD缺失的菌株中生产这些物质，构建了载体。结果示于下表7中。
基于pECCG117_Ptrc_Ava_ABCD作为模板利用SEQ ID NO:30和31以及SEQ ID NO:30和32的引物对，对Ptrc_Ava_AB和Ptrc_Ava_ABC进行PCR。通过将PCR片段连接到用BamHI和SpeI限制酶处理的pECCG117 Prc GFP载体，制备了pECCG117_Ptrc_Ava_AB和pECCG117_Ptrc_Ava_ABC。以同样的方式，通过基于pECCG117_PCJ1_Ava_ABCD作为模板利用SEQ IDNO:30和31以及SEQ ID NO:30和32的引物对将获得的PCR片段连接到用BamHI和SpeI限制酶处理的pECCG117 Pcj1 GFP载体，制备了pECCG117_PCJ1_Ava_AB和pECCG117_PCJ1_Ava_ABC。Ava_AB和Ava_ABC的核苷酸序列和氨基酸序列被指定为SEQ ID NO:85至SEQ ID NO:88。
[表7]
通过常用的电脉冲法将由此制备的载体转化到实施例8中使用的W3110ΔaroDΔfhuA::Pn-aroG-Pn-ppsA-Pn-tktA菌株中，并且将每种菌株平皿接种在LB固体培养基上并在37℃培养箱中培养过夜。将在LB固体培养基中培养过夜的菌株接种到实施例4的25mL的效价培养基中，以及然后在37℃下在培养箱中以200rpm的速率培养48小时。培养完成后，通过液体高速色谱法测量MAAs的生产，并且测试的对于每种菌株的培养物中MAAs的浓度示于下表8中。
[表8]
如表8中所示，当Ava_AB和Ava_ABC基因被引入时，确认产生了4-deoxygadusol和霉孢菌素甘氨酸，且其量随着启动子强度的增强而增加。
<衍生自谷氨酸棒杆菌的MAA生产重组微生物的构建及使用其的MAA的生产>
实施例10：引入有shinorine生物合成基因过表达载体的菌株的shinorine生产能 力的评价
为了确认谷氨酸棒杆菌的MAA生产能力，将实施例1中制备的四种质粒引入到谷氨酸棒杆菌13032菌株中，以制备具有增强的shinorine生物合成的菌株，并且将菌株平皿接种在含有卡那霉素的BHIS固体培养基上并在30培养箱中培养过夜。将一个铂环的在BHIS固体培养基中过夜培养的每种菌株接种到25ml的效价培养基[培养基组成：40g/L的葡萄糖、1g/L的KH2PO4、10g/L的(NH4)2SO4、5g/L的MgSO4·7H2O、5g/L的NaCl、5g/L的酵母提取物、30g/L的碳酸钙：pH7.0]中，以及然后在37℃下的培养箱中以200rpm的速率培养48小时。结果示于下表9中。
[表9]
菌株的名称 OD(600nm) Shinorine浓度(mg/L)
c.gl 13032 72.1 -
c.gl 13032/pECCG117_Ptrc_Ava_ABCD 71.5 132
c.gl 13032/pECCG117_PCJ1_Ava_ABCD 69.8 496
c.gl 13032/pECCG117_Ptrc_Npr_ABCD 70.9 103
c.gl 13032/pECCG117_PCJ1_Npr_ABCD 71.4 421
如表9中所示，确认了当shinorine生物合成基因被引入到谷氨酸棒杆菌13032中时，shinorine生产是可能的，并且根据启动子的强度其生产可增加了高达375％。
实施例11：染色体上引入有shinorine生物合成基因的载体和菌株的构建
为了将shinorine生物合成基因引入到谷氨酸棒杆菌中，制备了pDCΔN1021_Ava_ABCD质粒。基于pECCG117_Ptrc_Ava_ABCD作为模板利用SEQ ID NO:33(正向)和SEQ ID NO:34(反向)的引物对，对Ava_ABCD进行PCR。使用HD克隆试剂盒(ClontechLaboratories,Inc.)将约7kb PCR片段连接至用NdeI限制酶处理的pDCΔN1021载体，以制备pDCΔN1021_Ava_ABCD。随后，分别利用SEQ ID NO:35和36、SEQ ID NO:37和38以及SEQID NO:39和40的正向和反向引物对，对CJ7、Lysc8和O2启动子片段进行PCR，以控制Ava-ABCD的表达。然后，使用/>HD克隆试剂盒(Clontech Laboratories,Inc.)将片段连接至用NdeI限制酶处理的pDCΔN1021_Ava_ABCD载体，以制备pDCΔN1021_Pcj7_Ava_ABCD、pDCΔN1021_Plysc8_Ava_ABCD和pDCΔN1021_PO2_Ava_ABCD。
通过电穿孔将重组质粒转化到谷氨酸棒杆菌13032中(van der Rest等人，1999)，并且通过一次重组(交叉)将重组质粒引入到染色体中，然后通过二次重组(交叉)从染色体切除质粒。
通过使用SEQ ID NO:33(正向)和SEQ ID NO:34(反向)的基因特异性引物对的PCR，在其中完成了二次重组的谷氨酸棒杆菌转化的菌株中确认了Ava_ABCD基因的引入。
实施例12：染色体上引入有shinorine生物合成基因的菌株的shinorine生产能力 的评价
将所有菌株平皿接种在BHIS固体培养基上，以及然后在30培养箱中培养过夜，以确认shinorine生产能力。将一个铂环的在BHIS固体培养基中培养过夜的每种菌株接种到25mL的实施例11的效价培养基中，以及然后在37℃下的培养箱中以200rpm的速率培养48小时。结果示于下表10中。
[表10]
菌株的名称 OD(600nm) Shinorine浓度(mg/L)
c.gl 13032 70.2 -
c.gl 13032△N1021_PCJ7_Ava_ABCD 76.1 36
c.gl 13032△N1021_Plysc8_Ava_ABCD 79.8 75
c.gl 13032△N1021_PO2_Ava_ABCD 72.5 173
如表10中所示，确认了当单拷贝的shinorine生物合成基因被引入到野生型谷氨酸棒杆菌中时，可以生产从36mg至173mg的量的shinorine。
实施例13：棒杆菌(Corynebacterium)aroD(3-脱氢奎尼酸脱水酶)缺失载体和菌 株的构建
如实施例3中提到的，制备了具有缺失的菌株，以确认是否可以通过aroD(3-脱氢奎尼酸脱水酶)的缺失来增加shinorine生产。为了制备位点特异性aroD基因(SEQ ID NO:79和80)缺失的谷氨酸棒杆菌菌株，构建了其中aroD开放阅读框被内源地缺失的pDC-ΔaroD质粒。通过基于谷氨酸棒杆菌ATCC 13032的基因组DNA作为模板，利用SEQ ID NO:41和42以及SEQ ID NO:43和44的正向和反向引物对，通过将交叉PCR产生的基因片段引入到pDC载体中，产生了pDC-ΔaroD的内源基因缺失。通过电穿孔将重组质粒转化到谷氨酸棒杆菌13032△N1021_PO2_Ava_ABCD中(van der Rest等人,1999)，并且通过一次重组(交叉)将质粒引入到染色体中，随后通过二次重组(交叉)从染色体切除质粒。
通过使用SEQ ID NO:41和44的基因特异性引物对的PCR，在其中完成了二次重组的谷氨酸棒杆菌转化菌株中确认了aroD基因的缺失。
实施例14：aroD(3-脱氢奎尼酸脱水酶)缺失的评价
将由于在谷氨酸棒杆菌13032△N1021_PO2_Ava_ABCD菌株中3-脱氢奎尼酸脱水酶的缺失而预期具有DHQ积累的菌株平皿接种在BHIS固体培养基上，并且在30培养箱中培养过夜。将一个铂环的在BHIS固体培养基中培养过夜的每种菌株的接种到25mL的效价培养基[培养基组成：40g/L的葡萄糖、1g/L的KH2PO4、10g/L的(NH4)2SO4、5g/L的MgSO4·7H2O、5g/L的NaCl、5g/L的酵母提取物、30g/L的碳酸钙：pH7.0]中，以及然后在37℃下的培养箱中以200rpm的速率培养48小时。结果示于下文表11中。
[表11]
如表11中所示，确认了缺失aroD基因时，shinorine的浓度与对照组相比提高了239％，并且随着通过pECCG117_PCJ1_Ava_ABCD进一步增强shinorine的生物合成，shinorine的浓度增加了。因此，aroD缺失菌株c.gl 13032N1021_PO2_Ava_ABCD_△aroD被命名为CB06-0019，并于2017年6月26日根据布达佩斯条约保藏在韩国微生物保藏中心(KCCM)，保藏号为KCCM12046P。
<衍生自酵母的MAA生产重组微生物的构建和利用其的MAAs生产>
实施例15：衍生自微观藻类的shinorine生物合成基因过表达酵母载体的构建
构建了基于多变鱼腥藻ATCC29413和点状念珠藻ATCC29133的基因组DNA，引入有shinorine生物合成基因的酿酒酵母(S.cerevisiae)载体。使用酿酒酵母的ADH、TEF和GPD启动子制备了载体，并且用于制备总共24种shinorine生物合成基因表达载体的模板和引物示于下表12中。Ava_A、Ava_B、Ava_C、Ava_D、Npr_A、Npr_B、Npr_C和Npr_D的核苷酸序列和氨基酸序列以酶的顺序被指定为SEQ ID NO:89至104。
[表12]
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利用T4连接酶(NEB)将使用上述模板和引物的组合通过PCR获得的基因片段连接至用BamH1/XhoI限制酶处理的p413/414/415/416-pADH/pTEF/pGPD-CYC1_终止子载体，从而制备了24种p413/414/415/416-pADH/pTEF/pGPD-A,B,C,D载体。通过测序技术确认了其每种表达载体是否得到构建及其基因序列。将由此制备的表达载体引入到野生型酿酒酵母CEN.PK-1D菌株中，以制备能够生产shinorine的菌株。
实施例16：引入有shinorine生物合成基因过表达载体的菌株的shinorine生产能 力的评价
为了确认酵母的MAA生产能力，将实施例15中制备的24种质粒引入到酿酒酵母CEN.PK-1D菌株(酿酒酵母CEN.PK-1D)中，以制备具有增强的shinorine生物合成的菌株，并且将菌株平皿接种在不包括Leu、Trp、Ura和His的SC(合成完全(synthetic complete))固体培养基上，并且在30培养箱中培养过夜。将一个铂环的培养过夜的每种菌株接种到25mL的表13中所示的效价培养基中，以及然后在30下以150rpm的速率在培养箱中培养24小时。结果示于下表14中。
[表13]
[表14]
基于以上结果，确认了酿酒酵母CEN.PK-1D(即，酵母菌株)显示了Npr A、B、C、D基因的活性比Ava A、B、C、D基因高。另外，确认了根据启动子的强度控制基因的表达水平，并因此shinorine的生产被改变。特别地，确认了当引入基于GPD(甘油醛-3-磷酸脱氢酶)启动子的Npr A、B、C、D载体时，shinorine的生产量是最高的(521mg/L)。
实施例17：通过在酿酒酵母中ARO1缺失和大肠杆菌aroB的引入的shinorine生产 量的增加
为了确定脱氢奎尼酸脱水酶的失活是否提高了酵母中shinorine生产能力，在酿酒酵母CEN.PK-1D中使ARO1基因缺失。酿酒酵母的ARO1基因是具有5种功能的基因，并且在ARO1基因缺失时，对应于大肠杆菌aroB的3-脱氢奎尼酸合酶的功能丧失，使3-DHQ的合成变得不可能。因此，在染色体上作为大肠杆菌aroB同源物(homologue)的酿酒酵母ARO1基因(SEQ ID NO:81和82)的缺失后，基于GPD启动子在相同位置插入大肠杆菌aroB基因(SEQ IDNO:83和84)。所用模板和引物示于表15中。将实施例15中制备的24种质粒引入到其中缺失ARO1基因且引入大肠杆菌aroB基因的酿酒酵母CEN.PK-1D菌株中，并且将菌株平皿接种在不包括Leu、Trp、Ura和His的SC(合成完全)固体培养基上，并且在30培养箱中培养过夜。将一个铂环的培养过夜的每种菌株接种到25mL的表13中所示的效价培养基中，以及然后在30下以150rpm的速率在培养箱中培养24小时。结果示于下表16中。
[表15]
[表16]
基于表16中所示的结果，确认了与WT菌株相比，在其中通过ARO1缺失和大肠杆菌aroB引入而增强DHQ生产能力的菌株中，shinorine生产增加了3倍。另外，确认了菌株显示了与Ava A、B、C、D基因相比，Npr A、B、C、D基因的活性高，且随着启动子强度的增加，shinorine生产量增加。特别地，确认了当引入基于GPD启动子的Npr A、B、C、D载体时，shinorine的生产量是最高的(1.6g/L)。
本领域的技术人员将认识到，在不脱离本公开的精神或必要特征的情况下，本公开可以以其它具体形式体现。描述的实施方式在所有方面仅被认为是说明性的而不是限制性的。因此，本公开的范围由所附权利要求书而不是由前述描述表明。在权利要求的等同的含义和范围内的所有变化都被包含在本公开的范围内。
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<110> CJ第一制糖株式会社
<120> 生产霉孢菌素样氨基酸的微生物及利用其生产霉孢菌素样氨基酸的方法
<130> OPA18235-PCT
<150> KR10-2017-0103795
<151> 2017-08-16
<160> 104
<170> KopatentIn 2.0
<210> 1
<211> 6461
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 1
atgagtatcg tccaagcaaa gtttgaagct aaggaaacat cttttcatgt agaaggttac 60
gaaaagattg agtatgattt ggtgtatgta gatggtattt ttgaaatcca gaattctgca 120
ctagcagatg tatatcaagg ttttggacga tgcttggcga ttgtagatgc taacgtcagt 180
cggttgtatg gtaatcaaat tcaggcatat ttccagtatt atggtataga actgaggcta 240
tttcctatta ccattactga accagataag actattcaaa ctttcgagag agttatagat 300
gtctttgcag atttcaaatt agtccgcaaa gaaccagtat tagtcgtggg tggcggttta 360
attacagatg ttgtcggctt tgcttgttct acatatcgtc gcagcagcaa ttacatccgc 420
attcctacta cattgattgg attaattgat gccagtgtag caattaaggt agcagttaat 480
catcgcaaac tgaaaaaccg tttgggtgct tatcatgctt ctcgcaaagt atttttagat 540
ttctccttgt tgcgtactct ccctacagac caagtacgta acgggatggc ggaattggta 600
aaaatcgctg tagtagcgca tcaagaagtt tttgaattgt tggagaagta cggcgaagaa 660
ttactacgta ctcattttgg caatatagat gcaactccag agattaaaga aatagcccat 720
cgtttgactt acaaagctat ccataagatg ttggaattgg aagttcccaa cctgcatgag 780
ttagacctag atagggtgat tgcttacggt cacacttgga gtcccacctt ggaacttgcg 840
cctcgtctac ccatgttcca cggacacgcc gttaatgtag atatggcttt ctcggcaacg 900
atcgccgccc gtagaggata tattacaatt gcagaacgcg atcgtatttt aggattaatg 960
agtcgcgttg gtctatccct cgaccatccc atgttggata tagatatttt gtggcgtggt 1020
actgaatcta tcacattaac tcgtgatggt ttgttaagag ctgctatgcc aaaacccatt 1080
ggtgattgtg tcttcgtcaa tgacctgaca agagaagaat tagcagccgc attagctgac 1140
cacaaagaac tttgtaccag ttatccccgt ggtggtgaag gtgtggatgt gtatcccgtt 1200
tatcaaaaag aattaatcgg gagtgttaaa taatgacttt tttgaattca aaatgcaaaa 1260
tactccacgg atacactgcg cgagcgcggt agcatttctg ttcgcggagc gtcccgtagg 1320
gaaagagaag gctacgcaaa taatcggaca ctaattgtct ttaattttga attttgaatt 1380
ttgaattttg aattggagcg aagcgacttg acaaatgtga ttgtccaacc aacagctaga 1440
cctgttacac cattgggaat tttaaccaag cagttagaag ccatagtcca agaggttaag 1500
caacatccag atttacctgg ggaattgata gcaaacatcc atcaggcttg gcgtttagcc 1560
gcaggtatag acccttattt ggaagaatgc accactccag aatctcctga actcgctgca 1620
ttggcaaaaa ccacagccac cgaagcctgg ggagaacact tccacggagg tacaaccgtc 1680
cgtcctctag aacaagagat gctttctggt catatcgaag gacaaacctt aaagatgttt 1740
gttcacatga ccaaagctaa aaaagtctta gaaattggga tgtttaccgg ttattcggcg 1800
ctggcgatgg cggaagcatt accagaggat ggactgcttg tggcttgtga agttgaccct 1860
tacgcggcgg aaattggaca gaaagccttt caacaatctc cccacggtgg aaagattcgt 1920
gtggaattgg atgcagcctt agcaactctt gataagttag cagaagctgg ggagtctttt 1980
gacttggtat ttatcgacgc agataaaaaa gagtatgtag cctattttca caagttgcta 2040
ggtagcagtt tgttagcacc agatggcttt atttgtgtag ataacacctt attacaaggg 2100
gaagtttatc taccagcaga ggaacgtagc gtcaatggtg aagcgatcgc gcaatttaat 2160
catacagtag ctatagaccc ccgtgtagaa caggttttgt tgccgttgcg agatggttta 2220
acaattatcc gcagaataca accttaattg tccaatcgac tatggcacaa tcccttcccc 2280
tttcttccgc acctgctaca ccgtctcttc cttcccagac gaaaatagcc gcaattatcc 2340
aaaatatctg cactttggct ttgttattac tagcattgcc cattaatgcc accattgttt 2400
ttatatcctt gttagtcttc cgaccgcaaa aggtcaaagc agcaaacccc caaaccattc 2460
ttatcagtgg cggtaagatg accaaagctt tacaactagc aaggtcattc cacgcggctg 2520
gacatagagt tgtcttggtg gaaacccata aatactggtt gactggtcat cgtttttccc 2580
aagcagtgga taagttttac acagtccccg caccccagga caatccccaa gcttacattc 2640
aggctttggt agatatcgtc aaacaagaaa acatcgatgt ttatattccc gtcaccagtc 2700
cagtgggtag ctactacgac tcattagcca aaccagagtt atcccattat tgcgaagtgt 2760
ttcactttga cgcagatatt acccaaatgt tggatgataa atttgcgttg acacaaaaag 2820
cgcgatcgct tggtttatca gtacccaaat cctttaaaat tacctcacca gaacaagtca 2880
tcaacttcga tttttctgga gagacacgta aatacatcct caaaagcatt ccctacgact 2940
cagtgcggcg gttggactta accaaactcc cctgtgctac tccagaggaa acagcagcat 3000
tcgtcagaag tttgccaatt actcccgaaa aaccgtggat tatgcaggaa tttatccccg 3060
gtaaggaatt ctgcacccat agcaccgttc ggaatgggga actcagactg cattgctgtt 3120
gcgaatcttc agccttccaa gttaattatg agaatgtaaa taacccgcaa attaccgaat 3180
gggtacagca ttttgtcaag gaactgaaac tgacaggaca gatttccttt gactttatcc 3240
aagccgaaga cggaacagtt tacgccatcg agtgtaaccc ccgcacacat tcagcaatta 3300
ccacatttta cgaccacccc caggtagcag aagcgtactt gagtcaagca ccgacgactg 3360
aaaccataca accactaacg acaagcaagc ctacctattg gacttatcac gaagtttggc 3420
gtttaactgg tatccgttct ttcacccagt tgcaaagatg gctggggaat atttggcgcg 3480
ggactgatgc gatttatcag ccagatgacc ccttaccgtt tttgatggta catcattggc 3540
aaattcccct actgttattg aataatttgc gtcgtcttaa aggttggacg cggatagatt 3600
tcaatattgg gaagttggtg gaattggggg gagattagtt tttaaacgca gagggacgct 3660
gaggttagcg cagcgaaaag ttctggagga gggtttccct ccgtaggaaa cttttcaaga 3720
gagagggacg cggagtgtgt tttctctgcg tctctgcgtg agaaattttt tattattgag 3780
caaagttaga agatatgcag actatagatt ttaatattcg taagttactt gtagagtgga 3840
acgcgaccca cagagattat gatctttccc agagtttaca tgaactaatt gtagctcaag 3900
tagaacgaac acctgaggcg atcgctgtca cctttgacaa gcaacaacta acttatcaag 3960
aactaaatca taaagcaaac cagctaggac attatttaca aacattagga gtccagccag 4020
aaaccctggt aggcgtttgt ttagaacgtt ccttagaaat ggttatctgt cttttaggaa 4080
tcctcaaagc tgggggtgct tatgttccta ttgaccctga atatcctcaa gaacgcatag 4140
cttatatgct agaagattct caggtgaagg tactactaac tcaagaaaaa ttactcaatc 4200
aaattcccca ccatcaagca caaactatct gtgtagatag ggaatgggag aaaatttcca 4260
cacaagctaa taccaatccc aaaagtaata taaaaacgga taatcttgct tatgtaattt 4320
acacctctgg ttccactggt aaaccaaaag gtgcaatgaa cacccacaaa ggtatctgta 4380
atcgcttatt gtggatgcag gaagcttatc aaatcgattc cacagatagc attttacaaa 4440
aaaccccctt tagttttgat gtttccgttt gggagttctt ttggacttta ttaactggcg 4500
cacgtttggt aatagccaaa ccaggcggac ataaagatag tgcttacctc atcgatttaa 4560
ttactcaaga acaaatcact acgttgcatt ttgtcccctc aatgctgcaa gtgtttttac 4620
aaaatcgcca tgtaagcaaa tgcagctctc taaaaagagt tatttgtagc ggtgaagctt 4680
tatctataga tttacaaaat agatttttcc agcatttgca atgtgaatta cataacctct 4740
atggcccgac agaagcagca attgatgtca cattttggca atgtagaaaa gatagtaatt 4800
taaagagtgt acctattggt cgtcccattg ctaatactca aatttatatt cttgatgccg 4860
atttacaacc agtaaatatt ggtgtcactg gtgaaattta tattggtggt gtaggggttg 4920
ctcgtggtta tttgaataaa gaagaattga ccaaagaaaa atttattatt aatccctttc 4980
ccaattctga gtttaagcga ctttataaaa caggtgattt agctcgttat ttacccgatg 5040
gaaatattga atatcttggt agaacagatt atcaagtaaa aattcggggt tatagaattg 5100
aaattggcga gattgaaaat gttttatctt cacacccaca agtcagagaa gctgtagtca 5160
tagcgcggga tgataacgct caagaaaaac aaatcatcgc ttatattacc tataactcca 5220
tcaaacctca gcttgataat ctgcgtgatt tcctaaaagc aaggctacct gattttatga 5280
ttccagccgc ttttgtgatg ctggagcatc ttcctttaac tcccagtggt aaagtagacc 5340
gtaaggcatt acctaagcct gatttattta attatagtga acataattcc tatgtagcgc 5400
ctcggaatga agttgaagaa aaattagtac aaatctggtc gaatattctg catttaccta 5460
aagtaggtgt gacagaaaac tttttcgcta ttggtggtaa ttccctcaaa gctctacatt 5520
taatttctca aattgaagag ttatttgcta aagagatatc cttagcaaca cttttaacaa 5580
atccagtaat tgcagattta gccaaggtta ttcaagcaaa caaccaaatc cataattcac 5640
ccctagttcc aattcaacca caaggtaagc agcagccttt cttttgtata catcctgctg 5700
gtggtcatgt tttatgctat tttaaactcg cacaatatat aggaactgac caaccatttt 5760
atggcttaca agctcaagga ttttatggag atgaagcacc cttgacgcga gttgaagata 5820
tggctagtct ctacgtcaaa actattagag aatttcaacc ccaagggcct tatcgtgtcg 5880
gggggtggtc atttggtgga gtcgtagctt atgaagtagc acagcagtta catagacaag 5940
gacaagaagt atctttacta gcaatattag attcttacgt accgattctg ctggataaac 6000
aaaaacccat tgatgacgtt tatttagttg gtgttctctc cagagttttt ggcggtatgt 6060
ttggtcaaga taatctagtc acacctgaag aaatagaaaa tttaactgta gaagaaaaaa 6120
ttaattacat cattgataaa gcacggagcg ctagaatatt cccgcctggt gtagaacgtc 6180
aaaataatcg ccgtattctt gatgttttgg tgggaacttt aaaagcaact tattcctata 6240
taagacaacc atatccagga aaagtcactg tatttcgagc cagggaaaaa catattatgg 6300
ctcctgaccc gaccttagtt tgggtagaat tattttctgt aatggcggct caagaaatta 6360
agattattga tgtccctgga aaccattatt cgtttgttct agaaccccat gtacaggttt 6420
tagcacagcg tttacaagat tgtctggaaa ataattcata a 6461
<210> 2
<211> 2035
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 2
Met Ser Ile Val Gln Ala Lys Phe Glu Ala Lys Glu Thr Ser Phe His
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly
20 25 30
Ile Phe Glu Ile Gln Asn Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe
35 40 45
Gly Arg Cys Leu Ala Ile Val Asp Ala Asn Val Ser Arg Leu Tyr Gly
50 55 60
Asn Gln Ile Gln Ala Tyr Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu
65 70 75 80
Phe Pro Ile Thr Ile Thr Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu
85 90 95
Arg Val Ile Asp Val Phe Ala Asp Phe Lys Leu Val Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Ile Thr Asp Val Val Gly Phe Ala
115 120 125
Cys Ser Thr Tyr Arg Arg Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Ser Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Ser Arg Lys
165 170 175
Val Phe Leu Asp Phe Ser Leu Leu Arg Thr Leu Pro Thr Asp Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala His Gln
195 200 205
Glu Val Phe Glu Leu Leu Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr
210 215 220
His Phe Gly Asn Ile Asp Ala Thr Pro Glu Ile Lys Glu Ile Ala His
225 230 235 240
Arg Leu Thr Tyr Lys Ala Ile His Lys Met Leu Glu Leu Glu Val Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Arg Leu Pro Met Phe His Gly
275 280 285
His Ala Val Asn Val Asp Met Ala Phe Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ile Ala Glu Arg Asp Arg Ile Leu Gly Leu Met
305 310 315 320
Ser Arg Val Gly Leu Ser Leu Asp His Pro Met Leu Asp Ile Asp Ile
325 330 335
Leu Trp Arg Gly Thr Glu Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Asp Cys Val Phe Val Asn Asp
355 360 365
Leu Thr Arg Glu Glu Leu Ala Ala Ala Leu Ala Asp His Lys Glu Leu
370 375 380
Cys Thr Ser Tyr Pro Arg Gly Gly Glu Gly Val Asp Val Tyr Pro Val
385 390 395 400
Tyr Gln Lys Glu Leu Ile Gly Ser Val Lys Leu Thr Asn Val Ile Val
405 410 415
Gln Pro Thr Ala Arg Pro Val Thr Pro Leu Gly Ile Leu Thr Lys Gln
420 425 430
Leu Glu Ala Ile Val Gln Glu Val Lys Gln His Pro Asp Leu Pro Gly
435 440 445
Glu Leu Ile Ala Asn Ile His Gln Ala Trp Arg Leu Ala Ala Gly Ile
450 455 460
Asp Pro Tyr Leu Glu Glu Cys Thr Thr Pro Glu Ser Pro Glu Leu Ala
465 470 475 480
Ala Leu Ala Lys Thr Thr Ala Thr Glu Ala Trp Gly Glu His Phe His
485 490 495
Gly Gly Thr Thr Val Arg Pro Leu Glu Gln Glu Met Leu Ser Gly His
500 505 510
Ile Glu Gly Gln Thr Leu Lys Met Phe Val His Met Thr Lys Ala Lys
515 520 525
Lys Val Leu Glu Ile Gly Met Phe Thr Gly Tyr Ser Ala Leu Ala Met
530 535 540
Ala Glu Ala Leu Pro Glu Asp Gly Leu Leu Val Ala Cys Glu Val Asp
545 550 555 560
Pro Tyr Ala Ala Glu Ile Gly Gln Lys Ala Phe Gln Gln Ser Pro His
565 570 575
Gly Gly Lys Ile Arg Val Glu Leu Asp Ala Ala Leu Ala Thr Leu Asp
580 585 590
Lys Leu Ala Glu Ala Gly Glu Ser Phe Asp Leu Val Phe Ile Asp Ala
595 600 605
Asp Lys Lys Glu Tyr Val Ala Tyr Phe His Lys Leu Leu Gly Ser Ser
610 615 620
Leu Leu Ala Pro Asp Gly Phe Ile Cys Val Asp Asn Thr Leu Leu Gln
625 630 635 640
Gly Glu Val Tyr Leu Pro Ala Glu Glu Arg Ser Val Asn Gly Glu Ala
645 650 655
Ile Ala Gln Phe Asn His Thr Val Ala Ile Asp Pro Arg Val Glu Gln
660 665 670
Val Leu Leu Pro Leu Arg Asp Gly Leu Thr Ile Ile Arg Arg Ile Gln
675 680 685
Pro Met Ala Gln Ser Leu Pro Leu Ser Ser Ala Pro Ala Thr Pro Ser
690 695 700
Leu Pro Ser Gln Thr Lys Ile Ala Ala Ile Ile Gln Asn Ile Cys Thr
705 710 715 720
Leu Ala Leu Leu Leu Leu Ala Leu Pro Ile Asn Ala Thr Ile Val Phe
725 730 735
Ile Ser Leu Leu Val Phe Arg Pro Gln Lys Val Lys Ala Ala Asn Pro
740 745 750
Gln Thr Ile Leu Ile Ser Gly Gly Lys Met Thr Lys Ala Leu Gln Leu
755 760 765
Ala Arg Ser Phe His Ala Ala Gly His Arg Val Val Leu Val Glu Thr
770 775 780
His Lys Tyr Trp Leu Thr Gly His Arg Phe Ser Gln Ala Val Asp Lys
785 790 795 800
Phe Tyr Thr Val Pro Ala Pro Gln Asp Asn Pro Gln Ala Tyr Ile Gln
805 810 815
Ala Leu Val Asp Ile Val Lys Gln Glu Asn Ile Asp Val Tyr Ile Pro
820 825 830
Val Thr Ser Pro Val Gly Ser Tyr Tyr Asp Ser Leu Ala Lys Pro Glu
835 840 845
Leu Ser His Tyr Cys Glu Val Phe His Phe Asp Ala Asp Ile Thr Gln
850 855 860
Met Leu Asp Asp Lys Phe Ala Leu Thr Gln Lys Ala Arg Ser Leu Gly
865 870 875 880
Leu Ser Val Pro Lys Ser Phe Lys Ile Thr Ser Pro Glu Gln Val Ile
885 890 895
Asn Phe Asp Phe Ser Gly Glu Thr Arg Lys Tyr Ile Leu Lys Ser Ile
900 905 910
Pro Tyr Asp Ser Val Arg Arg Leu Asp Leu Thr Lys Leu Pro Cys Ala
915 920 925
Thr Pro Glu Glu Thr Ala Ala Phe Val Arg Ser Leu Pro Ile Thr Pro
930 935 940
Glu Lys Pro Trp Ile Met Gln Glu Phe Ile Pro Gly Lys Glu Phe Cys
945 950 955 960
Thr His Ser Thr Val Arg Asn Gly Glu Leu Arg Leu His Cys Cys Cys
965 970 975
Glu Ser Ser Ala Phe Gln Val Asn Tyr Glu Asn Val Asn Asn Pro Gln
980 985 990
Ile Thr Glu Trp Val Gln His Phe Val Lys Glu Leu Lys Leu Thr Gly
995 1000 1005
Gln Ile Ser Phe Asp Phe Ile Gln Ala Glu Asp Gly Thr Val Tyr Ala
1010 1015 1020
Ile Glu Cys Asn Pro Arg Thr His Ser Ala Ile Thr Thr Phe Tyr Asp
1025 1030 1035 1040
His Pro Gln Val Ala Glu Ala Tyr Leu Ser Gln Ala Pro Thr Thr Glu
1045 1050 1055
Thr Ile Gln Pro Leu Thr Thr Ser Lys Pro Thr Tyr Trp Thr Tyr His
1060 1065 1070
Glu Val Trp Arg Leu Thr Gly Ile Arg Ser Phe Thr Gln Leu Gln Arg
1075 1080 1085
Trp Leu Gly Asn Ile Trp Arg Gly Thr Asp Ala Ile Tyr Gln Pro Asp
1090 1095 1100
Asp Pro Leu Pro Phe Leu Met Val His His Trp Gln Ile Pro Leu Leu
1105 1110 1115 1120
Leu Leu Asn Asn Leu Arg Arg Leu Lys Gly Trp Thr Arg Ile Asp Phe
1125 1130 1135
Asn Ile Gly Lys Leu Val Glu Leu Gly Gly Asp Met Gln Thr Ile Asp
1140 1145 1150
Phe Asn Ile Arg Lys Leu Leu Val Glu Trp Asn Ala Thr His Arg Asp
1155 1160 1165
Tyr Asp Leu Ser Gln Ser Leu His Glu Leu Ile Val Ala Gln Val Glu
1170 1175 1180
Arg Thr Pro Glu Ala Ile Ala Val Thr Phe Asp Lys Gln Gln Leu Thr
1185 1190 1195 1200
Tyr Gln Glu Leu Asn His Lys Ala Asn Gln Leu Gly His Tyr Leu Gln
1205 1210 1215
Thr Leu Gly Val Gln Pro Glu Thr Leu Val Gly Val Cys Leu Glu Arg
1220 1225 1230
Ser Leu Glu Met Val Ile Cys Leu Leu Gly Ile Leu Lys Ala Gly Gly
1235 1240 1245
Ala Tyr Val Pro Ile Asp Pro Glu Tyr Pro Gln Glu Arg Ile Ala Tyr
1250 1255 1260
Met Leu Glu Asp Ser Gln Val Lys Val Leu Leu Thr Gln Glu Lys Leu
1265 1270 1275 1280
Leu Asn Gln Ile Pro His His Gln Ala Gln Thr Ile Cys Val Asp Arg
1285 1290 1295
Glu Trp Glu Lys Ile Ser Thr Gln Ala Asn Thr Asn Pro Lys Ser Asn
1300 1305 1310
Ile Lys Thr Asp Asn Leu Ala Tyr Val Ile Tyr Thr Ser Gly Ser Thr
1315 1320 1325
Gly Lys Pro Lys Gly Ala Met Asn Thr His Lys Gly Ile Cys Asn Arg
1330 1335 1340
Leu Leu Trp Met Gln Glu Ala Tyr Gln Ile Asp Ser Thr Asp Ser Ile
1345 1350 1355 1360
Leu Gln Lys Thr Pro Phe Ser Phe Asp Val Ser Val Trp Glu Phe Phe
1365 1370 1375
Trp Thr Leu Leu Thr Gly Ala Arg Leu Val Ile Ala Lys Pro Gly Gly
1380 1385 1390
His Lys Asp Ser Ala Tyr Leu Ile Asp Leu Ile Thr Gln Glu Gln Ile
1395 1400 1405
Thr Thr Leu His Phe Val Pro Ser Met Leu Gln Val Phe Leu Gln Asn
1410 1415 1420
Arg His Val Ser Lys Cys Ser Ser Leu Lys Arg Val Ile Cys Ser Gly
1425 1430 1435 1440
Glu Ala Leu Ser Ile Asp Leu Gln Asn Arg Phe Phe Gln His Leu Gln
1445 1450 1455
Cys Glu Leu His Asn Leu Tyr Gly Pro Thr Glu Ala Ala Ile Asp Val
1460 1465 1470
Thr Phe Trp Gln Cys Arg Lys Asp Ser Asn Leu Lys Ser Val Pro Ile
1475 1480 1485
Gly Arg Pro Ile Ala Asn Thr Gln Ile Tyr Ile Leu Asp Ala Asp Leu
1490 1495 1500
Gln Pro Val Asn Ile Gly Val Thr Gly Glu Ile Tyr Ile Gly Gly Val
1505 1510 1515 1520
Gly Val Ala Arg Gly Tyr Leu Asn Lys Glu Glu Leu Thr Lys Glu Lys
1525 1530 1535
Phe Ile Ile Asn Pro Phe Pro Asn Ser Glu Phe Lys Arg Leu Tyr Lys
1540 1545 1550
Thr Gly Asp Leu Ala Arg Tyr Leu Pro Asp Gly Asn Ile Glu Tyr Leu
1555 1560 1565
Gly Arg Thr Asp Tyr Gln Val Lys Ile Arg Gly Tyr Arg Ile Glu Ile
1570 1575 1580
Gly Glu Ile Glu Asn Val Leu Ser Ser His Pro Gln Val Arg Glu Ala
1585 1590 1595 1600
Val Val Ile Ala Arg Asp Asp Asn Ala Gln Glu Lys Gln Ile Ile Ala
1605 1610 1615
Tyr Ile Thr Tyr Asn Ser Ile Lys Pro Gln Leu Asp Asn Leu Arg Asp
1620 1625 1630
Phe Leu Lys Ala Arg Leu Pro Asp Phe Met Ile Pro Ala Ala Phe Val
1635 1640 1645
Met Leu Glu His Leu Pro Leu Thr Pro Ser Gly Lys Val Asp Arg Lys
1650 1655 1660
Ala Leu Pro Lys Pro Asp Leu Phe Asn Tyr Ser Glu His Asn Ser Tyr
1665 1670 1675 1680
Val Ala Pro Arg Asn Glu Val Glu Glu Lys Leu Val Gln Ile Trp Ser
1685 1690 1695
Asn Ile Leu His Leu Pro Lys Val Gly Val Thr Glu Asn Phe Phe Ala
1700 1705 1710
Ile Gly Gly Asn Ser Leu Lys Ala Leu His Leu Ile Ser Gln Ile Glu
1715 1720 1725
Glu Leu Phe Ala Lys Glu Ile Ser Leu Ala Thr Leu Leu Thr Asn Pro
1730 1735 1740
Val Ile Ala Asp Leu Ala Lys Val Ile Gln Ala Asn Asn Gln Ile His
1745 1750 1755 1760
Asn Ser Pro Leu Val Pro Ile Gln Pro Gln Gly Lys Gln Gln Pro Phe
1765 1770 1775
Phe Cys Ile His Pro Ala Gly Gly His Val Leu Cys Tyr Phe Lys Leu
1780 1785 1790
Ala Gln Tyr Ile Gly Thr Asp Gln Pro Phe Tyr Gly Leu Gln Ala Gln
1795 1800 1805
Gly Phe Tyr Gly Asp Glu Ala Pro Leu Thr Arg Val Glu Asp Met Ala
1810 1815 1820
Ser Leu Tyr Val Lys Thr Ile Arg Glu Phe Gln Pro Gln Gly Pro Tyr
1825 1830 1835 1840
Arg Val Gly Gly Trp Ser Phe Gly Gly Val Val Ala Tyr Glu Val Ala
1845 1850 1855
Gln Gln Leu His Arg Gln Gly Gln Glu Val Ser Leu Leu Ala Ile Leu
1860 1865 1870
Asp Ser Tyr Val Pro Ile Leu Leu Asp Lys Gln Lys Pro Ile Asp Asp
1875 1880 1885
Val Tyr Leu Val Gly Val Leu Ser Arg Val Phe Gly Gly Met Phe Gly
1890 1895 1900
Gln Asp Asn Leu Val Thr Pro Glu Glu Ile Glu Asn Leu Thr Val Glu
1905 1910 1915 1920
Glu Lys Ile Asn Tyr Ile Ile Asp Lys Ala Arg Ser Ala Arg Ile Phe
1925 1930 1935
Pro Pro Gly Val Glu Arg Gln Asn Asn Arg Arg Ile Leu Asp Val Leu
1940 1945 1950
Val Gly Thr Leu Lys Ala Thr Tyr Ser Tyr Ile Arg Gln Pro Tyr Pro
1955 1960 1965
Gly Lys Val Thr Val Phe Arg Ala Arg Glu Lys His Ile Met Ala Pro
1970 1975 1980
Asp Pro Thr Leu Val Trp Val Glu Leu Phe Ser Val Met Ala Ala Gln
1985 1990 1995 2000
Glu Ile Lys Ile Ile Asp Val Pro Gly Asn His Tyr Ser Phe Val Leu
2005 2010 2015
Glu Pro His Val Gln Val Leu Ala Gln Arg Leu Gln Asp Cys Leu Glu
2020 2025 2030
Asn Asn Ser
2035
<210> 3
<211> 4528
<212> DNA
<213> 点状念珠藻ATCC29133
<400> 3
atgagtaatg ttcaagcatc gtttgaagca acggaagctg aattccgcgt ggaaggttac 60
gaaaaaattg agtttagtct tgtctatgta aatggtgcat ttgatatcag taacagagaa 120
attgcagaca gctatgagaa gtttggccgt tgtctgactg tgattgatgc taatgtcaac 180
agattatatg gcaagcaaat caagtcatat tttagacact atggtattga tctgaccgta 240
gttcccattg tgattactga gcctactaaa acccttgcaa cctttgagaa aattgttgat 300
gctttttctg actttggttt aatccgcaag gaaccagtat tagtagtggg tggtggttta 360
accactgatg tagctgggtt tgcgtgtgct gcttaccgtc gtaagagtaa ctatattcgg 420
gttccgacaa cgctgattgg tctgattgat gcaggtgtag cgattaaggt tgcagtcaac 480
catcgcaagt taaaaaatcg cttgggtgca tatcatgctc ctttgaaagt catcctcgat 540
ttctccttct tgcaaacatt accaacggct caagttcgta atgggatggc agagttggtc 600
aaaattgctg ttgtggcgaa ctctgaagtc tttgaattgt tgtatgaata tggcgaagag 660
ttgctttcca ctcactttgg atatgtgaat ggtacaaagg aactgaaagc gatcgcacat 720
aaactcaatt acgaggcaat aaaaactatg ctggagttgg aaactccaaa cttgcatgag 780
ttagacctcg atcgcgtcat tgcctacggt cacacttgga gtccgacctt agaattagct 840
ccgatgatac cgttgtttca cggtcatgcc gtcaatatag acatggcttt gtctgcaact 900
attgcggcaa gacgtggcta cattacatca ggagaacgcg atcgcatttt gagcttgatg 960
agtcgtatag gtttatcaat cgatcatcct ctactagatg gcgatttgct ctggtatgct 1020
acccaatcta ttagcttgac gcgagacggg aaacaacgcg cagctatgcc taaacccatt 1080
ggtgagtgct tctttgtcaa cgatttcacc cgtgaagaac tagatgcagc tttagctgaa 1140
cacaaacgtc tgtgtgctac ataccctcgt ggtggagatg gcattgacgc ttacatagaa 1200
actcaagaag aatccaaact attgggagtg tgaaaacatg accagtattt taggacgaga 1260
taccgcaaga ccaataacgc cacatagcat tctggtagca cagctacaaa aaaccctcag 1320
aatggcagag gaaagtaata ttccttcaga gatactgact tctctgcgcc aagggttgca 1380
attagcagca ggtttagatc cctatctgga tgattgcact acccctgaat cgaccgcatt 1440
gacagcacta gcgcagaaga caagcattga agactggagt aaacgcttca gtgatggtga 1500
aacagtgcgt caattagagc aagaaatgct ctcaggacat cttgaaggac aaacactaaa 1560
gatgtttgtg catatcacta aggctaaaag catcctagaa gtgggaatgt tcacaggata 1620
ttcagctttg gcaatggcag aggcgttacc agatgatggg cgactgattg cttgtgaagt 1680
agactcctat gtggccgagt ttgctcaaac ttgctttcaa gagtctcccc acggccgcaa 1740
gattgttgta gaagtggcac ctgcactaga gacgctgcac aagctggtgg ctaaaaaaga 1800
atcctttgat ctgatcttca ttgatgcgga taaaaaggag tatatagaat acttccaaat 1860
tatcttggat agccatttac tagctcccga cggattaatc tgtgtagata atactttgtt 1920
gcaaggacaa gtttacctgc catcagaaca gcgtacagcc aatggtgaag cgatcgctca 1980
attcaaccgc attgtcgccg cagatcctcg tgtagagcaa gttctgttac ccatacgaga 2040
tggtataacc ctgattagac gcttggtata agcggccgcg agctcctcga gatggcacaa 2100
tcaatctctt tatctcttcc tcaatccaca acgccatcaa agggtgtgag gctaaaaata 2160
gcagctctac tgaagactat cgggactcta atactactgc tgatagcctt gccgcttaat 2220
gctttgatag tattgatatc tctgatgtgt aggccgttta caaaaaaacc tgccgtagcc 2280
actcatcccc agaatatctt ggtcagtggc ggcaaaatga ccaaagcatt gcaacttgcc 2340
cgctccttcc atgcagccgg acacagagtt attctgattg aaggtcataa atactggtta 2400
tcagggcata gattctcaaa ttctgtgagt cgtttttata cagttcctgc accacaagac 2460
gacccagaag gctataccca agcgctattg gaaattgtca aacgagagaa gattgacgtt 2520
tatgtacccg tatgcagccc tgtagctagt tattacgact ctttggcaaa gtctgcacta 2580
tcagaatatt gtgaggtttt tcactttgat gctgatataa ccaagatgct ggatgataaa 2640
tttgccttta ccgatcgggc gcgatcgctt ggtttatcag ccccgaaatc ttttaaaatt 2700
accgatcctg aacaagttat caacttcgat tttagtaaag agacgcgcaa atatattctt 2760
aagagtattt cttacgactc agttcgccgc ttaaatttaa ccaaacttcc ttgtgatacc 2820
ccagaagaga cagcagcgtt tgtcaagagt ttacccatca gcccagaaaa accttggatt 2880
atgcaagaat ttattcctgg gaaagaatta tgcacccata gcacagtccg agacggcgaa 2940
ttaaggttgc attgctgttc aaattcttca gcgtttcaga ttaactatga aaatgtcgaa 3000
aatccccaaa ttcaagaatg ggtacaacat ttcgtcaaaa gtttacggct gactggacaa 3060
atatctcttg actttatcca agctgaagat ggtacagctt atgccattga atgtaatcct 3120
cgcacccatt cggcgatcac aatgttctac aatcacccag gtgttgcaga agcctatctt 3180
ggtaaaactc ctctagctgc acctttggaa cctcttgcag atagcaagcc cacttactgg 3240
atatatcacg aaatctggcg attgactggg attcgctctg gacaacaatt acaaacttgg 3300
tttgggagat tagtcagagg tacagatgcc atttatcgcc tggacgatcc gataccattt 3360
ttaactttgc accattggca gattacttta cttttgctac aaaatttgca acgactcaaa 3420
ggttgggtaa agatcgattt taatatcggt aaactcgtgg aattaggggg cgactaaaaa 3480
catgccagta cttaatatcc ttcatttagt tgggtctgca cacgataagt tttactgtga 3540
tttatcacgt ctttatgccc aagactgttt agctgcaaca gcagatccat cgctttataa 3600
ctttcaaatt gcatatatca cacccgatcg gcagtggcga tttcctgact ctctcagtcg 3660
agaagatatt gctcttacca aaccgattcc tgtgtttgat gccatacaat ttctaacagg 3720
ccaaaacatt gacatgatgt taccacaaat gttttgtatt cctggaatga ctcagtaccg 3780
tgccctattc gatctgctca agatccctta tataggaaat accccagata ttatggcgat 3840
cgcggcccac aaagccagag ccaaagcaat tgtcgaagca gcaggggtaa aagtgcctcg 3900
tggagaattg cttcgccaag gagatattcc aacaattaca cctccagcag tcgtcaaacc 3960
tgtaagttct gacaactctt taggagtagt cttagttaaa gatgtgactg aatatgatgc 4020
tgccttaaag aaagcatttg aatatgcttc ggaggtcatc gtagaagcat tcatcgaact 4080
tggtcgagaa gtcagatgcg gcatcattgt aaaagacggt gagctaatag gtttacccct 4140
tgaagagtat ctggtagacc cacacgataa acctatccgt aactatgctg ataaactcca 4200
acaaactgac gatggcgact tgcatttgac tgctaaagat aatatcaagg cttggatttt 4260
agaccctaac gacccaatca cccaaaaggt tcagcaagtg gctaaaaggt gtcatcaggc 4320
tttgggttgt cgccactaca gtttatttga cttccgaatc gatccaaagg gacaaccttg 4380
gttcttagaa gctggattat attgttcttt tgcccccaaa agtgtgattt cttctatggc 4440
gaaagcagcc ggaatccctc taaatgattt attaataacc gctattaatg aaacattggg 4500
tagtaataaa aaggtgttac aaaattga 4528
<210> 4
<211> 1496
<212> PRT
<213> 点状念珠藻ATCC29133
<400> 4
Met Ser Asn Val Gln Ala Ser Phe Glu Ala Thr Glu Ala Glu Phe Arg
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Phe Ser Leu Val Tyr Val Asn Gly
20 25 30
Ala Phe Asp Ile Ser Asn Arg Glu Ile Ala Asp Ser Tyr Glu Lys Phe
35 40 45
Gly Arg Cys Leu Thr Val Ile Asp Ala Asn Val Asn Arg Leu Tyr Gly
50 55 60
Lys Gln Ile Lys Ser Tyr Phe Arg His Tyr Gly Ile Asp Leu Thr Val
65 70 75 80
Val Pro Ile Val Ile Thr Glu Pro Thr Lys Thr Leu Ala Thr Phe Glu
85 90 95
Lys Ile Val Asp Ala Phe Ser Asp Phe Gly Leu Ile Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Thr Thr Asp Val Ala Gly Phe Ala
115 120 125
Cys Ala Ala Tyr Arg Arg Lys Ser Asn Tyr Ile Arg Val Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Gly Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Pro Leu Lys
165 170 175
Val Ile Leu Asp Phe Ser Phe Leu Gln Thr Leu Pro Thr Ala Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala Asn Ser
195 200 205
Glu Val Phe Glu Leu Leu Tyr Glu Tyr Gly Glu Glu Leu Leu Ser Thr
210 215 220
His Phe Gly Tyr Val Asn Gly Thr Lys Glu Leu Lys Ala Ile Ala His
225 230 235 240
Lys Leu Asn Tyr Glu Ala Ile Lys Thr Met Leu Glu Leu Glu Thr Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Met Ile Pro Leu Phe His Gly
275 280 285
His Ala Val Asn Ile Asp Met Ala Leu Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ser Gly Glu Arg Asp Arg Ile Leu Ser Leu Met
305 310 315 320
Ser Arg Ile Gly Leu Ser Ile Asp His Pro Leu Leu Asp Gly Asp Leu
325 330 335
Leu Trp Tyr Ala Thr Gln Ser Ile Ser Leu Thr Arg Asp Gly Lys Gln
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Glu Cys Phe Phe Val Asn Asp
355 360 365
Phe Thr Arg Glu Glu Leu Asp Ala Ala Leu Ala Glu His Lys Arg Leu
370 375 380
Cys Ala Thr Tyr Pro Arg Gly Gly Asp Gly Ile Asp Ala Tyr Ile Glu
385 390 395 400
Thr Gln Glu Glu Ser Lys Leu Leu Gly Val Met Thr Ser Ile Leu Gly
405 410 415
Arg Asp Thr Ala Arg Pro Ile Thr Pro His Ser Ile Leu Val Ala Gln
420 425 430
Leu Gln Lys Thr Leu Arg Met Ala Glu Glu Ser Asn Ile Pro Ser Glu
435 440 445
Ile Leu Thr Ser Leu Arg Gln Gly Leu Gln Leu Ala Ala Gly Leu Asp
450 455 460
Pro Tyr Leu Asp Asp Cys Thr Thr Pro Glu Ser Thr Ala Leu Thr Ala
465 470 475 480
Leu Ala Gln Lys Thr Ser Ile Glu Asp Trp Ser Lys Arg Phe Ser Asp
485 490 495
Gly Glu Thr Val Arg Gln Leu Glu Gln Glu Met Leu Ser Gly His Leu
500 505 510
Glu Gly Gln Thr Leu Lys Met Phe Val His Ile Thr Lys Ala Lys Ser
515 520 525
Ile Leu Glu Val Gly Met Phe Thr Gly Tyr Ser Ala Leu Ala Met Ala
530 535 540
Glu Ala Leu Pro Asp Asp Gly Arg Leu Ile Ala Cys Glu Val Asp Ser
545 550 555 560
Tyr Val Ala Glu Phe Ala Gln Thr Cys Phe Gln Glu Ser Pro His Gly
565 570 575
Arg Lys Ile Val Val Glu Val Ala Pro Ala Leu Glu Thr Leu His Lys
580 585 590
Leu Val Ala Lys Lys Glu Ser Phe Asp Leu Ile Phe Ile Asp Ala Asp
595 600 605
Lys Lys Glu Tyr Ile Glu Tyr Phe Gln Ile Ile Leu Asp Ser His Leu
610 615 620
Leu Ala Pro Asp Gly Leu Ile Cys Val Asp Asn Thr Leu Leu Gln Gly
625 630 635 640
Gln Val Tyr Leu Pro Ser Glu Gln Arg Thr Ala Asn Gly Glu Ala Ile
645 650 655
Ala Gln Phe Asn Arg Ile Val Ala Ala Asp Pro Arg Val Glu Gln Val
660 665 670
Leu Leu Pro Ile Arg Asp Gly Ile Thr Leu Ile Arg Arg Leu Val Met
675 680 685
Ala Gln Ser Ile Ser Leu Ser Leu Pro Gln Ser Thr Thr Pro Ser Lys
690 695 700
Gly Val Arg Leu Lys Ile Ala Ala Leu Leu Lys Thr Ile Gly Thr Leu
705 710 715 720
Ile Leu Leu Leu Ile Ala Leu Pro Leu Asn Ala Leu Ile Val Leu Ile
725 730 735
Ser Leu Met Cys Arg Pro Phe Thr Lys Lys Pro Ala Val Ala Thr His
740 745 750
Pro Gln Asn Ile Leu Val Ser Gly Gly Lys Met Thr Lys Ala Leu Gln
755 760 765
Leu Ala Arg Ser Phe His Ala Ala Gly His Arg Val Ile Leu Ile Glu
770 775 780
Gly His Lys Tyr Trp Leu Ser Gly His Arg Phe Ser Asn Ser Val Ser
785 790 795 800
Arg Phe Tyr Thr Val Pro Ala Pro Gln Asp Asp Pro Glu Gly Tyr Thr
805 810 815
Gln Ala Leu Leu Glu Ile Val Lys Arg Glu Lys Ile Asp Val Tyr Val
820 825 830
Pro Val Cys Ser Pro Val Ala Ser Tyr Tyr Asp Ser Leu Ala Lys Ser
835 840 845
Ala Leu Ser Glu Tyr Cys Glu Val Phe His Phe Asp Ala Asp Ile Thr
850 855 860
Lys Met Leu Asp Asp Lys Phe Ala Phe Thr Asp Arg Ala Arg Ser Leu
865 870 875 880
Gly Leu Ser Ala Pro Lys Ser Phe Lys Ile Thr Asp Pro Glu Gln Val
885 890 895
Ile Asn Phe Asp Phe Ser Lys Glu Thr Arg Lys Tyr Ile Leu Lys Ser
900 905 910
Ile Ser Tyr Asp Ser Val Arg Arg Leu Asn Leu Thr Lys Leu Pro Cys
915 920 925
Asp Thr Pro Glu Glu Thr Ala Ala Phe Val Lys Ser Leu Pro Ile Ser
930 935 940
Pro Glu Lys Pro Trp Ile Met Gln Glu Phe Ile Pro Gly Lys Glu Leu
945 950 955 960
Cys Thr His Ser Thr Val Arg Asp Gly Glu Leu Arg Leu His Cys Cys
965 970 975
Ser Asn Ser Ser Ala Phe Gln Ile Asn Tyr Glu Asn Val Glu Asn Pro
980 985 990
Gln Ile Gln Glu Trp Val Gln His Phe Val Lys Ser Leu Arg Leu Thr
995 1000 1005
Gly Gln Ile Ser Leu Asp Phe Ile Gln Ala Glu Asp Gly Thr Ala Tyr
1010 1015 1020
Ala Ile Glu Cys Asn Pro Arg Thr His Ser Ala Ile Thr Met Phe Tyr
1025 1030 1035 1040
Asn His Pro Gly Val Ala Glu Ala Tyr Leu Gly Lys Thr Pro Leu Ala
1045 1050 1055
Ala Pro Leu Glu Pro Leu Ala Asp Ser Lys Pro Thr Tyr Trp Ile Tyr
1060 1065 1070
His Glu Ile Trp Arg Leu Thr Gly Ile Arg Ser Gly Gln Gln Leu Gln
1075 1080 1085
Thr Trp Phe Gly Arg Leu Val Arg Gly Thr Asp Ala Ile Tyr Arg Leu
1090 1095 1100
Asp Asp Pro Ile Pro Phe Leu Thr Leu His His Trp Gln Ile Thr Leu
1105 1110 1115 1120
Leu Leu Leu Gln Asn Leu Gln Arg Leu Lys Gly Trp Val Lys Ile Asp
1125 1130 1135
Phe Asn Ile Gly Lys Leu Val Glu Leu Gly Gly Asp Met Pro Val Leu
1140 1145 1150
Asn Ile Leu His Leu Val Gly Ser Ala His Asp Lys Phe Tyr Cys Asp
1155 1160 1165
Leu Ser Arg Leu Tyr Ala Gln Asp Cys Leu Ala Ala Thr Ala Asp Pro
1170 1175 1180
Ser Leu Tyr Asn Phe Gln Ile Ala Tyr Ile Thr Pro Asp Arg Gln Trp
1185 1190 1195 1200
Arg Phe Pro Asp Ser Leu Ser Arg Glu Asp Ile Ala Leu Thr Lys Pro
1205 1210 1215
Ile Pro Val Phe Asp Ala Ile Gln Phe Leu Thr Gly Gln Asn Ile Asp
1220 1225 1230
Met Met Leu Pro Gln Met Phe Cys Ile Pro Gly Met Thr Gln Tyr Arg
1235 1240 1245
Ala Leu Phe Asp Leu Leu Lys Ile Pro Tyr Ile Gly Asn Thr Pro Asp
1250 1255 1260
Ile Met Ala Ile Ala Ala His Lys Ala Arg Ala Lys Ala Ile Val Glu
1265 1270 1275 1280
Ala Ala Gly Val Lys Val Pro Arg Gly Glu Leu Leu Arg Gln Gly Asp
1285 1290 1295
Ile Pro Thr Ile Thr Pro Pro Ala Val Val Lys Pro Val Ser Ser Asp
1300 1305 1310
Asn Ser Leu Gly Val Val Leu Val Lys Asp Val Thr Glu Tyr Asp Ala
1315 1320 1325
Ala Leu Lys Lys Ala Phe Glu Tyr Ala Ser Glu Val Ile Val Glu Ala
1330 1335 1340
Phe Ile Glu Leu Gly Arg Glu Val Arg Cys Gly Ile Ile Val Lys Asp
1345 1350 1355 1360
Gly Glu Leu Ile Gly Leu Pro Leu Glu Glu Tyr Leu Val Asp Pro His
1365 1370 1375
Asp Lys Pro Ile Arg Asn Tyr Ala Asp Lys Leu Gln Gln Thr Asp Asp
1380 1385 1390
Gly Asp Leu His Leu Thr Ala Lys Asp Asn Ile Lys Ala Trp Ile Leu
1395 1400 1405
Asp Pro Asn Asp Pro Ile Thr Gln Lys Val Gln Gln Val Ala Lys Arg
1410 1415 1420
Cys His Gln Ala Leu Gly Cys Arg His Tyr Ser Leu Phe Asp Phe Arg
1425 1430 1435 1440
Ile Asp Pro Lys Gly Gln Pro Trp Phe Leu Glu Ala Gly Leu Tyr Cys
1445 1450 1455
Ser Phe Ala Pro Lys Ser Val Ile Ser Ser Met Ala Lys Ala Ala Gly
1460 1465 1470
Ile Pro Leu Asn Asp Leu Leu Ile Thr Ala Ile Asn Glu Thr Leu Gly
1475 1480 1485
Ser Asn Lys Lys Val Leu Gln Asn
1490 1495
<210> 5
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_ABCD的正向引物
<400> 5
acaatttcac acaggaaaga tatcatgagt atcgtccaag caaag 45
<210> 6
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_ABCD的反向引物
<400> 6
ctcatccgcc aaaacagctc tagattatga attattttcc agaca 45
<210> 7
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_ABCD的正向引物
<400> 7
gatcgatatc atgagtaatg ttcaagcatc g 31
<210> 8
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_ABCD的反向引物
<400> 8
gatctctaga tcaattttgt aacacctttt tattac 36
<210> 9
<211> 71
<212> DNA
<213> 人工序列
<220>
<223> 用于aroD缺失盒的正向引物
<400> 9
tcatggggtt cggtgcctga caggctgacc gcgtgcagaa agggtaaaaa gctggagctg 60
cttcgaagtt c 71
<210> 10
<211> 79
<212> DNA
<213> 人工序列
<220>
<223> 用于aroD缺失盒的反向引物
<400> 10
atatattttt tagttcggcg gggagggtgt tcccgccgaa atattattgc gccatggtcc 60
atatgaatat cctccttag 79
<210> 11
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 用于aroD缺失的正向引物
<400> 11
caaagatttc cctctggaat atg 23
<210> 12
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 用于aroD缺失的反向引物
<400> 12
cagatgtgat tttccctacg c 21
<210> 13
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于aroG的正向引物
<400> 13
aaggagctca ctagtggtac ggatgctcct gttatggtcg 40
<210> 14
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 用于aroG的反向引物
<400> 14
ctctcttcga caatttccac ttacccgcga cgcgctttta c 41
<210> 15
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 用于ppsA的正向引物
<400> 15
gtaaaagcgc gtcgcgggta agtggaaatt gtcgaagaga g 41
<210> 16
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 用于ppsA的反向引物
<400> 16
gccctagtgg atctgatggg ttatttcttc agttcagcca gg 42
<210> 17
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 用于tktA的正向引物
<400> 17
cctggctgaa ctgaagaaat aacccatcag atccactagg gc 42
<210> 18
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 用于tktA的反向引物
<400> 18
cgacaccggt cagcagggtg ttacagcagt tcttttgctt tc 42
<210> 19
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 用于pSKH130-ΔfhuA-Pn-aroG-Pn-ppsA-Pn-tktA质粒的确认的正向引物
<400> 19
gaaaatgccg atgggtaccg 20
<210> 20
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 用于pSKH130-ΔfhuA-Pn-aroG-Pn-ppsA-Pn-tktA质粒的确认的反向引物
<400> 20
cgttagaacg cggctacaat taatac 26
<210> 21
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_ABCD的正向引物
<400> 21
catctcacac cgagattatt ttagtactat gagtatcgtc caagcaaag 49
<210> 22
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_ABCD的反向引物
<400> 22
caatttcgta tagagtctca ctagtaccgc aaaaaggcca tccgtc 46
<210> 23
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 用于Ptrc启动子片断的正向引物
<400> 23
tctcacaccg agattatttt agtcgactgc acggtgcacc aat 43
<210> 24
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 用于Ptrc启动子片断的反向引物
<400> 24
tttgcttgga cgatactcat agtctgtttc ctgtgtgaaa ttgttatccg 50
<210> 25
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 用于PCJ1或PCJ2.2启动子片断的正向引物
<400> 25
tctcacaccg agattatttt agtcaccgcg ggcttattcc att 43
<210> 26
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 用于PCJ1或PCJ2.2启动子片断的反向引物
<400> 26
ttgcttggac gatactcata gtatcttaat ctcctagatt gggtttcac 49
<210> 27
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> 用于PCJ2.2启动子片断的反向引物
<400> 27
tttgcttgga cgatactcat agtatcttaa tctcctagat ttggtttcac 50
<210> 28
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava-ABCD整合的确认的正向引物
<400> 28
cagaatgagt gaacaaccac gg 22
<210> 29
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava-ABCD整合的确认的反向引物
<400> 29
catatccaat aacccagaca aaacc 25
<210> 30
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Ptrc/PCJ1-Ava_AB或Ptrc/PCJ1-Ava_ABC的正向引物
<400> 30
gatcggatcc atgagtatcg tccaagcaaa g 31
<210> 31
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 用于Ptrc/PCJ1-Ava_AB的反向引物
<400> 31
gatcactagt ttaaggttgt attctgcgg 29
<210> 32
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 用于Ptrc/PCJ1-Ava_ABC的反向引物
<400> 32
gatcactagt ctaatctccc cccaattcc 29
<210> 33
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_ABCD的正向引物
<400> 33
ccaaacacca acaaaaggct ctacccatat gatgagtatc gtccaagcaa agtttgaagc 60
60
<210> 34
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_ABCD的反向引物
<400> 34
agagggtgac gctagtcaga actagtttat gaattatttt ccagacaatc ttgtaaacgc 60
60
<210> 35
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 用于CJ7启动子片断的正向引物
<400> 35
ccaaacacca acaaaaggct ctacccatat gagaaacatc ccagcgctac taataggg 58
<210> 36
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 用于CJ7启动子片断的反向引物
<400> 36
caaactttgc ttggacgata ctcatagtgt ttcctttcgt tgggtacg 48
<210> 37
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 用于lysc8启动子片断的正向引物
<400> 37
caccaacaaa aggctctacc catatgaaca ctcctctggc taggtag 47
<210> 38
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 用于lysc8启动子片断的反向引物
<400> 38
caaactttgc ttggacgata ctcatctttg tgcacctttc gatctacg 48
<210> 39
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 用于O2启动子片断的正向引物
<400> 39
ccaaacacca acaaaaggct ctacccatat gcaataatcg tgaattttgg cagc 54
<210> 40
<211> 53
<212> DNA
<213> 人工序列
<220>
<223> 用于O2启动子片断的反向引物
<400> 40
caaactttgc ttggacgata ctcattgttt tgatctcctc caataatcta tgc 53
<210> 41
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 用于构建pDC Δ aroD的aroD基因的正向引物
<400> 41
gatcggatcc cactggacgt ttgggtgaga cc 32
<210> 42
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 用于构建pDC Δ aroD的aroD基因的反向引物
<400> 42
gatcggatcc actagtttta ggttccattt ctaattg 37
<210> 43
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 用于构建pDC Δ aroD的aroD基因的正向引物
<400> 43
gatcactagt atgaacgaca gtattctcc 29
<210> 44
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 用于构建pDC Δ aroD的aroD基因的反向引物
<400> 44
gatcaagctt gttacatcct gacgttgtgg 30
<210> 45
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_A的正向引物
<400> 45
cgcggatcca tgagtatcgt ccaagcaaag 30
<210> 46
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_A的反向引物
<400> 46
cgcctcgagt tatttaacac tcccgattaa ttc 33
<210> 47
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_B的正向引物
<400> 47
cgcggatcca tgacaaatgt gattgtccaa c 31
<210> 48
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_B的反向引物
<400> 48
cgcctcgagt taaggttgta ttctgcggat aattg 35
<210> 49
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_C的正向引物
<400> 49
cgcggatcca tggcacaatc ccttcccctt tc 32
<210> 50
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_C的反向引物
<400> 50
cgcctcgagc taatctcccc ccaattccac c 31
<210> 51
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_D的正向引物
<400> 51
cgcggatcca tgcagactat agattttaat attc 34
<210> 52
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 用于Ava_D的反向引物
<400> 52
cgcctcgagt tatgaattat tttccagaca atc 33
<210> 53
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_A的正向引物
<400> 53
cgcggatcca tgagtaatgt tcaagcatcg 30
<210> 54
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_A的反向引物
<400> 54
cgcctcgagt cacactccca atagtttgg 29
<210> 55
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_B的正向引物
<400> 55
cgcggatcca tgaccagtat tttaggacga g 31
<210> 56
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_B的反向引物
<400> 56
cgcctcgagt tataccaagc gtctaatc 28
<210> 57
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_C的正向引物
<400> 57
cgcggatcca tggcacaatc aatctcttta tc 32
<210> 58
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_C的反向引物
<400> 58
cgcctcgagt tagtcgcccc ctaattccac g 31
<210> 59
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_D的正向引物
<400> 59
cgcggatcca tgccagtact taatatcctt c 31
<210> 60
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 用于Npr_D的反向引物
<400> 60
cgcctcgagt caattttgta acaccttttt attac 35
<210> 61
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 用于pGPD的正向引物
<400> 61
acgtaagata attgtatatt acgcagttta tcattatcaa tactcgcc 48
<210> 62
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于pGPD的反向引物
<400> 62
gtaacgacaa tcctctccat gtggtcttga atcaaagctg 40
<210> 63
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于LoxP(Ura)的正向引物
<400> 63
ccgattgtca atcagcgtaa cagctgaagc ttcgtacgc 39
<210> 64
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 用于LoxP(Ura)的反向引物
<400> 64
ttgttttggc cagtcggcaa gggcataggc cactagtgga tctg 44
<210> 65
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于aroB的正向引物
<400> 65
cagctttgat tcaagaccac atggagagga ttgtcgttac 40
<210> 66
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于aroB的反向引物
<400> 66
gcgtacgaag cttcagctgt tacgctgatt gacaatcgg 39
<210> 67
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 用于ARO1区段1的正向引物
<400> 67
accattcaag agtatatgag agaaggaaaa ct 32
<210> 68
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 用于ARO1区段1的反向引物
<400> 68
ggcgagtatt gataatgata aactgcgtaa tatacaatta tcttacg 47
<210> 69
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 用于ARO1区段2的正向引物
<400> 69
cagatccact agtggcctat gcccttgccg actggccaaa acaa 44
<210> 70
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 用于ARO1区段2的反向引物
<400> 70
caaatatcaa tggcacgttg 20
<210> 71
<211> 759
<212> DNA
<213> 大肠杆菌W3110
<400> 71
atgaaaaccg taactgtaaa agatctcgtc attggtacgg gcgcacctaa aatcatcgtc 60
tcgctgatgg cgaaagatat cgccagcgtg aaatccgaag ctctcgccta tcgtgaagcg 120
gactttgata ttctggaatg gcgtgtggac cactatgccg acctctccaa tgtggagtct 180
gtcatggcgg cagcaaaaat tctccgtgag accatgccag aaaaaccgct gctgtttacc 240
ttccgcagtg ccaaagaagg cggcgagcag gcgatttcca ccgaggctta tattgcactc 300
aatcgtgcag ccatcgacag cggcctggtt gatatgatcg atctggagtt atttaccggt 360
gatgatcagg ttaaagaaac cgtcgcctac gcccacgcgc atgatgtgaa agtagtcatg 420
tccaaccatg acttccataa aacgccggaa gccgaagaaa tcattgcccg tctgcgcaaa 480
atgcaatcct tcgacgccga tattcctaag attgcgctga tgccgcaaag taccagcgat 540
gtgctgacgt tgcttgccgc gaccctggag atgcaggagc agtatgccga tcgtccaatt 600
atcacgatgt cgatggcaaa aactggcgta atttctcgtc tggctggtga agtatttggc 660
tcggcggcaa cttttggtgc ggtaaaaaaa gcgtctgcgc cagggcaaat ctcggtaaat 720
gatttgcgca cggtattaac tattttacac caggcataa 759
<210> 72
<211> 252
<212> PRT
<213> 大肠杆菌W3110
<400> 72
Met Lys Thr Val Thr Val Lys Asp Leu Val Ile Gly Thr Gly Ala Pro
1 5 10 15
Lys Ile Ile Val Ser Leu Met Ala Lys Asp Ile Ala Ser Val Lys Ser
20 25 30
Glu Ala Leu Ala Tyr Arg Glu Ala Asp Phe Asp Ile Leu Glu Trp Arg
35 40 45
Val Asp His Tyr Ala Asp Leu Ser Asn Val Glu Ser Val Met Ala Ala
50 55 60
Ala Lys Ile Leu Arg Glu Thr Met Pro Glu Lys Pro Leu Leu Phe Thr
65 70 75 80
Phe Arg Ser Ala Lys Glu Gly Gly Glu Gln Ala Ile Ser Thr Glu Ala
85 90 95
Tyr Ile Ala Leu Asn Arg Ala Ala Ile Asp Ser Gly Leu Val Asp Met
100 105 110
Ile Asp Leu Glu Leu Phe Thr Gly Asp Asp Gln Val Lys Glu Thr Val
115 120 125
Ala Tyr Ala His Ala His Asp Val Lys Val Val Met Ser Asn His Asp
130 135 140
Phe His Lys Thr Pro Glu Ala Glu Glu Ile Ile Ala Arg Leu Arg Lys
145 150 155 160
Met Gln Ser Phe Asp Ala Asp Ile Pro Lys Ile Ala Leu Met Pro Gln
165 170 175
Ser Thr Ser Asp Val Leu Thr Leu Leu Ala Ala Thr Leu Glu Met Gln
180 185 190
Glu Gln Tyr Ala Asp Arg Pro Ile Ile Thr Met Ser Met Ala Lys Thr
195 200 205
Gly Val Ile Ser Arg Leu Ala Gly Glu Val Phe Gly Ser Ala Ala Thr
210 215 220
Phe Gly Ala Val Lys Lys Ala Ser Ala Pro Gly Gln Ile Ser Val Asn
225 230 235 240
Asp Leu Arg Thr Val Leu Thr Ile Leu His Gln Ala
245 250
<210> 73
<211> 1053
<212> DNA
<213> 大肠杆菌W3110
<400> 73
atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60
gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120
aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180
tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240
gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300
acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360
gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420
gcaggtgagt ttctcgatat gatcacccca caatatctcg ctgacctgat gagctggggc 480
gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540
tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600
aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660
gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720
tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780
caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840
gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900
gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960
ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020
ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053
<210> 74
<211> 350
<212> PRT
<213> 大肠杆菌W3110
<400> 74
Met Asn Tyr Gln Asn Asp Asp Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asp Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
325 330 335
Leu Leu Arg Gln Leu Ala Asn Ala Val Lys Ala Arg Arg Gly
340 345 350
<210> 75
<211> 2379
<212> DNA
<213> 大肠杆菌W3110
<400> 75
atgtccaaca atggctcgtc accgctggtg ctttggtata accaactcgg catgaatgat 60
gtagacaggg ttgggggcaa aaatgcctcc ctgggtgaaa tgattactaa tctttccgga 120
atgggtgttt ccgttccgaa tggtttcgcc acaaccgccg acgcgtttaa ccagtttctg 180
gaccaaagcg gcgtaaacca gcgcatttat gaactgctgg ataaaacgga tattgacgat 240
gttactcagc ttgcgaaagc gggcgcgcaa atccgccagt ggattatcga cactcccttc 300
cagcctgagc tggaaaacgc catccgcgaa gcctatgcac agctttccgc cgatgacgaa 360
aacgcctctt ttgcggtgcg ctcctccgcc accgcagaag atatgccgga cgcttctttt 420
gccggtcagc aggaaacctt cctcaacgtt cagggttttg acgccgttct cgtggcagtg 480
aaacatgtat ttgcttctct gtttaacgat cgcgccatct cttatcgtgt gcaccagggt 540
tacgatcacc gtggtgtggc gctctccgcc ggtgttcaac ggatggtgcg ctctgacctc 600
gcatcatctg gcgtgatgtt ctccattgat accgaatccg gctttgacca ggtggtgttt 660
atcacttccg catggggcct tggtgagatg gtcgtgcagg gtgcggttaa cccggatgag 720
ttttacgtgc ataaaccgac actggcggcg aatcgcccgg ctatcgtgcg ccgcaccatg 780
gggtcgaaaa aaatccgcat ggtttacgcg ccgacccagg agcacggcaa gcaggttaaa 840
atcgaagacg taccgcagga acagcgtgac atcttctcgc tgaccaacga agaagtgcag 900
gaactggcaa aacaggccgt acaaattgag aaacactacg gtcgcccgat ggatattgag 960
tgggcgaaag atggccacac cggtaaactg ttcattgtgc aggcgcgtcc ggaaaccgtg 1020
cgctcacgcg gtcaggtcat ggagcgttat acgctgcatt cacagggtaa gattatcgcc 1080
gaaggccgtg ctatcggtca tcgcatcggt gcgggtccgg tgaaagtcat ccatgacatc 1140
agcgaaatga accgcatcga acctggcgac gtgctggtta ctgacatgac cgacccggac 1200
tgggaaccga tcatgaagaa agcatctgcc atcgtcacca accgtggcgg tcgtacctgt 1260
cacgcggcga tcatcgctcg tgaactgggc attccggcgg tagtgggctg tggagatgca 1320
acagaacgga tgaaagacgg tgagaacgtc actgtttctt gtgccgaagg tgataccggt 1380
tacgtctatg cggagttgct ggaatttagc gtgaaaagct ccagcgtaga aacgatgccg 1440
gatctgccgt tgaaagtgat gatgaacgtc ggtaacccgg accgtgcttt cgacttcgcc 1500
tgcctaccga acgaaggcgt gggccttgcg cgtctggaat ttatcatcaa ccgtatgatt 1560
ggcgtccacc cacgcgcact gcttgagttt gacgatcagg aaccgcagtt gcaaaacgaa 1620
atccgcgaga tgatgaaagg ttttgattct ccgcgtgaat tttacgttgg tcgtctgact 1680
gaagggatcg cgacgctggg tgccgcgttt tatccgaagc gcgtcattgt ccgtctctct 1740
gattttaaat cgaacgaata tgccaacctg gtcggtggtg agcgttacga gccagatgaa 1800
gagaacccga tgctcggctt ccgtggcgcg ggccgctatg tttccgacag cttccgcgac 1860
tgtttcgcgc tggagtgtga agcagtgaaa cgtgtgcgca acgacatggg actgaccaac 1920
gttgagatca tgatcccgtt cgtgcgtacc gtagatcagg cgaaagcggt ggttgaagaa 1980
ctggcgcgtc aggggctgaa acgtggcgag aacgggctga aaatcatcat gatgtgtgaa 2040
atcccgtcca acgccttgct ggccgagcag ttcctcgaat atttcgacgg cttctcaatt 2100
ggctcaaacg atatgacgca gctggcgctc ggtctggacc gtgactccgg cgtggtgtct 2160
gaattgttcg atgagcgcaa cgatgcggtg aaagcactgc tgtcgatggc tatccgtgcc 2220
gcgaagaaac agggcaaata tgtcgggatt tgcggtcagg gtccgtccga ccacgaagac 2280
tttgccgcat ggttgatgga agaggggatc gatagcctgt ctctgaaccc ggacaccgtg 2340
gtgcaaacct ggttaagcct ggctgaactg aagaaataa 2379
<210> 76
<211> 792
<212> PRT
<213> 大肠杆菌W3110
<400> 76
Met Ser Asn Asn Gly Ser Ser Pro Leu Val Leu Trp Tyr Asn Gln Leu
1 5 10 15
Gly Met Asn Asp Val Asp Arg Val Gly Gly Lys Asn Ala Ser Leu Gly
20 25 30
Glu Met Ile Thr Asn Leu Ser Gly Met Gly Val Ser Val Pro Asn Gly
35 40 45
Phe Ala Thr Thr Ala Asp Ala Phe Asn Gln Phe Leu Asp Gln Ser Gly
50 55 60
Val Asn Gln Arg Ile Tyr Glu Leu Leu Asp Lys Thr Asp Ile Asp Asp
65 70 75 80
Val Thr Gln Leu Ala Lys Ala Gly Ala Gln Ile Arg Gln Trp Ile Ile
85 90 95
Asp Thr Pro Phe Gln Pro Glu Leu Glu Asn Ala Ile Arg Glu Ala Tyr
100 105 110
Ala Gln Leu Ser Ala Asp Asp Glu Asn Ala Ser Phe Ala Val Arg Ser
115 120 125
Ser Ala Thr Ala Glu Asp Met Pro Asp Ala Ser Phe Ala Gly Gln Gln
130 135 140
Glu Thr Phe Leu Asn Val Gln Gly Phe Asp Ala Val Leu Val Ala Val
145 150 155 160
Lys His Val Phe Ala Ser Leu Phe Asn Asp Arg Ala Ile Ser Tyr Arg
165 170 175
Val His Gln Gly Tyr Asp His Arg Gly Val Ala Leu Ser Ala Gly Val
180 185 190
Gln Arg Met Val Arg Ser Asp Leu Ala Ser Ser Gly Val Met Phe Ser
195 200 205
Ile Asp Thr Glu Ser Gly Phe Asp Gln Val Val Phe Ile Thr Ser Ala
210 215 220
Trp Gly Leu Gly Glu Met Val Val Gln Gly Ala Val Asn Pro Asp Glu
225 230 235 240
Phe Tyr Val His Lys Pro Thr Leu Ala Ala Asn Arg Pro Ala Ile Val
245 250 255
Arg Arg Thr Met Gly Ser Lys Lys Ile Arg Met Val Tyr Ala Pro Thr
260 265 270
Gln Glu His Gly Lys Gln Val Lys Ile Glu Asp Val Pro Gln Glu Gln
275 280 285
Arg Asp Ile Phe Ser Leu Thr Asn Glu Glu Val Gln Glu Leu Ala Lys
290 295 300
Gln Ala Val Gln Ile Glu Lys His Tyr Gly Arg Pro Met Asp Ile Glu
305 310 315 320
Trp Ala Lys Asp Gly His Thr Gly Lys Leu Phe Ile Val Gln Ala Arg
325 330 335
Pro Glu Thr Val Arg Ser Arg Gly Gln Val Met Glu Arg Tyr Thr Leu
340 345 350
His Ser Gln Gly Lys Ile Ile Ala Glu Gly Arg Ala Ile Gly His Arg
355 360 365
Ile Gly Ala Gly Pro Val Lys Val Ile His Asp Ile Ser Glu Met Asn
370 375 380
Arg Ile Glu Pro Gly Asp Val Leu Val Thr Asp Met Thr Asp Pro Asp
385 390 395 400
Trp Glu Pro Ile Met Lys Lys Ala Ser Ala Ile Val Thr Asn Arg Gly
405 410 415
Gly Arg Thr Cys His Ala Ala Ile Ile Ala Arg Glu Leu Gly Ile Pro
420 425 430
Ala Val Val Gly Cys Gly Asp Ala Thr Glu Arg Met Lys Asp Gly Glu
435 440 445
Asn Val Thr Val Ser Cys Ala Glu Gly Asp Thr Gly Tyr Val Tyr Ala
450 455 460
Glu Leu Leu Glu Phe Ser Val Lys Ser Ser Ser Val Glu Thr Met Pro
465 470 475 480
Asp Leu Pro Leu Lys Val Met Met Asn Val Gly Asn Pro Asp Arg Ala
485 490 495
Phe Asp Phe Ala Cys Leu Pro Asn Glu Gly Val Gly Leu Ala Arg Leu
500 505 510
Glu Phe Ile Ile Asn Arg Met Ile Gly Val His Pro Arg Ala Leu Leu
515 520 525
Glu Phe Asp Asp Gln Glu Pro Gln Leu Gln Asn Glu Ile Arg Glu Met
530 535 540
Met Lys Gly Phe Asp Ser Pro Arg Glu Phe Tyr Val Gly Arg Leu Thr
545 550 555 560
Glu Gly Ile Ala Thr Leu Gly Ala Ala Phe Tyr Pro Lys Arg Val Ile
565 570 575
Val Arg Leu Ser Asp Phe Lys Ser Asn Glu Tyr Ala Asn Leu Val Gly
580 585 590
Gly Glu Arg Tyr Glu Pro Asp Glu Glu Asn Pro Met Leu Gly Phe Arg
595 600 605
Gly Ala Gly Arg Tyr Val Ser Asp Ser Phe Arg Asp Cys Phe Ala Leu
610 615 620
Glu Cys Glu Ala Val Lys Arg Val Arg Asn Asp Met Gly Leu Thr Asn
625 630 635 640
Val Glu Ile Met Ile Pro Phe Val Arg Thr Val Asp Gln Ala Lys Ala
645 650 655
Val Val Glu Glu Leu Ala Arg Gln Gly Leu Lys Arg Gly Glu Asn Gly
660 665 670
Leu Lys Ile Ile Met Met Cys Glu Ile Pro Ser Asn Ala Leu Leu Ala
675 680 685
Glu Gln Phe Leu Glu Tyr Phe Asp Gly Phe Ser Ile Gly Ser Asn Asp
690 695 700
Met Thr Gln Leu Ala Leu Gly Leu Asp Arg Asp Ser Gly Val Val Ser
705 710 715 720
Glu Leu Phe Asp Glu Arg Asn Asp Ala Val Lys Ala Leu Leu Ser Met
725 730 735
Ala Ile Arg Ala Ala Lys Lys Gln Gly Lys Tyr Val Gly Ile Cys Gly
740 745 750
Gln Gly Pro Ser Asp His Glu Asp Phe Ala Ala Trp Leu Met Glu Glu
755 760 765
Gly Ile Asp Ser Leu Ser Leu Asn Pro Asp Thr Val Val Gln Thr Trp
770 775 780
Leu Ser Leu Ala Glu Leu Lys Lys
785 790
<210> 77
<211> 1992
<212> DNA
<213> 大肠杆菌W3110
<400> 77
atgtcctcac gtaaagagct tgccaatgct attcgtgcgc tgagcatgga cgcagtacag 60
aaagccaaat ccggtcaccc gggtgcccct atgggtatgg ctgacattgc cgaagtcctg 120
tggcgtgatt tcctgaaaca caacccgcag aatccgtcct gggctgaccg tgaccgcttc 180
gtgctgtcca acggccacgg ctccatgctg atctacagcc tgctgcacct caccggttac 240
gatctgccga tggaagaact gaaaaacttc cgtcagctgc actctaaaac tccgggtcac 300
ccggaagtgg gttacaccgc tggtgtggaa accaccaccg gtccgctggg tcagggtatt 360
gccaacgcag tcggtatggc gattgcagaa aaaacgctgg cggcgcagtt taaccgtccg 420
ggccacgaca ttgtcgacca ctacacctac gccttcatgg gcgacggctg catgatggaa 480
ggcatctccc acgaagtttg ctctctggcg ggtacgctga agctgggtaa actgattgca 540
ttctacgatg acaacggtat ttctatcgat ggtcacgttg aaggctggtt caccgacgac 600
accgcaatgc gtttcgaagc ttacggctgg cacgttattc gcgacatcga cggtcatgac 660
gcggcatcta tcaaacgcgc agtagaagaa gcgcgcgcag tgactgacaa accttccctg 720
ctgatgtgca aaaccatcat cggtttcggt tccccgaaca aagccggtac ccacgactcc 780
cacggtgcgc cgctgggcga cgctgaaatt gccctgaccc gcgaacaact gggctggaaa 840
tatgcgccgt tcgaaatccc gtctgaaatc tatgctcagt gggatgcgaa agaagcaggc 900
caggcgaaag aatccgcatg gaacgagaaa ttcgctgctt acgcgaaagc ttatccgcag 960
gaagccgctg aatttacccg ccgtatgaaa ggcgaaatgc cgtctgactt cgacgctaaa 1020
gcgaaagagt tcatcgctaa actgcaggct aatccggcga aaatcgccag ccgtaaagcg 1080
tctcagaatg ctatcgaagc gttcggtccg ctgttgccgg aattcctcgg cggttctgct 1140
gacctggcgc cgtctaacct gaccctgtgg tctggttcta aagcaatcaa cgaagatgct 1200
gcgggtaact acatccacta cggtgttcgc gagttcggta tgaccgcgat tgctaacggt 1260
atctccctgc acggtggctt cctgccgtac acctccacct tcctgatgtt cgtggaatac 1320
gcacgtaacg ccgtacgtat ggctgcgctg atgaaacagc gtcaggtgat ggtttacacc 1380
cacgactcca tcggtctggg cgaagacggc ccgactcacc agccggttga gcaggtcgct 1440
tctctgcgcg taaccccgaa catgtctaca tggcgtccgt gtgaccaggt tgaatccgcg 1500
gtcgcgtgga aatacggtgt tgagcgtcag gacggcccga ccgcactgat cctctcccgt 1560
cagaacctgg cgcagcagga acgaactgaa gagcaactgg caaacatcgc gcgcggtggt 1620
tatgtgctga aagactgcgc cggtcagccg gaactgattt tcatcgctac cggttcagaa 1680
gttgaactgg ctgttgctgc ctacgaaaaa ctgactgccg aaggcgtgaa agcgcgcgtg 1740
gtgtccatgc cgtctaccga cgcatttgac aagcaggatg ctgcttaccg tgaatccgta 1800
ctgccgaaag cggttactgc acgcgttgct gtagaagcgg gtattgctga ctactggtac 1860
aagtatgttg gcctgaacgg tgctatcgtc ggtatgacca ccttcggtga atctgctccg 1920
gcagagctgc tgtttgaaga gttcggcttc actgttgata acgttgttgc gaaagcaaaa 1980
gaactgctgt aa 1992
<210> 78
<211> 663
<212> PRT
<213> 大肠杆菌W3110
<400> 78
Met Ser Ser Arg Lys Glu Leu Ala Asn Ala Ile Arg Ala Leu Ser Met
1 5 10 15
Asp Ala Val Gln Lys Ala Lys Ser Gly His Pro Gly Ala Pro Met Gly
20 25 30
Met Ala Asp Ile Ala Glu Val Leu Trp Arg Asp Phe Leu Lys His Asn
35 40 45
Pro Gln Asn Pro Ser Trp Ala Asp Arg Asp Arg Phe Val Leu Ser Asn
50 55 60
Gly His Gly Ser Met Leu Ile Tyr Ser Leu Leu His Leu Thr Gly Tyr
65 70 75 80
Asp Leu Pro Met Glu Glu Leu Lys Asn Phe Arg Gln Leu His Ser Lys
85 90 95
Thr Pro Gly His Pro Glu Val Gly Tyr Thr Ala Gly Val Glu Thr Thr
100 105 110
Thr Gly Pro Leu Gly Gln Gly Ile Ala Asn Ala Val Gly Met Ala Ile
115 120 125
Ala Glu Lys Thr Leu Ala Ala Gln Phe Asn Arg Pro Gly His Asp Ile
130 135 140
Val Asp His Tyr Thr Tyr Ala Phe Met Gly Asp Gly Cys Met Met Glu
145 150 155 160
Gly Ile Ser His Glu Val Cys Ser Leu Ala Gly Thr Leu Lys Leu Gly
165 170 175
Lys Leu Ile Ala Phe Tyr Asp Asp Asn Gly Ile Ser Ile Asp Gly His
180 185 190
Val Glu Gly Trp Phe Thr Asp Asp Thr Ala Met Arg Phe Glu Ala Tyr
195 200 205
Gly Trp His Val Ile Arg Asp Ile Asp Gly His Asp Ala Ala Ser Ile
210 215 220
Lys Arg Ala Val Glu Glu Ala Arg Ala Val Thr Asp Lys Pro Ser Leu
225 230 235 240
Leu Met Cys Lys Thr Ile Ile Gly Phe Gly Ser Pro Asn Lys Ala Gly
245 250 255
Thr His Asp Ser His Gly Ala Pro Leu Gly Asp Ala Glu Ile Ala Leu
260 265 270
Thr Arg Glu Gln Leu Gly Trp Lys Tyr Ala Pro Phe Glu Ile Pro Ser
275 280 285
Glu Ile Tyr Ala Gln Trp Asp Ala Lys Glu Ala Gly Gln Ala Lys Glu
290 295 300
Ser Ala Trp Asn Glu Lys Phe Ala Ala Tyr Ala Lys Ala Tyr Pro Gln
305 310 315 320
Glu Ala Ala Glu Phe Thr Arg Arg Met Lys Gly Glu Met Pro Ser Asp
325 330 335
Phe Asp Ala Lys Ala Lys Glu Phe Ile Ala Lys Leu Gln Ala Asn Pro
340 345 350
Ala Lys Ile Ala Ser Arg Lys Ala Ser Gln Asn Ala Ile Glu Ala Phe
355 360 365
Gly Pro Leu Leu Pro Glu Phe Leu Gly Gly Ser Ala Asp Leu Ala Pro
370 375 380
Ser Asn Leu Thr Leu Trp Ser Gly Ser Lys Ala Ile Asn Glu Asp Ala
385 390 395 400
Ala Gly Asn Tyr Ile His Tyr Gly Val Arg Glu Phe Gly Met Thr Ala
405 410 415
Ile Ala Asn Gly Ile Ser Leu His Gly Gly Phe Leu Pro Tyr Thr Ser
420 425 430
Thr Phe Leu Met Phe Val Glu Tyr Ala Arg Asn Ala Val Arg Met Ala
435 440 445
Ala Leu Met Lys Gln Arg Gln Val Met Val Tyr Thr His Asp Ser Ile
450 455 460
Gly Leu Gly Glu Asp Gly Pro Thr His Gln Pro Val Glu Gln Val Ala
465 470 475 480
Ser Leu Arg Val Thr Pro Asn Met Ser Thr Trp Arg Pro Cys Asp Gln
485 490 495
Val Glu Ser Ala Val Ala Trp Lys Tyr Gly Val Glu Arg Gln Asp Gly
500 505 510
Pro Thr Ala Leu Ile Leu Ser Arg Gln Asn Leu Ala Gln Gln Glu Arg
515 520 525
Thr Glu Glu Gln Leu Ala Asn Ile Ala Arg Gly Gly Tyr Val Leu Lys
530 535 540
Asp Cys Ala Gly Gln Pro Glu Leu Ile Phe Ile Ala Thr Gly Ser Glu
545 550 555 560
Val Glu Leu Ala Val Ala Ala Tyr Glu Lys Leu Thr Ala Glu Gly Val
565 570 575
Lys Ala Arg Val Val Ser Met Pro Ser Thr Asp Ala Phe Asp Lys Gln
580 585 590
Asp Ala Ala Tyr Arg Glu Ser Val Leu Pro Lys Ala Val Thr Ala Arg
595 600 605
Val Ala Val Glu Ala Gly Ile Ala Asp Tyr Trp Tyr Lys Tyr Val Gly
610 615 620
Leu Asn Gly Ala Ile Val Gly Met Thr Thr Phe Gly Glu Ser Ala Pro
625 630 635 640
Ala Glu Leu Leu Phe Glu Glu Phe Gly Phe Thr Val Asp Asn Val Val
645 650 655
Ala Lys Ala Lys Glu Leu Leu
660
<210> 79
<211> 438
<212> DNA
<213> 谷氨酸棒杆菌ATCC 13032
<400> 79
atgcctggaa aaattctcct cctcaacggc ccaaacctga acatgctggg caaacgcgag 60
cctgacattt acggacacga caccttggaa gacgtcgtcg cgctggcaac cgctgaggct 120
gcgaagcacg gccttgaggt tgaggcgctg cagagcaatc acgaaggtga gctaatcgat 180
gcgctgcaca acgctcgcgg cacccacatc ggttgcgtga ttaaccccgg cggcctgact 240
cacacttcgg tggcgctttt ggatgctgtg aaggcgtctg agcttcctac cgttgaggtg 300
cacatttcca atccgcatgc ccgtgaagag ttccgccacc attcttacat ttccctcgcc 360
gcggtctccg ttatcgctgg cgctggcatc cagggttacc gtttcgcggt cgatatcctg 420
gcaaatctca aaaagtag 438
<210> 80
<211> 145
<212> PRT
<213> 谷氨酸棒杆菌ATCC 13032
<400> 80
Met Pro Gly Lys Ile Leu Leu Leu Asn Gly Pro Asn Leu Asn Met Leu
1 5 10 15
Gly Lys Arg Glu Pro Asp Ile Tyr Gly His Asp Thr Leu Glu Asp Val
20 25 30
Val Ala Leu Ala Thr Ala Glu Ala Ala Lys His Gly Leu Glu Val Glu
35 40 45
Ala Leu Gln Ser Asn His Glu Gly Glu Leu Ile Asp Ala Leu His Asn
50 55 60
Ala Arg Gly Thr His Ile Gly Cys Val Ile Asn Pro Gly Gly Leu Thr
65 70 75 80
His Thr Ser Val Ala Leu Leu Asp Ala Val Lys Ala Ser Glu Leu Pro
85 90 95
Thr Val Glu Val His Ile Ser Asn Pro His Ala Arg Glu Glu Phe Arg
100 105 110
His His Ser Tyr Ile Ser Leu Ala Ala Val Ser Val Ile Ala Gly Ala
115 120 125
Gly Ile Gln Gly Tyr Arg Phe Ala Val Asp Ile Leu Ala Asn Leu Lys
130 135 140
Lys
145
<210> 81
<211> 4767
<212> DNA
<213> 酿酒酵母
<400> 81
atggtgcagt tagccaaagt cccaattcta ggaaatgata ttatccacgt tgggtataac 60
attcatgacc atttggttga aaccataatt aaacattgtc cttcttcgac atacgttatt 120
tgcaatgata cgaacttgag taaagttcca tactaccagc aattagtcct ggaattcaag 180
gcttctttgc cagaaggctc tcgtttactt acttatgttg ttaaaccagg tgagacaagt 240
aaaagtagag aaaccaaagc gcagctagaa gattatcttt tagtggaagg atgtactcgt 300
gatacggtta tggtagcgat cggtggtggt gttattggtg acatgattgg gttcgttgca 360
tctacattta tgagaggtgt tcgtgttgtc caagtaccaa catccttatt ggcaatggtc 420
gattcctcca ttggtggtaa aactgctatt gacactcctc taggtaaaaa ctttattggt 480
gcattttggc aaccaaaatt tgtccttgta gatattaaat ggctagaaac gttagccaag 540
agagagttta tcaatgggat ggcagaagtt atcaagactg cttgtatttg gaacgctgac 600
gaatttacta gattagaatc aaacgcttcg ttgttcttaa atgttgttaa tggggcaaaa 660
aatgtcaagg ttaccaatca attgacaaac gagattgacg agatatcgaa tacagatatt 720
gaagctatgt tggatcatac atataagtta gttcttgaga gtattaaggt caaagcggaa 780
gttgtctctt cggatgaacg tgaatccagt ctaagaaacc ttttgaactt cggacattct 840
attggtcatg cttatgaagc tatactaacc ccacaagcat tacatggtga atgtgtgtcc 900
attggtatgg ttaaagaggc ggaattatcc cgttatttcg gtattctctc ccctacccaa 960
gttgcacgtc tatccaagat tttggttgcc tacgggttgc ctgtttcgcc tgatgagaaa 1020
tggtttaaag agctaacctt acataagaaa acaccattgg atatcttatt gaagaaaatg 1080
agtattgaca agaaaaacga gggttccaaa aagaaggtgg tcattttaga aagtattggt 1140
aagtgctatg gtgactccgc tcaatttgtt agcgatgaag acctgagatt tattctaaca 1200
gatgaaaccc tcgtttaccc cttcaaggac atccctgctg atcaacagaa agttgttatc 1260
ccccctggtt ctaagtccat ctccaatcgt gctttaattc ttgctgccct cggtgaaggt 1320
caatgtaaaa tcaagaactt attacattct gatgatacta aacatatgtt aaccgctgtt 1380
catgaattga aaggtgctac gatatcatgg gaagataatg gtgagacggt agtggtggaa 1440
ggacatggtg gttccacatt gtcagcttgt gctgacccct tatatctagg taatgcaggt 1500
actgcatcta gatttttgac ttccttggct gccttggtca attctacttc aagccaaaag 1560
tatatcgttt taactggtaa cgcaagaatg caacaaagac caattgctcc tttggtcgat 1620
tctttgcgtg ctaatggtac taaaattgag tacttgaata atgaaggttc cctgccaatc 1680
aaagtttata ctgattcggt attcaaaggt ggtagaattg aattagctgc tacagtttct 1740
tctcagtacg tatcctctat cttgatgtgt gccccatacg ctgaagaacc tgtaactttg 1800
gctcttgttg gtggtaagcc aatctctaaa ttgtacgtcg atatgacaat aaaaatgatg 1860
gaaaaattcg gtatcaatgt tgaaacttct actacagaac cttacactta ttatattcca 1920
aagggacatt atattaaccc atcagaatac gtcattgaaa gtgatgcctc aagtgctaca 1980
tacccattgg ccttcgccgc aatgactggt actaccgtaa cggttccaaa cattggtttt 2040
gagtcgttac aaggtgatgc cagatttgca agagatgtct tgaaacctat gggttgtaaa 2100
ataactcaaa cggcaacttc aactactgtt tcgggtcctc ctgtaggtac tttaaagcca 2160
ttaaaacatg ttgatatgga gccaatgact gatgcgttct taactgcatg tgttgttgcc 2220
gctatttcgc acgacagtga tccaaattct gcaaatacaa ccaccattga aggtattgca 2280
aaccagcgtg tcaaagagtg taacagaatt ttggccatgg ctacagagct cgccaaattt 2340
ggcgtcaaaa ctacagaatt accagatggt attcaagtcc atggtttaaa ctcgataaaa 2400
gatttgaagg ttccttccga ctcttctgga cctgtcggtg tatgcacata tgatgatcat 2460
cgtgtggcca tgagtttctc gcttcttgca ggaatggtaa attctcaaaa tgaacgtgac 2520
gaagttgcta atcctgtaag aatacttgaa agacattgta ctggtaaaac ctggcctggc 2580
tggtgggatg tgttacattc cgaactaggt gccaaattag atggtgcaga acctttagag 2640
tgcacatcca aaaagaactc aaagaaaagc gttgtcatta ttggcatgag agcagctggc 2700
aaaactacta taagtaaatg gtgcgcatcc gctctgggtt acaaattagt tgacctagac 2760
gagctgtttg agcaacagca taacaatcaa agtgttaaac aatttgttgt ggagaacggt 2820
tgggagaagt tccgtgagga agaaacaaga attttcaagg aagttattca aaattacggc 2880
gatgatggat atgttttctc aacaggtggc ggtattgttg aaagcgctga gtctagaaaa 2940
gccttaaaag attttgcctc atcaggtgga tacgttttac acttacatag ggatattgag 3000
gagacaattg tctttttaca aagtgatcct tcaagacctg cctatgtgga agaaattcgt 3060
gaagtttgga acagaaggga ggggtggtat aaagaatgct caaatttctc tttctttgct 3120
cctcattgct ccgcagaagc tgagttccaa gctctaagaa gatcgtttag taagtacatt 3180
gcaaccatta caggtgtcag agaaatagaa attccaagcg gaagatctgc ctttgtgtgt 3240
ttaacctttg atgacttaac tgaacaaact gagaatttga ctccaatctg ttatggttgt 3300
gaggctgtag aggtcagagt agaccatttg gctaattact ctgctgattt cgtgagtaaa 3360
cagttatcta tattgcgtaa agccactgac agtattccta tcatttttac tgtgcgaacc 3420
atgaagcaag gtggcaactt tcctgatgaa gagttcaaaa ccttgagaga gctatacgat 3480
attgccttga agaatggtgt tgaattcctt gacttagaac taactttacc tactgatatc 3540
caatatgagg ttattaacaa aaggggcaac accaagatca ttggttccca tcatgacttc 3600
caaggattat actcctggga cgacgctgaa tgggaaaaca gattcaatca agcgttaact 3660
cttgatgtgg atgttgtaaa atttgtgggt acggctgtta atttcgaaga taatttgaga 3720
ctggaacact ttagggatac acacaagaat aagcctttaa ttgcagttaa tatgacttct 3780
aaaggtagca tttctcgtgt tttgaataat gttttaacac ctgtgacatc agatttattg 3840
cctaactccg ctgcccctgg ccaattgaca gtagcacaaa ttaacaagat gtatacatct 3900
atgggaggta tcgagcctaa ggaactgttt gttgttggaa agccaattgg ccactctaga 3960
tcgccaattt tacataacac tggctatgaa attttaggtt tacctcacaa gttcgataaa 4020
tttgaaactg aatccgcaca attggtgaaa gaaaaacttt tggacggaaa caagaacttt 4080
ggcggtgctg cagtcacaat tcctctgaaa ttagatataa tgcagtacat ggatgaattg 4140
actgatgctg ctaaagttat tggtgctgta aacacagtta taccattggg taacaagaag 4200
tttaagggtg ataataccga ctggttaggt atccgtaatg ccttaattaa caatggcgtt 4260
cccgaatatg ttggtcatac cgctggtttg gttatcggtg caggtggcac ttctagagcc 4320
gccctttacg ccttgcacag tttaggttgc aaaaagatct tcataatcaa caggacaact 4380
tcgaaattga agccattaat agagtcactt ccatctgaat tcaacattat tggaatagag 4440
tccactaaat ctatagaaga gattaaggaa cacgttggcg ttgctgtcag ctgtgtacca 4500
gccgacaaac cattagatga cgaactttta agtaagctgg agagattcct tgtgaaaggt 4560
gcccatgctg cttttgtacc aaccttattg gaagccgcat acaaaccaag cgttactccc 4620
gttatgacaa tttcacaaga caaatatcaa tggcacgttg tccctggatc acaaatgtta 4680
gtacaccaag gtgtagctca gtttgaaaag tggacaggat tcaagggccc tttcaaggcc 4740
atttttgatg ccgttacgaa agagtag 4767
<210> 82
<211> 1588
<212> PRT
<213> 酿酒酵母
<400> 82
Met Val Gln Leu Ala Lys Val Pro Ile Leu Gly Asn Asp Ile Ile His
1 5 10 15
Val Gly Tyr Asn Ile His Asp His Leu Val Glu Thr Ile Ile Lys His
20 25 30
Cys Pro Ser Ser Thr Tyr Val Ile Cys Asn Asp Thr Asn Leu Ser Lys
35 40 45
Val Pro Tyr Tyr Gln Gln Leu Val Leu Glu Phe Lys Ala Ser Leu Pro
50 55 60
Glu Gly Ser Arg Leu Leu Thr Tyr Val Val Lys Pro Gly Glu Thr Ser
65 70 75 80
Lys Ser Arg Glu Thr Lys Ala Gln Leu Glu Asp Tyr Leu Leu Val Glu
85 90 95
Gly Cys Thr Arg Asp Thr Val Met Val Ala Ile Gly Gly Gly Val Ile
100 105 110
Gly Asp Met Ile Gly Phe Val Ala Ser Thr Phe Met Arg Gly Val Arg
115 120 125
Val Val Gln Val Pro Thr Ser Leu Leu Ala Met Val Asp Ser Ser Ile
130 135 140
Gly Gly Lys Thr Ala Ile Asp Thr Pro Leu Gly Lys Asn Phe Ile Gly
145 150 155 160
Ala Phe Trp Gln Pro Lys Phe Val Leu Val Asp Ile Lys Trp Leu Glu
165 170 175
Thr Leu Ala Lys Arg Glu Phe Ile Asn Gly Met Ala Glu Val Ile Lys
180 185 190
Thr Ala Cys Ile Trp Asn Ala Asp Glu Phe Thr Arg Leu Glu Ser Asn
195 200 205
Ala Ser Leu Phe Leu Asn Val Val Asn Gly Ala Lys Asn Val Lys Val
210 215 220
Thr Asn Gln Leu Thr Asn Glu Ile Asp Glu Ile Ser Asn Thr Asp Ile
225 230 235 240
Glu Ala Met Leu Asp His Thr Tyr Lys Leu Val Leu Glu Ser Ile Lys
245 250 255
Val Lys Ala Glu Val Val Ser Ser Asp Glu Arg Glu Ser Ser Leu Arg
260 265 270
Asn Leu Leu Asn Phe Gly His Ser Ile Gly His Ala Tyr Glu Ala Ile
275 280 285
Leu Thr Pro Gln Ala Leu His Gly Glu Cys Val Ser Ile Gly Met Val
290 295 300
Lys Glu Ala Glu Leu Ser Arg Tyr Phe Gly Ile Leu Ser Pro Thr Gln
305 310 315 320
Val Ala Arg Leu Ser Lys Ile Leu Val Ala Tyr Gly Leu Pro Val Ser
325 330 335
Pro Asp Glu Lys Trp Phe Lys Glu Leu Thr Leu His Lys Lys Thr Pro
340 345 350
Leu Asp Ile Leu Leu Lys Lys Met Ser Ile Asp Lys Lys Asn Glu Gly
355 360 365
Ser Lys Lys Lys Val Val Ile Leu Glu Ser Ile Gly Lys Cys Tyr Gly
370 375 380
Asp Ser Ala Gln Phe Val Ser Asp Glu Asp Leu Arg Phe Ile Leu Thr
385 390 395 400
Asp Glu Thr Leu Val Tyr Pro Phe Lys Asp Ile Pro Ala Asp Gln Gln
405 410 415
Lys Val Val Ile Pro Pro Gly Ser Lys Ser Ile Ser Asn Arg Ala Leu
420 425 430
Ile Leu Ala Ala Leu Gly Glu Gly Gln Cys Lys Ile Lys Asn Leu Leu
435 440 445
His Ser Asp Asp Thr Lys His Met Leu Thr Ala Val His Glu Leu Lys
450 455 460
Gly Ala Thr Ile Ser Trp Glu Asp Asn Gly Glu Thr Val Val Val Glu
465 470 475 480
Gly His Gly Gly Ser Thr Leu Ser Ala Cys Ala Asp Pro Leu Tyr Leu
485 490 495
Gly Asn Ala Gly Thr Ala Ser Arg Phe Leu Thr Ser Leu Ala Ala Leu
500 505 510
Val Asn Ser Thr Ser Ser Gln Lys Tyr Ile Val Leu Thr Gly Asn Ala
515 520 525
Arg Met Gln Gln Arg Pro Ile Ala Pro Leu Val Asp Ser Leu Arg Ala
530 535 540
Asn Gly Thr Lys Ile Glu Tyr Leu Asn Asn Glu Gly Ser Leu Pro Ile
545 550 555 560
Lys Val Tyr Thr Asp Ser Val Phe Lys Gly Gly Arg Ile Glu Leu Ala
565 570 575
Ala Thr Val Ser Ser Gln Tyr Val Ser Ser Ile Leu Met Cys Ala Pro
580 585 590
Tyr Ala Glu Glu Pro Val Thr Leu Ala Leu Val Gly Gly Lys Pro Ile
595 600 605
Ser Lys Leu Tyr Val Asp Met Thr Ile Lys Met Met Glu Lys Phe Gly
610 615 620
Ile Asn Val Glu Thr Ser Thr Thr Glu Pro Tyr Thr Tyr Tyr Ile Pro
625 630 635 640
Lys Gly His Tyr Ile Asn Pro Ser Glu Tyr Val Ile Glu Ser Asp Ala
645 650 655
Ser Ser Ala Thr Tyr Pro Leu Ala Phe Ala Ala Met Thr Gly Thr Thr
660 665 670
Val Thr Val Pro Asn Ile Gly Phe Glu Ser Leu Gln Gly Asp Ala Arg
675 680 685
Phe Ala Arg Asp Val Leu Lys Pro Met Gly Cys Lys Ile Thr Gln Thr
690 695 700
Ala Thr Ser Thr Thr Val Ser Gly Pro Pro Val Gly Thr Leu Lys Pro
705 710 715 720
Leu Lys His Val Asp Met Glu Pro Met Thr Asp Ala Phe Leu Thr Ala
725 730 735
Cys Val Val Ala Ala Ile Ser His Asp Ser Asp Pro Asn Ser Ala Asn
740 745 750
Thr Thr Thr Ile Glu Gly Ile Ala Asn Gln Arg Val Lys Glu Cys Asn
755 760 765
Arg Ile Leu Ala Met Ala Thr Glu Leu Ala Lys Phe Gly Val Lys Thr
770 775 780
Thr Glu Leu Pro Asp Gly Ile Gln Val His Gly Leu Asn Ser Ile Lys
785 790 795 800
Asp Leu Lys Val Pro Ser Asp Ser Ser Gly Pro Val Gly Val Cys Thr
805 810 815
Tyr Asp Asp His Arg Val Ala Met Ser Phe Ser Leu Leu Ala Gly Met
820 825 830
Val Asn Ser Gln Asn Glu Arg Asp Glu Val Ala Asn Pro Val Arg Ile
835 840 845
Leu Glu Arg His Cys Thr Gly Lys Thr Trp Pro Gly Trp Trp Asp Val
850 855 860
Leu His Ser Glu Leu Gly Ala Lys Leu Asp Gly Ala Glu Pro Leu Glu
865 870 875 880
Cys Thr Ser Lys Lys Asn Ser Lys Lys Ser Val Val Ile Ile Gly Met
885 890 895
Arg Ala Ala Gly Lys Thr Thr Ile Ser Lys Trp Cys Ala Ser Ala Leu
900 905 910
Gly Tyr Lys Leu Val Asp Leu Asp Glu Leu Phe Glu Gln Gln His Asn
915 920 925
Asn Gln Ser Val Lys Gln Phe Val Val Glu Asn Gly Trp Glu Lys Phe
930 935 940
Arg Glu Glu Glu Thr Arg Ile Phe Lys Glu Val Ile Gln Asn Tyr Gly
945 950 955 960
Asp Asp Gly Tyr Val Phe Ser Thr Gly Gly Gly Ile Val Glu Ser Ala
965 970 975
Glu Ser Arg Lys Ala Leu Lys Asp Phe Ala Ser Ser Gly Gly Tyr Val
980 985 990
Leu His Leu His Arg Asp Ile Glu Glu Thr Ile Val Phe Leu Gln Ser
995 1000 1005
Asp Pro Ser Arg Pro Ala Tyr Val Glu Glu Ile Arg Glu Val Trp Asn
1010 1015 1020
Arg Arg Glu Gly Trp Tyr Lys Glu Cys Ser Asn Phe Ser Phe Phe Ala
1025 1030 1035 1040
Pro His Cys Ser Ala Glu Ala Glu Phe Gln Ala Leu Arg Arg Ser Phe
1045 1050 1055
Ser Lys Tyr Ile Ala Thr Ile Thr Gly Val Arg Glu Ile Glu Ile Pro
1060 1065 1070
Ser Gly Arg Ser Ala Phe Val Cys Leu Thr Phe Asp Asp Leu Thr Glu
1075 1080 1085
Gln Thr Glu Asn Leu Thr Pro Ile Cys Tyr Gly Cys Glu Ala Val Glu
1090 1095 1100
Val Arg Val Asp His Leu Ala Asn Tyr Ser Ala Asp Phe Val Ser Lys
1105 1110 1115 1120
Gln Leu Ser Ile Leu Arg Lys Ala Thr Asp Ser Ile Pro Ile Ile Phe
1125 1130 1135
Thr Val Arg Thr Met Lys Gln Gly Gly Asn Phe Pro Asp Glu Glu Phe
1140 1145 1150
Lys Thr Leu Arg Glu Leu Tyr Asp Ile Ala Leu Lys Asn Gly Val Glu
1155 1160 1165
Phe Leu Asp Leu Glu Leu Thr Leu Pro Thr Asp Ile Gln Tyr Glu Val
1170 1175 1180
Ile Asn Lys Arg Gly Asn Thr Lys Ile Ile Gly Ser His His Asp Phe
1185 1190 1195 1200
Gln Gly Leu Tyr Ser Trp Asp Asp Ala Glu Trp Glu Asn Arg Phe Asn
1205 1210 1215
Gln Ala Leu Thr Leu Asp Val Asp Val Val Lys Phe Val Gly Thr Ala
1220 1225 1230
Val Asn Phe Glu Asp Asn Leu Arg Leu Glu His Phe Arg Asp Thr His
1235 1240 1245
Lys Asn Lys Pro Leu Ile Ala Val Asn Met Thr Ser Lys Gly Ser Ile
1250 1255 1260
Ser Arg Val Leu Asn Asn Val Leu Thr Pro Val Thr Ser Asp Leu Leu
1265 1270 1275 1280
Pro Asn Ser Ala Ala Pro Gly Gln Leu Thr Val Ala Gln Ile Asn Lys
1285 1290 1295
Met Tyr Thr Ser Met Gly Gly Ile Glu Pro Lys Glu Leu Phe Val Val
1300 1305 1310
Gly Lys Pro Ile Gly His Ser Arg Ser Pro Ile Leu His Asn Thr Gly
1315 1320 1325
Tyr Glu Ile Leu Gly Leu Pro His Lys Phe Asp Lys Phe Glu Thr Glu
1330 1335 1340
Ser Ala Gln Leu Val Lys Glu Lys Leu Leu Asp Gly Asn Lys Asn Phe
1345 1350 1355 1360
Gly Gly Ala Ala Val Thr Ile Pro Leu Lys Leu Asp Ile Met Gln Tyr
1365 1370 1375
Met Asp Glu Leu Thr Asp Ala Ala Lys Val Ile Gly Ala Val Asn Thr
1380 1385 1390
Val Ile Pro Leu Gly Asn Lys Lys Phe Lys Gly Asp Asn Thr Asp Trp
1395 1400 1405
Leu Gly Ile Arg Asn Ala Leu Ile Asn Asn Gly Val Pro Glu Tyr Val
1410 1415 1420
Gly His Thr Ala Gly Leu Val Ile Gly Ala Gly Gly Thr Ser Arg Ala
1425 1430 1435 1440
Ala Leu Tyr Ala Leu His Ser Leu Gly Cys Lys Lys Ile Phe Ile Ile
1445 1450 1455
Asn Arg Thr Thr Ser Lys Leu Lys Pro Leu Ile Glu Ser Leu Pro Ser
1460 1465 1470
Glu Phe Asn Ile Ile Gly Ile Glu Ser Thr Lys Ser Ile Glu Glu Ile
1475 1480 1485
Lys Glu His Val Gly Val Ala Val Ser Cys Val Pro Ala Asp Lys Pro
1490 1495 1500
Leu Asp Asp Glu Leu Leu Ser Lys Leu Glu Arg Phe Leu Val Lys Gly
1505 1510 1515 1520
Ala His Ala Ala Phe Val Pro Thr Leu Leu Glu Ala Ala Tyr Lys Pro
1525 1530 1535
Ser Val Thr Pro Val Met Thr Ile Ser Gln Asp Lys Tyr Gln Trp His
1540 1545 1550
Val Val Pro Gly Ser Gln Met Leu Val His Gln Gly Val Ala Gln Phe
1555 1560 1565
Glu Lys Trp Thr Gly Phe Lys Gly Pro Phe Lys Ala Ile Phe Asp Ala
1570 1575 1580
Val Thr Lys Glu
1585
<210> 83
<211> 1089
<212> DNA
<213> 大肠杆菌W3110
<400> 83
atggagagga ttgtcgttac tctcggggaa cgtagttacc caattaccat cgcatctggt 60
ttgtttaatg aaccagcttc attcttaccg ctgaaatcgg gcgagcaggt catgttggtc 120
accaacgaaa ccctggctcc tctgtatctc gataaggtcc gcggcgtact tgaacaggcg 180
ggtgttaacg tcgatagcgt tatcctccct gacggcgagc agtataaaag cctggctgta 240
ctcgataccg tctttacggc gttgttacaa aaaccgcatg gtcgcgatac tacgctggtg 300
gcgcttggcg gcggcgtagt gggcgatctg accggcttcg cggcggcgag ttatcagcgc 360
ggtgtccgtt tcattcaagt cccgacgacg ttactgtcgc aggtcgattc ctccgttggc 420
ggcaaaactg cggtcaacca tcccctcggt aaaaacatga ttggcgcgtt ctaccaacct 480
gcttcagtgg tggtggatct cgactgtctg aaaacgcttc ccccgcgtga gttagcgtcg 540
gggctggcag aagtcatcaa atacggcatt attcttgacg gtgcgttttt taactggctg 600
gaagagaatc tggatgcgtt gttgcgtctg gacggtccgg caatggcgta ctgtattcgc 660
cgttgttgtg aactgaaggc agaagttgtc gccgccgacg agcgcgaaac cgggttacgt 720
gctttactga atctgggaca cacctttggt catgccattg aagctgaaat ggggtatggc 780
aattggttac atggtgaagc ggtcgctgcg ggtatggtga tggcggcgcg gacgtcggaa 840
cgtctcgggc agtttagttc tgccgaaacg cagcgtatta taaccctgct caagcgggct 900
gggttaccgg tcaatgggcc gcgcgaaatg tccgcgcagg cgtatttacc gcatatgctg 960
cgtgacaaga aagtccttgc gggagagatg cgcttaattc ttccgttggc aattggtaag 1020
agtgaagttc gcagcggcgt ttcgcacgag cttgttctta acgccattgc cgattgtcaa 1080
tcagcgtaa 1089
<210> 84
<211> 362
<212> PRT
<213> 大肠杆菌W3110
<400> 84
Met Glu Arg Ile Val Val Thr Leu Gly Glu Arg Ser Tyr Pro Ile Thr
1 5 10 15
Ile Ala Ser Gly Leu Phe Asn Glu Pro Ala Ser Phe Leu Pro Leu Lys
20 25 30
Ser Gly Glu Gln Val Met Leu Val Thr Asn Glu Thr Leu Ala Pro Leu
35 40 45
Tyr Leu Asp Lys Val Arg Gly Val Leu Glu Gln Ala Gly Val Asn Val
50 55 60
Asp Ser Val Ile Leu Pro Asp Gly Glu Gln Tyr Lys Ser Leu Ala Val
65 70 75 80
Leu Asp Thr Val Phe Thr Ala Leu Leu Gln Lys Pro His Gly Arg Asp
85 90 95
Thr Thr Leu Val Ala Leu Gly Gly Gly Val Val Gly Asp Leu Thr Gly
100 105 110
Phe Ala Ala Ala Ser Tyr Gln Arg Gly Val Arg Phe Ile Gln Val Pro
115 120 125
Thr Thr Leu Leu Ser Gln Val Asp Ser Ser Val Gly Gly Lys Thr Ala
130 135 140
Val Asn His Pro Leu Gly Lys Asn Met Ile Gly Ala Phe Tyr Gln Pro
145 150 155 160
Ala Ser Val Val Val Asp Leu Asp Cys Leu Lys Thr Leu Pro Pro Arg
165 170 175
Glu Leu Ala Ser Gly Leu Ala Glu Val Ile Lys Tyr Gly Ile Ile Leu
180 185 190
Asp Gly Ala Phe Phe Asn Trp Leu Glu Glu Asn Leu Asp Ala Leu Leu
195 200 205
Arg Leu Asp Gly Pro Ala Met Ala Tyr Cys Ile Arg Arg Cys Cys Glu
210 215 220
Leu Lys Ala Glu Val Val Ala Ala Asp Glu Arg Glu Thr Gly Leu Arg
225 230 235 240
Ala Leu Leu Asn Leu Gly His Thr Phe Gly His Ala Ile Glu Ala Glu
245 250 255
Met Gly Tyr Gly Asn Trp Leu His Gly Glu Ala Val Ala Ala Gly Met
260 265 270
Val Met Ala Ala Arg Thr Ser Glu Arg Leu Gly Gln Phe Ser Ser Ala
275 280 285
Glu Thr Gln Arg Ile Ile Thr Leu Leu Lys Arg Ala Gly Leu Pro Val
290 295 300
Asn Gly Pro Arg Glu Met Ser Ala Gln Ala Tyr Leu Pro His Met Leu
305 310 315 320
Arg Asp Lys Lys Val Leu Ala Gly Glu Met Arg Leu Ile Leu Pro Leu
325 330 335
Ala Ile Gly Lys Ser Glu Val Arg Ser Gly Val Ser His Glu Leu Val
340 345 350
Leu Asn Ala Ile Ala Asp Cys Gln Ser Ala
355 360
<210> 85
<211> 2247
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 85
atgagtatcg tccaagcaaa gtttgaagct aaggaaacat cttttcatgt agaaggttac 60
gaaaagattg agtatgattt ggtgtatgta gatggtattt ttgaaatcca gaattctgca 120
ctagcagatg tatatcaagg ttttggacga tgcttggcga ttgtagatgc taacgtcagt 180
cggttgtatg gtaatcaaat tcaggcatat ttccagtatt atggtataga actgaggcta 240
tttcctatta ccattactga accagataag actattcaaa ctttcgagag agttatagat 300
gtctttgcag atttcaaatt agtccgcaaa gaaccagtat tagtcgtggg tggcggttta 360
attacagatg ttgtcggctt tgcttgttct acatatcgtc gcagcagcaa ttacatccgc 420
attcctacta cattgattgg attaattgat gccagtgtag caattaaggt agcagttaat 480
catcgcaaac tgaaaaaccg tttgggtgct tatcatgctt ctcgcaaagt atttttagat 540
ttctccttgt tgcgtactct ccctacagac caagtacgta acgggatggc ggaattggta 600
aaaatcgctg tagtagcgca tcaagaagtt tttgaattgt tggagaagta cggcgaagaa 660
ttactacgta ctcattttgg caatatagat gcaactccag agattaaaga aatagcccat 720
cgtttgactt acaaagctat ccataagatg ttggaattgg aagttcccaa cctgcatgag 780
ttagacctag atagggtgat tgcttacggt cacacttgga gtcccacctt ggaacttgcg 840
cctcgtctac ccatgttcca cggacacgcc gttaatgtag atatggcttt ctcggcaacg 900
atcgccgccc gtagaggata tattacaatt gcagaacgcg atcgtatttt aggattaatg 960
agtcgcgttg gtctatccct cgaccatccc atgttggata tagatatttt gtggcgtggt 1020
actgaatcta tcacattaac tcgtgatggt ttgttaagag ctgctatgcc aaaacccatt 1080
ggtgattgtg tcttcgtcaa tgacctgaca agagaagaat tagcagccgc attagctgac 1140
cacaaagaac tttgtaccag ttatccccgt ggtggtgaag gtgtggatgt gtatcccgtt 1200
tatcaaaaag aattaatcgg gagtgttaaa taatgacttt tttgaattca aaatgcaaaa 1260
tactccacgg atacactgcg cgagcgcggt agcatttctg ttcgcggagc gtcccgtagg 1320
gaaagagaag gctacgcaaa taatcggaca ctaattgtct ttaattttga attttgaatt 1380
ttgaattttg aattggagcg aagcgacttg acaaatgtga ttgtccaacc aacagctaga 1440
cctgttacac cattgggaat tttaaccaag cagttagaag ccatagtcca agaggttaag 1500
caacatccag atttacctgg ggaattgata gcaaacatcc atcaggcttg gcgtttagcc 1560
gcaggtatag acccttattt ggaagaatgc accactccag aatctcctga actcgctgca 1620
ttggcaaaaa ccacagccac cgaagcctgg ggagaacact tccacggagg tacaaccgtc 1680
cgtcctctag aacaagagat gctttctggt catatcgaag gacaaacctt aaagatgttt 1740
gttcacatga ccaaagctaa aaaagtctta gaaattggga tgtttaccgg ttattcggcg 1800
ctggcgatgg cggaagcatt accagaggat ggactgcttg tggcttgtga agttgaccct 1860
tacgcggcgg aaattggaca gaaagccttt caacaatctc cccacggtgg aaagattcgt 1920
gtggaattgg atgcagcctt agcaactctt gataagttag cagaagctgg ggagtctttt 1980
gacttggtat ttatcgacgc agataaaaaa gagtatgtag cctattttca caagttgcta 2040
ggtagcagtt tgttagcacc agatggcttt atttgtgtag ataacacctt attacaaggg 2100
gaagtttatc taccagcaga ggaacgtagc gtcaatggtg aagcgatcgc gcaatttaat 2160
catacagtag ctatagaccc ccgtgtagaa caggttttgt tgccgttgcg agatggttta 2220
acaattatcc gcagaataca accttaa 2247
<210> 86
<211> 820
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 86
Met Ser Ile Val Gln Ala Lys Phe Glu Ala Lys Glu Thr Ser Phe His
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly
20 25 30
Ile Phe Glu Ile Gln Asn Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe
35 40 45
Gly Arg Cys Leu Ala Ile Val Asp Ala Asn Val Ser Arg Leu Tyr Gly
50 55 60
Asn Gln Ile Gln Ala Tyr Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu
65 70 75 80
Phe Pro Ile Thr Ile Thr Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu
85 90 95
Arg Val Ile Asp Val Phe Ala Asp Phe Lys Leu Val Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Ile Thr Asp Val Val Gly Phe Ala
115 120 125
Cys Ser Thr Tyr Arg Arg Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Ser Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Ser Arg Lys
165 170 175
Val Phe Leu Asp Phe Ser Leu Leu Arg Thr Leu Pro Thr Asp Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala His Gln
195 200 205
Glu Val Phe Glu Leu Leu Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr
210 215 220
His Phe Gly Asn Ile Asp Ala Thr Pro Glu Ile Lys Glu Ile Ala His
225 230 235 240
Arg Leu Thr Tyr Lys Ala Ile His Lys Met Leu Glu Leu Glu Val Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Arg Leu Pro Met Phe His Gly
275 280 285
His Ala Val Asn Val Asp Met Ala Phe Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ile Ala Glu Arg Asp Arg Ile Leu Gly Leu Met
305 310 315 320
Ser Arg Val Gly Leu Ser Leu Asp His Pro Met Leu Asp Ile Asp Ile
325 330 335
Leu Trp Arg Gly Thr Glu Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Asp Cys Val Phe Val Asn Asp
355 360 365
Leu Thr Arg Glu Glu Leu Ala Ala Ala Leu Ala Asp His Lys Glu Leu
370 375 380
Cys Thr Ser Tyr Pro Arg Gly Gly Glu Gly Val Asp Val Tyr Pro Val
385 390 395 400
Tyr Gln Lys Glu Leu Ile Gly Ser Val Lys Met Ser Ile Val Gln Ala
405 410 415
Lys Phe Glu Ala Lys Glu Thr Ser Phe His Val Glu Gly Tyr Glu Lys
420 425 430
Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly Ile Phe Glu Ile Gln Asn
435 440 445
Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe Gly Arg Cys Leu Ala Ile
450 455 460
Val Asp Ala Asn Val Ser Arg Leu Tyr Gly Asn Gln Ile Gln Ala Tyr
465 470 475 480
Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu Phe Pro Ile Thr Ile Thr
485 490 495
Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu Arg Val Ile Asp Val Phe
500 505 510
Ala Asp Phe Lys Leu Val Arg Lys Glu Pro Val Leu Val Val Gly Gly
515 520 525
Gly Leu Ile Thr Asp Val Val Gly Phe Ala Cys Ser Thr Tyr Arg Arg
530 535 540
Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr Leu Ile Gly Leu Ile Asp
545 550 555 560
Ala Ser Val Ala Ile Lys Val Ala Val Asn His Arg Lys Leu Lys Asn
565 570 575
Arg Leu Gly Ala Tyr His Ala Ser Arg Lys Val Phe Leu Asp Phe Ser
580 585 590
Leu Leu Arg Thr Leu Pro Thr Asp Gln Val Arg Asn Gly Met Ala Glu
595 600 605
Leu Val Lys Ile Ala Val Val Ala His Gln Glu Val Phe Glu Leu Leu
610 615 620
Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr His Phe Gly Asn Ile Asp
625 630 635 640
Ala Thr Pro Glu Ile Lys Glu Ile Ala His Arg Leu Thr Tyr Lys Ala
645 650 655
Ile His Lys Met Leu Glu Leu Glu Val Pro Asn Leu His Glu Leu Asp
660 665 670
Leu Asp Arg Val Ile Ala Tyr Gly His Thr Trp Ser Pro Thr Leu Glu
675 680 685
Leu Ala Pro Arg Leu Pro Met Phe His Gly His Ala Val Asn Val Asp
690 695 700
Met Ala Phe Ser Ala Thr Ile Ala Ala Arg Arg Gly Tyr Ile Thr Ile
705 710 715 720
Ala Glu Arg Asp Arg Ile Leu Gly Leu Met Ser Arg Val Gly Leu Ser
725 730 735
Leu Asp His Pro Met Leu Asp Ile Asp Ile Leu Trp Arg Gly Thr Glu
740 745 750
Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu Arg Ala Ala Met Pro Lys
755 760 765
Pro Ile Gly Asp Cys Val Phe Val Asn Asp Leu Thr Arg Glu Glu Leu
770 775 780
Ala Ala Ala Leu Ala Asp His Lys Glu Leu Cys Thr Ser Tyr Pro Arg
785 790 795 800
Gly Gly Glu Gly Val Asp Val Tyr Pro Val Tyr Gln Lys Glu Leu Ile
805 810 815
Gly Ser Val Lys
820
<210> 87
<211> 3638
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 87
atgagtatcg tccaagcaaa gtttgaagct aaggaaacat cttttcatgt agaaggttac 60
gaaaagattg agtatgattt ggtgtatgta gatggtattt ttgaaatcca gaattctgca 120
ctagcagatg tatatcaagg ttttggacga tgcttggcga ttgtagatgc taacgtcagt 180
cggttgtatg gtaatcaaat tcaggcatat ttccagtatt atggtataga actgaggcta 240
tttcctatta ccattactga accagataag actattcaaa ctttcgagag agttatagat 300
gtctttgcag atttcaaatt agtccgcaaa gaaccagtat tagtcgtggg tggcggttta 360
attacagatg ttgtcggctt tgcttgttct acatatcgtc gcagcagcaa ttacatccgc 420
attcctacta cattgattgg attaattgat gccagtgtag caattaaggt agcagttaat 480
catcgcaaac tgaaaaaccg tttgggtgct tatcatgctt ctcgcaaagt atttttagat 540
ttctccttgt tgcgtactct ccctacagac caagtacgta acgggatggc ggaattggta 600
aaaatcgctg tagtagcgca tcaagaagtt tttgaattgt tggagaagta cggcgaagaa 660
ttactacgta ctcattttgg caatatagat gcaactccag agattaaaga aatagcccat 720
cgtttgactt acaaagctat ccataagatg ttggaattgg aagttcccaa cctgcatgag 780
ttagacctag atagggtgat tgcttacggt cacacttgga gtcccacctt ggaacttgcg 840
cctcgtctac ccatgttcca cggacacgcc gttaatgtag atatggcttt ctcggcaacg 900
atcgccgccc gtagaggata tattacaatt gcagaacgcg atcgtatttt aggattaatg 960
agtcgcgttg gtctatccct cgaccatccc atgttggata tagatatttt gtggcgtggt 1020
actgaatcta tcacattaac tcgtgatggt ttgttaagag ctgctatgcc aaaacccatt 1080
ggtgattgtg tcttcgtcaa tgacctgaca agagaagaat tagcagccgc attagctgac 1140
cacaaagaac tttgtaccag ttatccccgt ggtggtgaag gtgtggatgt gtatcccgtt 1200
tatcaaaaag aattaatcgg gagtgttaaa taatgacttt tttgaattca aaatgcaaaa 1260
tactccacgg atacactgcg cgagcgcggt agcatttctg ttcgcggagc gtcccgtagg 1320
gaaagagaag gctacgcaaa taatcggaca ctaattgtct ttaattttga attttgaatt 1380
ttgaattttg aattggagcg aagcgacttg acaaatgtga ttgtccaacc aacagctaga 1440
cctgttacac cattgggaat tttaaccaag cagttagaag ccatagtcca agaggttaag 1500
caacatccag atttacctgg ggaattgata gcaaacatcc atcaggcttg gcgtttagcc 1560
gcaggtatag acccttattt ggaagaatgc accactccag aatctcctga actcgctgca 1620
ttggcaaaaa ccacagccac cgaagcctgg ggagaacact tccacggagg tacaaccgtc 1680
cgtcctctag aacaagagat gctttctggt catatcgaag gacaaacctt aaagatgttt 1740
gttcacatga ccaaagctaa aaaagtctta gaaattggga tgtttaccgg ttattcggcg 1800
ctggcgatgg cggaagcatt accagaggat ggactgcttg tggcttgtga agttgaccct 1860
tacgcggcgg aaattggaca gaaagccttt caacaatctc cccacggtgg aaagattcgt 1920
gtggaattgg atgcagcctt agcaactctt gataagttag cagaagctgg ggagtctttt 1980
gacttggtat ttatcgacgc agataaaaaa gagtatgtag cctattttca caagttgcta 2040
ggtagcagtt tgttagcacc agatggcttt atttgtgtag ataacacctt attacaaggg 2100
gaagtttatc taccagcaga ggaacgtagc gtcaatggtg aagcgatcgc gcaatttaat 2160
catacagtag ctatagaccc ccgtgtagaa caggttttgt tgccgttgcg agatggttta 2220
acaattatcc gcagaataca accttaattg tccaatcgac tatggcacaa tcccttcccc 2280
tttcttccgc acctgctaca ccgtctcttc cttcccagac gaaaatagcc gcaattatcc 2340
aaaatatctg cactttggct ttgttattac tagcattgcc cattaatgcc accattgttt 2400
ttatatcctt gttagtcttc cgaccgcaaa aggtcaaagc agcaaacccc caaaccattc 2460
ttatcagtgg cggtaagatg accaaagctt tacaactagc aaggtcattc cacgcggctg 2520
gacatagagt tgtcttggtg gaaacccata aatactggtt gactggtcat cgtttttccc 2580
aagcagtgga taagttttac acagtccccg caccccagga caatccccaa gcttacattc 2640
aggctttggt agatatcgtc aaacaagaaa acatcgatgt ttatattccc gtcaccagtc 2700
cagtgggtag ctactacgac tcattagcca aaccagagtt atcccattat tgcgaagtgt 2760
ttcactttga cgcagatatt acccaaatgt tggatgataa atttgcgttg acacaaaaag 2820
cgcgatcgct tggtttatca gtacccaaat cctttaaaat tacctcacca gaacaagtca 2880
tcaacttcga tttttctgga gagacacgta aatacatcct caaaagcatt ccctacgact 2940
cagtgcggcg gttggactta accaaactcc cctgtgctac tccagaggaa acagcagcat 3000
tcgtcagaag tttgccaatt actcccgaaa aaccgtggat tatgcaggaa tttatccccg 3060
gtaaggaatt ctgcacccat agcaccgttc ggaatgggga actcagactg cattgctgtt 3120
gcgaatcttc agccttccaa gttaattatg agaatgtaaa taacccgcaa attaccgaat 3180
gggtacagca ttttgtcaag gaactgaaac tgacaggaca gatttccttt gactttatcc 3240
aagccgaaga cggaacagtt tacgccatcg agtgtaaccc ccgcacacat tcagcaatta 3300
ccacatttta cgaccacccc caggtagcag aagcgtactt gagtcaagca ccgacgactg 3360
aaaccataca accactaacg acaagcaagc ctacctattg gacttatcac gaagtttggc 3420
gtttaactgg tatccgttct ttcacccagt tgcaaagatg gctggggaat atttggcgcg 3480
ggactgatgc gatttatcag ccagatgacc ccttaccgtt tttgatggta catcattggc 3540
aaattcccct actgttattg aataatttgc gtcgtcttaa aggttggacg cggatagatt 3600
tcaatattgg gaagttggtg gaattggggg gagattag 3638
<210> 88
<211> 1099
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 88
Met Ser Ile Val Gln Ala Lys Phe Glu Ala Lys Glu Thr Ser Phe His
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly
20 25 30
Ile Phe Glu Ile Gln Asn Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe
35 40 45
Gly Arg Cys Leu Ala Ile Val Asp Ala Asn Val Ser Arg Leu Tyr Gly
50 55 60
Asn Gln Ile Gln Ala Tyr Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu
65 70 75 80
Phe Pro Ile Thr Ile Thr Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu
85 90 95
Arg Val Ile Asp Val Phe Ala Asp Phe Lys Leu Val Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Ile Thr Asp Val Val Gly Phe Ala
115 120 125
Cys Ser Thr Tyr Arg Arg Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Ser Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Ser Arg Lys
165 170 175
Val Phe Leu Asp Phe Ser Leu Leu Arg Thr Leu Pro Thr Asp Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala His Gln
195 200 205
Glu Val Phe Glu Leu Leu Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr
210 215 220
His Phe Gly Asn Ile Asp Ala Thr Pro Glu Ile Lys Glu Ile Ala His
225 230 235 240
Arg Leu Thr Tyr Lys Ala Ile His Lys Met Leu Glu Leu Glu Val Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Arg Leu Pro Met Phe His Gly
275 280 285
His Ala Val Asn Val Asp Met Ala Phe Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ile Ala Glu Arg Asp Arg Ile Leu Gly Leu Met
305 310 315 320
Ser Arg Val Gly Leu Ser Leu Asp His Pro Met Leu Asp Ile Asp Ile
325 330 335
Leu Trp Arg Gly Thr Glu Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Asp Cys Val Phe Val Asn Asp
355 360 365
Leu Thr Arg Glu Glu Leu Ala Ala Ala Leu Ala Asp His Lys Glu Leu
370 375 380
Cys Thr Ser Tyr Pro Arg Gly Gly Glu Gly Val Asp Val Tyr Pro Val
385 390 395 400
Tyr Gln Lys Glu Leu Ile Gly Ser Val Lys Met Ser Ile Val Gln Ala
405 410 415
Lys Phe Glu Ala Lys Glu Thr Ser Phe His Val Glu Gly Tyr Glu Lys
420 425 430
Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly Ile Phe Glu Ile Gln Asn
435 440 445
Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe Gly Arg Cys Leu Ala Ile
450 455 460
Val Asp Ala Asn Val Ser Arg Leu Tyr Gly Asn Gln Ile Gln Ala Tyr
465 470 475 480
Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu Phe Pro Ile Thr Ile Thr
485 490 495
Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu Arg Val Ile Asp Val Phe
500 505 510
Ala Asp Phe Lys Leu Val Arg Lys Glu Pro Val Leu Val Val Gly Gly
515 520 525
Gly Leu Ile Thr Asp Val Val Gly Phe Ala Cys Ser Thr Tyr Arg Arg
530 535 540
Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr Leu Ile Gly Leu Ile Asp
545 550 555 560
Ala Ser Val Ala Ile Lys Val Ala Val Asn His Arg Lys Leu Lys Asn
565 570 575
Arg Leu Gly Ala Tyr His Ala Ser Arg Lys Val Phe Leu Asp Phe Ser
580 585 590
Leu Leu Arg Thr Leu Pro Thr Asp Gln Val Arg Asn Gly Met Ala Glu
595 600 605
Leu Val Lys Ile Ala Val Val Ala His Gln Glu Val Phe Glu Leu Leu
610 615 620
Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr His Phe Gly Asn Ile Asp
625 630 635 640
Ala Thr Pro Glu Ile Lys Glu Ile Ala His Arg Leu Thr Tyr Lys Ala
645 650 655
Ile His Lys Met Leu Glu Leu Glu Val Pro Asn Leu His Glu Leu Asp
660 665 670
Leu Asp Arg Val Ile Ala Tyr Gly His Thr Trp Ser Pro Thr Leu Glu
675 680 685
Leu Ala Pro Arg Leu Pro Met Phe His Gly His Ala Val Asn Val Asp
690 695 700
Met Ala Phe Ser Ala Thr Ile Ala Ala Arg Arg Gly Tyr Ile Thr Ile
705 710 715 720
Ala Glu Arg Asp Arg Ile Leu Gly Leu Met Ser Arg Val Gly Leu Ser
725 730 735
Leu Asp His Pro Met Leu Asp Ile Asp Ile Leu Trp Arg Gly Thr Glu
740 745 750
Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu Arg Ala Ala Met Pro Lys
755 760 765
Pro Ile Gly Asp Cys Val Phe Val Asn Asp Leu Thr Arg Glu Glu Leu
770 775 780
Ala Ala Ala Leu Ala Asp His Lys Glu Leu Cys Thr Ser Tyr Pro Arg
785 790 795 800
Gly Gly Glu Gly Val Asp Val Tyr Pro Val Tyr Gln Lys Glu Leu Ile
805 810 815
Gly Ser Val Lys Met Thr Asn Val Ile Val Gln Pro Thr Ala Arg Pro
820 825 830
Val Thr Pro Leu Gly Ile Leu Thr Lys Gln Leu Glu Ala Ile Val Gln
835 840 845
Glu Val Lys Gln His Pro Asp Leu Pro Gly Glu Leu Ile Ala Asn Ile
850 855 860
His Gln Ala Trp Arg Leu Ala Ala Gly Ile Asp Pro Tyr Leu Glu Glu
865 870 875 880
Cys Thr Thr Pro Glu Ser Pro Glu Leu Ala Ala Leu Ala Lys Thr Thr
885 890 895
Ala Thr Glu Ala Trp Gly Glu His Phe His Gly Gly Thr Thr Val Arg
900 905 910
Pro Leu Glu Gln Glu Met Leu Ser Gly His Ile Glu Gly Gln Thr Leu
915 920 925
Lys Met Phe Val His Met Thr Lys Ala Lys Lys Val Leu Glu Ile Gly
930 935 940
Met Phe Thr Gly Tyr Ser Ala Leu Ala Met Ala Glu Ala Leu Pro Glu
945 950 955 960
Asp Gly Leu Leu Val Ala Cys Glu Val Asp Pro Tyr Ala Ala Glu Ile
965 970 975
Gly Gln Lys Ala Phe Gln Gln Ser Pro His Gly Gly Lys Ile Arg Val
980 985 990
Glu Leu Asp Ala Ala Leu Ala Thr Leu Asp Lys Leu Ala Glu Ala Gly
995 1000 1005
Glu Ser Phe Asp Leu Val Phe Ile Asp Ala Asp Lys Lys Glu Tyr Val
1010 1015 1020
Ala Tyr Phe His Lys Leu Leu Gly Ser Ser Leu Leu Ala Pro Asp Gly
1025 1030 1035 1040
Phe Ile Cys Val Asp Asn Thr Leu Leu Gln Gly Glu Val Tyr Leu Pro
1045 1050 1055
Ala Glu Glu Arg Ser Val Asn Gly Glu Ala Ile Ala Gln Phe Asn His
1060 1065 1070
Thr Val Ala Ile Asp Pro Arg Val Glu Gln Val Leu Leu Pro Leu Arg
1075 1080 1085
Asp Gly Leu Thr Ile Ile Arg Arg Ile Gln Pro
1090 1095
<210> 89
<211> 1233
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 89
atgagtatcg tccaagcaaa gtttgaagct aaggaaacat cttttcatgt agaaggttac 60
gaaaagattg agtatgattt ggtgtatgta gatggtattt ttgaaatcca gaattctgca 120
ctagcagatg tatatcaagg ttttggacga tgcttggcga ttgtagatgc taacgtcagt 180
cggttgtatg gtaatcaaat tcaggcatat ttccagtatt atggtataga actgaggcta 240
tttcctatta ccattactga accagataag actattcaaa ctttcgagag agttatagat 300
gtctttgcag atttcaaatt agtccgcaaa gaaccagtat tagtcgtggg tggcggttta 360
attacagatg ttgtcggctt tgcttgttct acatatcgtc gcagcagcaa ttacatccgc 420
attcctacta cattgattgg attaattgat gccagtgtag caattaaggt agcagttaat 480
catcgcaaac tgaaaaaccg tttgggtgct tatcatgctt ctcgcaaagt atttttagat 540
ttctccttgt tgcgtactct ccctacagac caagtacgta acgggatggc ggaattggta 600
aaaatcgctg tagtagcgca tcaagaagtt tttgaattgt tggagaagta cggcgaagaa 660
ttactacgta ctcattttgg caatatagat gcaactccag agattaaaga aatagcccat 720
cgtttgactt acaaagctat ccataagatg ttggaattgg aagttcccaa cctgcatgag 780
ttagacctag atagggtgat tgcttacggt cacacttgga gtcccacctt ggaacttgcg 840
cctcgtctac ccatgttcca cggacacgcc gttaatgtag atatggcttt ctcggcaacg 900
atcgccgccc gtagaggata tattacaatt gcagaacgcg atcgtatttt aggattaatg 960
agtcgcgttg gtctatccct cgaccatccc atgttggata tagatatttt gtggcgtggt 1020
actgaatcta tcacattaac tcgtgatggt ttgttaagag ctgctatgcc aaaacccatt 1080
ggtgattgtg tcttcgtcaa tgacctgaca agagaagaat tagcagccgc attagctgac 1140
cacaaagaac tttgtaccag ttatccccgt ggtggtgaag gtgtggatgt gtatcccgtt 1200
tatcaaaaag aattaatcgg gagtgttaaa taa 1233
<210> 90
<211> 410
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 90
Met Ser Ile Val Gln Ala Lys Phe Glu Ala Lys Glu Thr Ser Phe His
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly
20 25 30
Ile Phe Glu Ile Gln Asn Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe
35 40 45
Gly Arg Cys Leu Ala Ile Val Asp Ala Asn Val Ser Arg Leu Tyr Gly
50 55 60
Asn Gln Ile Gln Ala Tyr Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu
65 70 75 80
Phe Pro Ile Thr Ile Thr Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu
85 90 95
Arg Val Ile Asp Val Phe Ala Asp Phe Lys Leu Val Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Ile Thr Asp Val Val Gly Phe Ala
115 120 125
Cys Ser Thr Tyr Arg Arg Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Ser Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Ser Arg Lys
165 170 175
Val Phe Leu Asp Phe Ser Leu Leu Arg Thr Leu Pro Thr Asp Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala His Gln
195 200 205
Glu Val Phe Glu Leu Leu Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr
210 215 220
His Phe Gly Asn Ile Asp Ala Thr Pro Glu Ile Lys Glu Ile Ala His
225 230 235 240
Arg Leu Thr Tyr Lys Ala Ile His Lys Met Leu Glu Leu Glu Val Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Arg Leu Pro Met Phe His Gly
275 280 285
His Ala Val Asn Val Asp Met Ala Phe Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ile Ala Glu Arg Asp Arg Ile Leu Gly Leu Met
305 310 315 320
Ser Arg Val Gly Leu Ser Leu Asp His Pro Met Leu Asp Ile Asp Ile
325 330 335
Leu Trp Arg Gly Thr Glu Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Asp Cys Val Phe Val Asn Asp
355 360 365
Leu Thr Arg Glu Glu Leu Ala Ala Ala Leu Ala Asp His Lys Glu Leu
370 375 380
Cys Thr Ser Tyr Pro Arg Gly Gly Glu Gly Val Asp Val Tyr Pro Val
385 390 395 400
Tyr Gln Lys Glu Leu Ile Gly Ser Val Lys
405 410
<210> 91
<211> 840
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 91
ttgacaaatg tgattgtcca accaacagct agacctgtta caccattggg aattttaacc 60
aagcagttag aagccatagt ccaagaggtt aagcaacatc cagatttacc tggggaattg 120
atagcaaaca tccatcaggc ttggcgttta gccgcaggta tagaccctta tttggaagaa 180
tgcaccactc cagaatctcc tgaactcgct gcattggcaa aaaccacagc caccgaagcc 240
tggggagaac acttccacgg aggtacaacc gtccgtcctc tagaacaaga gatgctttct 300
ggtcatatcg aaggacaaac cttaaagatg tttgttcaca tgaccaaagc taaaaaagtc 360
ttagaaattg ggatgtttac cggttattcg gcgctggcga tggcggaagc attaccagag 420
gatggactgc ttgtggcttg tgaagttgac ccttacgcgg cggaaattgg acagaaagcc 480
tttcaacaat ctccccacgg tggaaagatt cgtgtggaat tggatgcagc cttagcaact 540
cttgataagt tagcagaagc tggggagtct tttgacttgg tatttatcga cgcagataaa 600
aaagagtatg tagcctattt tcacaagttg ctaggtagca gtttgttagc accagatggc 660
tttatttgtg tagataacac cttattacaa ggggaagttt atctaccagc agaggaacgt 720
agcgtcaatg gtgaagcgat cgcgcaattt aatcatacag tagctataga cccccgtgta 780
gaacaggttt tgttgccgtt gcgagatggt ttaacaatta tccgcagaat acaaccttaa 840
840
<210> 92
<211> 410
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 92
Met Ser Ile Val Gln Ala Lys Phe Glu Ala Lys Glu Thr Ser Phe His
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Tyr Asp Leu Val Tyr Val Asp Gly
20 25 30
Ile Phe Glu Ile Gln Asn Ser Ala Leu Ala Asp Val Tyr Gln Gly Phe
35 40 45
Gly Arg Cys Leu Ala Ile Val Asp Ala Asn Val Ser Arg Leu Tyr Gly
50 55 60
Asn Gln Ile Gln Ala Tyr Phe Gln Tyr Tyr Gly Ile Glu Leu Arg Leu
65 70 75 80
Phe Pro Ile Thr Ile Thr Glu Pro Asp Lys Thr Ile Gln Thr Phe Glu
85 90 95
Arg Val Ile Asp Val Phe Ala Asp Phe Lys Leu Val Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Ile Thr Asp Val Val Gly Phe Ala
115 120 125
Cys Ser Thr Tyr Arg Arg Ser Ser Asn Tyr Ile Arg Ile Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Ser Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Ser Arg Lys
165 170 175
Val Phe Leu Asp Phe Ser Leu Leu Arg Thr Leu Pro Thr Asp Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala His Gln
195 200 205
Glu Val Phe Glu Leu Leu Glu Lys Tyr Gly Glu Glu Leu Leu Arg Thr
210 215 220
His Phe Gly Asn Ile Asp Ala Thr Pro Glu Ile Lys Glu Ile Ala His
225 230 235 240
Arg Leu Thr Tyr Lys Ala Ile His Lys Met Leu Glu Leu Glu Val Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Arg Leu Pro Met Phe His Gly
275 280 285
His Ala Val Asn Val Asp Met Ala Phe Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ile Ala Glu Arg Asp Arg Ile Leu Gly Leu Met
305 310 315 320
Ser Arg Val Gly Leu Ser Leu Asp His Pro Met Leu Asp Ile Asp Ile
325 330 335
Leu Trp Arg Gly Thr Glu Ser Ile Thr Leu Thr Arg Asp Gly Leu Leu
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Asp Cys Val Phe Val Asn Asp
355 360 365
Leu Thr Arg Glu Glu Leu Ala Ala Ala Leu Ala Asp His Lys Glu Leu
370 375 380
Cys Thr Ser Tyr Pro Arg Gly Gly Glu Gly Val Asp Val Tyr Pro Val
385 390 395 400
Tyr Gln Lys Glu Leu Ile Gly Ser Val Lys
405 410
<210> 93
<211> 840
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 93
ttgacaaatg tgattgtcca accaacagct agacctgtta caccattggg aattttaacc 60
aagcagttag aagccatagt ccaagaggtt aagcaacatc cagatttacc tggggaattg 120
atagcaaaca tccatcaggc ttggcgttta gccgcaggta tagaccctta tttggaagaa 180
tgcaccactc cagaatctcc tgaactcgct gcattggcaa aaaccacagc caccgaagcc 240
tggggagaac acttccacgg aggtacaacc gtccgtcctc tagaacaaga gatgctttct 300
ggtcatatcg aaggacaaac cttaaagatg tttgttcaca tgaccaaagc taaaaaagtc 360
ttagaaattg ggatgtttac cggttattcg gcgctggcga tggcggaagc attaccagag 420
gatggactgc ttgtggcttg tgaagttgac ccttacgcgg cggaaattgg acagaaagcc 480
tttcaacaat ctccccacgg tggaaagatt cgtgtggaat tggatgcagc cttagcaact 540
cttgataagt tagcagaagc tggggagtct tttgacttgg tatttatcga cgcagataaa 600
aaagagtatg tagcctattt tcacaagttg ctaggtagca gtttgttagc accagatggc 660
tttatttgtg tagataacac cttattacaa ggggaagttt atctaccagc agaggaacgt 720
agcgtcaatg gtgaagcgat cgcgcaattt aatcatacag tagctataga cccccgtgta 780
gaacaggttt tgttgccgtt gcgagatggt ttaacaatta tccgcagaat acaaccttaa 840
840
<210> 94
<211> 279
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 94
Met Thr Asn Val Ile Val Gln Pro Thr Ala Arg Pro Val Thr Pro Leu
1 5 10 15
Gly Ile Leu Thr Lys Gln Leu Glu Ala Ile Val Gln Glu Val Lys Gln
20 25 30
His Pro Asp Leu Pro Gly Glu Leu Ile Ala Asn Ile His Gln Ala Trp
35 40 45
Arg Leu Ala Ala Gly Ile Asp Pro Tyr Leu Glu Glu Cys Thr Thr Pro
50 55 60
Glu Ser Pro Glu Leu Ala Ala Leu Ala Lys Thr Thr Ala Thr Glu Ala
65 70 75 80
Trp Gly Glu His Phe His Gly Gly Thr Thr Val Arg Pro Leu Glu Gln
85 90 95
Glu Met Leu Ser Gly His Ile Glu Gly Gln Thr Leu Lys Met Phe Val
100 105 110
His Met Thr Lys Ala Lys Lys Val Leu Glu Ile Gly Met Phe Thr Gly
115 120 125
Tyr Ser Ala Leu Ala Met Ala Glu Ala Leu Pro Glu Asp Gly Leu Leu
130 135 140
Val Ala Cys Glu Val Asp Pro Tyr Ala Ala Glu Ile Gly Gln Lys Ala
145 150 155 160
Phe Gln Gln Ser Pro His Gly Gly Lys Ile Arg Val Glu Leu Asp Ala
165 170 175
Ala Leu Ala Thr Leu Asp Lys Leu Ala Glu Ala Gly Glu Ser Phe Asp
180 185 190
Leu Val Phe Ile Asp Ala Asp Lys Lys Glu Tyr Val Ala Tyr Phe His
195 200 205
Lys Leu Leu Gly Ser Ser Leu Leu Ala Pro Asp Gly Phe Ile Cys Val
210 215 220
Asp Asn Thr Leu Leu Gln Gly Glu Val Tyr Leu Pro Ala Glu Glu Arg
225 230 235 240
Ser Val Asn Gly Glu Ala Ile Ala Gln Phe Asn His Thr Val Ala Ile
245 250 255
Asp Pro Arg Val Glu Gln Val Leu Leu Pro Leu Arg Asp Gly Leu Thr
260 265 270
Ile Ile Arg Arg Ile Gln Pro
275
<210> 95
<211> 2667
<212> DNA
<213> 多变鱼腥藻ATCC29413
<400> 95
atgcagacta tagattttaa tattcgtaag ttacttgtag agtggaacgc gacccacaga 60
gattatgatc tttcccagag tttacatgaa ctaattgtag ctcaagtaga acgaacacct 120
gaggcgatcg ctgtcacctt tgacaagcaa caactaactt atcaagaact aaatcataaa 180
gcaaaccagc taggacatta tttacaaaca ttaggagtcc agccagaaac cctggtaggc 240
gtttgtttag aacgttcctt agaaatggtt atctgtcttt taggaatcct caaagctggg 300
ggtgcttatg ttcctattga ccctgaatat cctcaagaac gcatagctta tatgctagaa 360
gattctcagg tgaaggtact actaactcaa gaaaaattac tcaatcaaat tccccaccat 420
caagcacaaa ctatctgtgt agatagggaa tgggagaaaa tttccacaca agctaatacc 480
aatcccaaaa gtaatataaa aacggataat cttgcttatg taatttacac ctctggttcc 540
actggtaaac caaaaggtgc aatgaacacc cacaaaggta tctgtaatcg cttattgtgg 600
atgcaggaag cttatcaaat cgattccaca gatagcattt tacaaaaaac cccctttagt 660
tttgatgttt ccgtttggga gttcttttgg actttattaa ctggcgcacg tttggtaata 720
gccaaaccag gcggacataa agatagtgct tacctcatcg atttaattac tcaagaacaa 780
atcactacgt tgcattttgt cccctcaatg ctgcaagtgt ttttacaaaa tcgccatgta 840
agcaaatgca gctctctaaa aagagttatt tgtagcggtg aagctttatc tatagattta 900
caaaatagat ttttccagca tttgcaatgt gaattacata acctctatgg cccgacagaa 960
gcagcaattg atgtcacatt ttggcaatgt agaaaagata gtaatttaaa gagtgtacct 1020
attggtcgtc ccattgctaa tactcaaatt tatattcttg atgccgattt acaaccagta 1080
aatattggtg tcactggtga aatttatatt ggtggtgtag gggttgctcg tggttatttg 1140
aataaagaag aattgaccaa agaaaaattt attattaatc cctttcccaa ttctgagttt 1200
aagcgacttt ataaaacagg tgatttagct cgttatttac ccgatggaaa tattgaatat 1260
cttggtagaa cagattatca agtaaaaatt cggggttata gaattgaaat tggcgagatt 1320
gaaaatgttt tatcttcaca cccacaagtc agagaagctg tagtcatagc gcgggatgat 1380
aacgctcaag aaaaacaaat catcgcttat attacctata actccatcaa acctcagctt 1440
gataatctgc gtgatttcct aaaagcaagg ctacctgatt ttatgattcc agccgctttt 1500
gtgatgctgg agcatcttcc tttaactccc agtggtaaag tagaccgtaa ggcattacct 1560
aagcctgatt tatttaatta tagtgaacat aattcctatg tagcgcctcg gaatgaagtt 1620
gaagaaaaat tagtacaaat ctggtcgaat attctgcatt tacctaaagt aggtgtgaca 1680
gaaaactttt tcgctattgg tggtaattcc ctcaaagctc tacatttaat ttctcaaatt 1740
gaagagttat ttgctaaaga gatatcctta gcaacacttt taacaaatcc agtaattgca 1800
gatttagcca aggttattca agcaaacaac caaatccata attcacccct agttccaatt 1860
caaccacaag gtaagcagca gcctttcttt tgtatacatc ctgctggtgg tcatgtttta 1920
tgctatttta aactcgcaca atatatagga actgaccaac cattttatgg cttacaagct 1980
caaggatttt atggagatga agcacccttg acgcgagttg aagatatggc tagtctctac 2040
gtcaaaacta ttagagaatt tcaaccccaa gggccttatc gtgtcggggg gtggtcattt 2100
ggtggagtcg tagcttatga agtagcacag cagttacata gacaaggaca agaagtatct 2160
ttactagcaa tattagattc ttacgtaccg attctgctgg ataaacaaaa acccattgat 2220
gacgtttatt tagttggtgt tctctccaga gtttttggcg gtatgtttgg tcaagataat 2280
ctagtcacac ctgaagaaat agaaaattta actgtagaag aaaaaattaa ttacatcatt 2340
gataaagcac ggagcgctag aatattcccg cctggtgtag aacgtcaaaa taatcgccgt 2400
attcttgatg ttttggtggg aactttaaaa gcaacttatt cctatataag acaaccatat 2460
ccaggaaaag tcactgtatt tcgagccagg gaaaaacata ttatggctcc tgacccgacc 2520
ttagtttggg tagaattatt ttctgtaatg gcggctcaag aaattaagat tattgatgtc 2580
cctggaaacc attattcgtt tgttctagaa ccccatgtac aggttttagc acagcgttta 2640
caagattgtc tggaaaataa ttcataa 2667
<210> 96
<211> 888
<212> PRT
<213> 多变鱼腥藻ATCC29413
<400> 96
Met Gln Thr Ile Asp Phe Asn Ile Arg Lys Leu Leu Val Glu Trp Asn
1 5 10 15
Ala Thr His Arg Asp Tyr Asp Leu Ser Gln Ser Leu His Glu Leu Ile
20 25 30
Val Ala Gln Val Glu Arg Thr Pro Glu Ala Ile Ala Val Thr Phe Asp
35 40 45
Lys Gln Gln Leu Thr Tyr Gln Glu Leu Asn His Lys Ala Asn Gln Leu
50 55 60
Gly His Tyr Leu Gln Thr Leu Gly Val Gln Pro Glu Thr Leu Val Gly
65 70 75 80
Val Cys Leu Glu Arg Ser Leu Glu Met Val Ile Cys Leu Leu Gly Ile
85 90 95
Leu Lys Ala Gly Gly Ala Tyr Val Pro Ile Asp Pro Glu Tyr Pro Gln
100 105 110
Glu Arg Ile Ala Tyr Met Leu Glu Asp Ser Gln Val Lys Val Leu Leu
115 120 125
Thr Gln Glu Lys Leu Leu Asn Gln Ile Pro His His Gln Ala Gln Thr
130 135 140
Ile Cys Val Asp Arg Glu Trp Glu Lys Ile Ser Thr Gln Ala Asn Thr
145 150 155 160
Asn Pro Lys Ser Asn Ile Lys Thr Asp Asn Leu Ala Tyr Val Ile Tyr
165 170 175
Thr Ser Gly Ser Thr Gly Lys Pro Lys Gly Ala Met Asn Thr His Lys
180 185 190
Gly Ile Cys Asn Arg Leu Leu Trp Met Gln Glu Ala Tyr Gln Ile Asp
195 200 205
Ser Thr Asp Ser Ile Leu Gln Lys Thr Pro Phe Ser Phe Asp Val Ser
210 215 220
Val Trp Glu Phe Phe Trp Thr Leu Leu Thr Gly Ala Arg Leu Val Ile
225 230 235 240
Ala Lys Pro Gly Gly His Lys Asp Ser Ala Tyr Leu Ile Asp Leu Ile
245 250 255
Thr Gln Glu Gln Ile Thr Thr Leu His Phe Val Pro Ser Met Leu Gln
260 265 270
Val Phe Leu Gln Asn Arg His Val Ser Lys Cys Ser Ser Leu Lys Arg
275 280 285
Val Ile Cys Ser Gly Glu Ala Leu Ser Ile Asp Leu Gln Asn Arg Phe
290 295 300
Phe Gln His Leu Gln Cys Glu Leu His Asn Leu Tyr Gly Pro Thr Glu
305 310 315 320
Ala Ala Ile Asp Val Thr Phe Trp Gln Cys Arg Lys Asp Ser Asn Leu
325 330 335
Lys Ser Val Pro Ile Gly Arg Pro Ile Ala Asn Thr Gln Ile Tyr Ile
340 345 350
Leu Asp Ala Asp Leu Gln Pro Val Asn Ile Gly Val Thr Gly Glu Ile
355 360 365
Tyr Ile Gly Gly Val Gly Val Ala Arg Gly Tyr Leu Asn Lys Glu Glu
370 375 380
Leu Thr Lys Glu Lys Phe Ile Ile Asn Pro Phe Pro Asn Ser Glu Phe
385 390 395 400
Lys Arg Leu Tyr Lys Thr Gly Asp Leu Ala Arg Tyr Leu Pro Asp Gly
405 410 415
Asn Ile Glu Tyr Leu Gly Arg Thr Asp Tyr Gln Val Lys Ile Arg Gly
420 425 430
Tyr Arg Ile Glu Ile Gly Glu Ile Glu Asn Val Leu Ser Ser His Pro
435 440 445
Gln Val Arg Glu Ala Val Val Ile Ala Arg Asp Asp Asn Ala Gln Glu
450 455 460
Lys Gln Ile Ile Ala Tyr Ile Thr Tyr Asn Ser Ile Lys Pro Gln Leu
465 470 475 480
Asp Asn Leu Arg Asp Phe Leu Lys Ala Arg Leu Pro Asp Phe Met Ile
485 490 495
Pro Ala Ala Phe Val Met Leu Glu His Leu Pro Leu Thr Pro Ser Gly
500 505 510
Lys Val Asp Arg Lys Ala Leu Pro Lys Pro Asp Leu Phe Asn Tyr Ser
515 520 525
Glu His Asn Ser Tyr Val Ala Pro Arg Asn Glu Val Glu Glu Lys Leu
530 535 540
Val Gln Ile Trp Ser Asn Ile Leu His Leu Pro Lys Val Gly Val Thr
545 550 555 560
Glu Asn Phe Phe Ala Ile Gly Gly Asn Ser Leu Lys Ala Leu His Leu
565 570 575
Ile Ser Gln Ile Glu Glu Leu Phe Ala Lys Glu Ile Ser Leu Ala Thr
580 585 590
Leu Leu Thr Asn Pro Val Ile Ala Asp Leu Ala Lys Val Ile Gln Ala
595 600 605
Asn Asn Gln Ile His Asn Ser Pro Leu Val Pro Ile Gln Pro Gln Gly
610 615 620
Lys Gln Gln Pro Phe Phe Cys Ile His Pro Ala Gly Gly His Val Leu
625 630 635 640
Cys Tyr Phe Lys Leu Ala Gln Tyr Ile Gly Thr Asp Gln Pro Phe Tyr
645 650 655
Gly Leu Gln Ala Gln Gly Phe Tyr Gly Asp Glu Ala Pro Leu Thr Arg
660 665 670
Val Glu Asp Met Ala Ser Leu Tyr Val Lys Thr Ile Arg Glu Phe Gln
675 680 685
Pro Gln Gly Pro Tyr Arg Val Gly Gly Trp Ser Phe Gly Gly Val Val
690 695 700
Ala Tyr Glu Val Ala Gln Gln Leu His Arg Gln Gly Gln Glu Val Ser
705 710 715 720
Leu Leu Ala Ile Leu Asp Ser Tyr Val Pro Ile Leu Leu Asp Lys Gln
725 730 735
Lys Pro Ile Asp Asp Val Tyr Leu Val Gly Val Leu Ser Arg Val Phe
740 745 750
Gly Gly Met Phe Gly Gln Asp Asn Leu Val Thr Pro Glu Glu Ile Glu
755 760 765
Asn Leu Thr Val Glu Glu Lys Ile Asn Tyr Ile Ile Asp Lys Ala Arg
770 775 780
Ser Ala Arg Ile Phe Pro Pro Gly Val Glu Arg Gln Asn Asn Arg Arg
785 790 795 800
Ile Leu Asp Val Leu Val Gly Thr Leu Lys Ala Thr Tyr Ser Tyr Ile
805 810 815
Arg Gln Pro Tyr Pro Gly Lys Val Thr Val Phe Arg Ala Arg Glu Lys
820 825 830
His Ile Met Ala Pro Asp Pro Thr Leu Val Trp Val Glu Leu Phe Ser
835 840 845
Val Met Ala Ala Gln Glu Ile Lys Ile Ile Asp Val Pro Gly Asn His
850 855 860
Tyr Ser Phe Val Leu Glu Pro His Val Gln Val Leu Ala Gln Arg Leu
865 870 875 880
Gln Asp Cys Leu Glu Asn Asn Ser
885
<210> 97
<211> 1233
<212> DNA
<213> 点状念珠藻ATCC29133
<400> 97
atgagtaatg ttcaagcatc gtttgaagca acggaagctg aattccgcgt ggaaggttac 60
gaaaaaattg agtttagtct tgtctatgta aatggtgcat ttgatatcag taacagagaa 120
attgcagaca gctatgagaa gtttggccgt tgtctgactg tgattgatgc taatgtcaac 180
agattatatg gcaagcaaat caagtcatat tttagacact atggtattga tctgaccgta 240
gttcccattg tgattactga gcctactaaa acccttgcaa cctttgagaa aattgttgat 300
gctttttctg actttggttt aatccgcaag gaaccagtat tagtagtggg tggtggttta 360
accactgatg tagctgggtt tgcgtgtgct gcttaccgtc gtaagagtaa ctatattcgg 420
gttccgacaa cgctgattgg tctgattgat gcaggtgtag cgattaaggt tgcagtcaac 480
catcgcaagt taaaaaatcg cttgggtgca tatcatgctc ctttgaaagt catcctcgat 540
ttctccttct tgcaaacatt accaacggct caagttcgta atgggatggc agagttggtc 600
aaaattgctg ttgtggcgaa ctctgaagtc tttgaattgt tgtatgaata tggcgaagag 660
ttgctttcca ctcactttgg atatgtgaat ggtacaaagg aactgaaagc gatcgcacat 720
aaactcaatt acgaggcaat aaaaactatg ctggagttgg aaactccaaa cttgcatgag 780
ttagacctcg atcgcgtcat tgcctacggt cacacttgga gtccgacctt agaattagct 840
ccgatgatac cgttgtttca cggtcatgcc gtcaatatag acatggcttt gtctgcaact 900
attgcggcaa gacgtggcta cattacatca ggagaacgcg atcgcatttt gagcttgatg 960
agtcgtatag gtttatcaat cgatcatcct ctactagatg gcgatttgct ctggtatgct 1020
acccaatcta ttagcttgac gcgagacggg aaacaacgcg cagctatgcc taaacccatt 1080
ggtgagtgct tctttgtcaa cgatttcacc cgtgaagaac tagatgcagc tttagctgaa 1140
cacaaacgtc tgtgtgctac ataccctcgt ggtggagatg gcattgacgc ttacatagaa 1200
actcaagaag aatccaaact attgggagtg tga 1233
<210> 98
<211> 410
<212> PRT
<213> 点状念珠藻ATCC29133
<400> 98
Met Ser Asn Val Gln Ala Ser Phe Glu Ala Thr Glu Ala Glu Phe Arg
1 5 10 15
Val Glu Gly Tyr Glu Lys Ile Glu Phe Ser Leu Val Tyr Val Asn Gly
20 25 30
Ala Phe Asp Ile Ser Asn Arg Glu Ile Ala Asp Ser Tyr Glu Lys Phe
35 40 45
Gly Arg Cys Leu Thr Val Ile Asp Ala Asn Val Asn Arg Leu Tyr Gly
50 55 60
Lys Gln Ile Lys Ser Tyr Phe Arg His Tyr Gly Ile Asp Leu Thr Val
65 70 75 80
Val Pro Ile Val Ile Thr Glu Pro Thr Lys Thr Leu Ala Thr Phe Glu
85 90 95
Lys Ile Val Asp Ala Phe Ser Asp Phe Gly Leu Ile Arg Lys Glu Pro
100 105 110
Val Leu Val Val Gly Gly Gly Leu Thr Thr Asp Val Ala Gly Phe Ala
115 120 125
Cys Ala Ala Tyr Arg Arg Lys Ser Asn Tyr Ile Arg Val Pro Thr Thr
130 135 140
Leu Ile Gly Leu Ile Asp Ala Gly Val Ala Ile Lys Val Ala Val Asn
145 150 155 160
His Arg Lys Leu Lys Asn Arg Leu Gly Ala Tyr His Ala Pro Leu Lys
165 170 175
Val Ile Leu Asp Phe Ser Phe Leu Gln Thr Leu Pro Thr Ala Gln Val
180 185 190
Arg Asn Gly Met Ala Glu Leu Val Lys Ile Ala Val Val Ala Asn Ser
195 200 205
Glu Val Phe Glu Leu Leu Tyr Glu Tyr Gly Glu Glu Leu Leu Ser Thr
210 215 220
His Phe Gly Tyr Val Asn Gly Thr Lys Glu Leu Lys Ala Ile Ala His
225 230 235 240
Lys Leu Asn Tyr Glu Ala Ile Lys Thr Met Leu Glu Leu Glu Thr Pro
245 250 255
Asn Leu His Glu Leu Asp Leu Asp Arg Val Ile Ala Tyr Gly His Thr
260 265 270
Trp Ser Pro Thr Leu Glu Leu Ala Pro Met Ile Pro Leu Phe His Gly
275 280 285
His Ala Val Asn Ile Asp Met Ala Leu Ser Ala Thr Ile Ala Ala Arg
290 295 300
Arg Gly Tyr Ile Thr Ser Gly Glu Arg Asp Arg Ile Leu Ser Leu Met
305 310 315 320
Ser Arg Ile Gly Leu Ser Ile Asp His Pro Leu Leu Asp Gly Asp Leu
325 330 335
Leu Trp Tyr Ala Thr Gln Ser Ile Ser Leu Thr Arg Asp Gly Lys Gln
340 345 350
Arg Ala Ala Met Pro Lys Pro Ile Gly Glu Cys Phe Phe Val Asn Asp
355 360 365
Phe Thr Arg Glu Glu Leu Asp Ala Ala Leu Ala Glu His Lys Arg Leu
370 375 380
Cys Ala Thr Tyr Pro Arg Gly Gly Asp Gly Ile Asp Ala Tyr Ile Glu
385 390 395 400
Thr Gln Glu Glu Ser Lys Leu Leu Gly Val
405 410
<210> 99
<211> 834
<212> DNA
<213> 点状念珠藻ATCC29133
<400> 99
atgaccagta ttttaggacg agataccgca agaccaataa cgccacatag cattctggta 60
gcacagctac aaaaaaccct cagaatggca gaggaaagta atattccttc agagatactg 120
acttctctgc gccaagggtt gcaattagca gcaggtttag atccctatct ggatgattgc 180
actacccctg aatcgaccgc attgacagca ctagcgcaga agacaagcat tgaagactgg 240
agtaaacgct tcagtgatgg tgaaacagtg cgtcaattag agcaagaaat gctctcagga 300
catcttgaag gacaaacact aaagatgttt gtgcatatca ctaaggctaa aagcatccta 360
gaagtgggaa tgttcacagg atattcagct ttggcaatgg cagaggcgtt accagatgat 420
gggcgactga ttgcttgtga agtagactcc tatgtggccg agtttgctca aacttgcttt 480
caagagtctc cccacggccg caagattgtt gtagaagtgg cacctgcact agagacgctg 540
cacaagctgg tggctaaaaa agaatccttt gatctgatct tcattgatgc ggataaaaag 600
gagtatatag aatacttcca aattatcttg gatagccatt tactagctcc cgacggatta 660
atctgtgtag ataatacttt gttgcaagga caagtttacc tgccatcaga acagcgtaca 720
gccaatggtg aagcgatcgc tcaattcaac cgcattgtcg ccgcagatcc tcgtgtagag 780
caagttctgt tacccatacg agatggtata accctgatta gacgcttggt ataa 834
<210> 100
<211> 277
<212> PRT
<213> 点状念珠藻ATCC29133
<400> 100
Met Thr Ser Ile Leu Gly Arg Asp Thr Ala Arg Pro Ile Thr Pro His
1 5 10 15
Ser Ile Leu Val Ala Gln Leu Gln Lys Thr Leu Arg Met Ala Glu Glu
20 25 30
Ser Asn Ile Pro Ser Glu Ile Leu Thr Ser Leu Arg Gln Gly Leu Gln
35 40 45
Leu Ala Ala Gly Leu Asp Pro Tyr Leu Asp Asp Cys Thr Thr Pro Glu
50 55 60
Ser Thr Ala Leu Thr Ala Leu Ala Gln Lys Thr Ser Ile Glu Asp Trp
65 70 75 80
Ser Lys Arg Phe Ser Asp Gly Glu Thr Val Arg Gln Leu Glu Gln Glu
85 90 95
Met Leu Ser Gly His Leu Glu Gly Gln Thr Leu Lys Met Phe Val His
100 105 110
Ile Thr Lys Ala Lys Ser Ile Leu Glu Val Gly Met Phe Thr Gly Tyr
115 120 125
Ser Ala Leu Ala Met Ala Glu Ala Leu Pro Asp Asp Gly Arg Leu Ile
130 135 140
Ala Cys Glu Val Asp Ser Tyr Val Ala Glu Phe Ala Gln Thr Cys Phe
145 150 155 160
Gln Glu Ser Pro His Gly Arg Lys Ile Val Val Glu Val Ala Pro Ala
165 170 175
Leu Glu Thr Leu His Lys Leu Val Ala Lys Lys Glu Ser Phe Asp Leu
180 185 190
Ile Phe Ile Asp Ala Asp Lys Lys Glu Tyr Ile Glu Tyr Phe Gln Ile
195 200 205
Ile Leu Asp Ser His Leu Leu Ala Pro Asp Gly Leu Ile Cys Val Asp
210 215 220
Asn Thr Leu Leu Gln Gly Gln Val Tyr Leu Pro Ser Glu Gln Arg Thr
225 230 235 240
Ala Asn Gly Glu Ala Ile Ala Gln Phe Asn Arg Ile Val Ala Ala Asp
245 250 255
Pro Arg Val Glu Gln Val Leu Leu Pro Ile Arg Asp Gly Ile Thr Leu
260 265 270
Ile Arg Arg Leu Val
275
<210> 101
<211> 1386
<212> DNA
<213> 点状念珠藻ATCC29133
<400> 101
atggcacaat caatctcttt atctcttcct caatccacaa cgccatcaaa gggtgtgagg 60
ctaaaaatag cagctctact gaagactatc gggactctaa tactactgct gatagccttg 120
ccgcttaatg ctttgatagt attgatatct ctgatgtgta ggccgtttac aaaaaaacct 180
gccgtagcca ctcatcccca gaatatcttg gtcagtggcg gcaaaatgac caaagcattg 240
caacttgccc gctccttcca tgcagccgga cacagagtta ttctgattga aggtcataaa 300
tactggttat cagggcatag attctcaaat tctgtgagtc gtttttatac agttcctgca 360
ccacaagacg acccagaagg ctatacccaa gcgctattgg aaattgtcaa acgagagaag 420
attgacgttt atgtacccgt atgcagccct gtagctagtt attacgactc tttggcaaag 480
tctgcactat cagaatattg tgaggttttt cactttgatg ctgatataac caagatgctg 540
gatgataaat ttgcctttac cgatcgggcg cgatcgcttg gtttatcagc cccgaaatct 600
tttaaaatta ccgatcctga acaagttatc aacttcgatt ttagtaaaga gacgcgcaaa 660
tatattctta agagtatttc ttacgactca gttcgccgct taaatttaac caaacttcct 720
tgtgataccc cagaagagac agcagcgttt gtcaagagtt tacccatcag cccagaaaaa 780
ccttggatta tgcaagaatt tattcctggg aaagaattat gcacccatag cacagtccga 840
gacggcgaat taaggttgca ttgctgttca aattcttcag cgtttcagat taactatgaa 900
aatgtcgaaa atccccaaat tcaagaatgg gtacaacatt tcgtcaaaag tttacggctg 960
actggacaaa tatctcttga ctttatccaa gctgaagatg gtacagctta tgccattgaa 1020
tgtaatcctc gcacccattc ggcgatcaca atgttctaca atcacccagg tgttgcagaa 1080
gcctatcttg gtaaaactcc tctagctgca cctttggaac ctcttgcaga tagcaagccc 1140
acttactgga tatatcacga aatctggcga ttgactggga ttcgctctgg acaacaatta 1200
caaacttggt ttgggagatt agtcagaggt acagatgcca tttatcgcct ggacgatccg 1260
ataccatttt taactttgca ccattggcag attactttac ttttgctaca aaatttgcaa 1320
cgactcaaag gttgggtaaa gatcgatttt aatatcggta aactcgtgga attagggggc 1380
gactaa 1386
<210> 102
<211> 461
<212> PRT
<213> 点状念珠藻ATCC29133
<400> 102
Met Ala Gln Ser Ile Ser Leu Ser Leu Pro Gln Ser Thr Thr Pro Ser
1 5 10 15
Lys Gly Val Arg Leu Lys Ile Ala Ala Leu Leu Lys Thr Ile Gly Thr
20 25 30
Leu Ile Leu Leu Leu Ile Ala Leu Pro Leu Asn Ala Leu Ile Val Leu
35 40 45
Ile Ser Leu Met Cys Arg Pro Phe Thr Lys Lys Pro Ala Val Ala Thr
50 55 60
His Pro Gln Asn Ile Leu Val Ser Gly Gly Lys Met Thr Lys Ala Leu
65 70 75 80
Gln Leu Ala Arg Ser Phe His Ala Ala Gly His Arg Val Ile Leu Ile
85 90 95
Glu Gly His Lys Tyr Trp Leu Ser Gly His Arg Phe Ser Asn Ser Val
100 105 110
Ser Arg Phe Tyr Thr Val Pro Ala Pro Gln Asp Asp Pro Glu Gly Tyr
115 120 125
Thr Gln Ala Leu Leu Glu Ile Val Lys Arg Glu Lys Ile Asp Val Tyr
130 135 140
Val Pro Val Cys Ser Pro Val Ala Ser Tyr Tyr Asp Ser Leu Ala Lys
145 150 155 160
Ser Ala Leu Ser Glu Tyr Cys Glu Val Phe His Phe Asp Ala Asp Ile
165 170 175
Thr Lys Met Leu Asp Asp Lys Phe Ala Phe Thr Asp Arg Ala Arg Ser
180 185 190
Leu Gly Leu Ser Ala Pro Lys Ser Phe Lys Ile Thr Asp Pro Glu Gln
195 200 205
Val Ile Asn Phe Asp Phe Ser Lys Glu Thr Arg Lys Tyr Ile Leu Lys
210 215 220
Ser Ile Ser Tyr Asp Ser Val Arg Arg Leu Asn Leu Thr Lys Leu Pro
225 230 235 240
Cys Asp Thr Pro Glu Glu Thr Ala Ala Phe Val Lys Ser Leu Pro Ile
245 250 255
Ser Pro Glu Lys Pro Trp Ile Met Gln Glu Phe Ile Pro Gly Lys Glu
260 265 270
Leu Cys Thr His Ser Thr Val Arg Asp Gly Glu Leu Arg Leu His Cys
275 280 285
Cys Ser Asn Ser Ser Ala Phe Gln Ile Asn Tyr Glu Asn Val Glu Asn
290 295 300
Pro Gln Ile Gln Glu Trp Val Gln His Phe Val Lys Ser Leu Arg Leu
305 310 315 320
Thr Gly Gln Ile Ser Leu Asp Phe Ile Gln Ala Glu Asp Gly Thr Ala
325 330 335
Tyr Ala Ile Glu Cys Asn Pro Arg Thr His Ser Ala Ile Thr Met Phe
340 345 350
Tyr Asn His Pro Gly Val Ala Glu Ala Tyr Leu Gly Lys Thr Pro Leu
355 360 365
Ala Ala Pro Leu Glu Pro Leu Ala Asp Ser Lys Pro Thr Tyr Trp Ile
370 375 380
Tyr His Glu Ile Trp Arg Leu Thr Gly Ile Arg Ser Gly Gln Gln Leu
385 390 395 400
Gln Thr Trp Phe Gly Arg Leu Val Arg Gly Thr Asp Ala Ile Tyr Arg
405 410 415
Leu Asp Asp Pro Ile Pro Phe Leu Thr Leu His His Trp Gln Ile Thr
420 425 430
Leu Leu Leu Leu Gln Asn Leu Gln Arg Leu Lys Gly Trp Val Lys Ile
435 440 445
Asp Phe Asn Ile Gly Lys Leu Val Glu Leu Gly Gly Asp
450 455 460
<210> 103
<211> 1047
<212> DNA
<213> 点状念珠藻ATCC29133
<400> 103
atgccagtac ttaatatcct tcatttagtt gggtctgcac acgataagtt ttactgtgat 60
ttatcacgtc tttatgccca agactgttta gctgcaacag cagatccatc gctttataac 120
tttcaaattg catatatcac acccgatcgg cagtggcgat ttcctgactc tctcagtcga 180
gaagatattg ctcttaccaa accgattcct gtgtttgatg ccatacaatt tctaacaggc 240
caaaacattg acatgatgtt accacaaatg ttttgtattc ctggaatgac tcagtaccgt 300
gccctattcg atctgctcaa gatcccttat ataggaaata ccccagatat tatggcgatc 360
gcggcccaca aagccagagc caaagcaatt gtcgaagcag caggggtaaa agtgcctcgt 420
ggagaattgc ttcgccaagg agatattcca acaattacac ctccagcagt cgtcaaacct 480
gtaagttctg acaactcttt aggagtagtc ttagttaaag atgtgactga atatgatgct 540
gccttaaaga aagcatttga atatgcttcg gaggtcatcg tagaagcatt catcgaactt 600
ggtcgagaag tcagatgcgg catcattgta aaagacggtg agctaatagg tttacccctt 660
gaagagtatc tggtagaccc acacgataaa cctatccgta actatgctga taaactccaa 720
caaactgacg atggcgactt gcatttgact gctaaagata atatcaaggc ttggatttta 780
gaccctaacg acccaatcac ccaaaaggtt cagcaagtgg ctaaaaggtg tcatcaggct 840
ttgggttgtc gccactacag tttatttgac ttccgaatcg atccaaaggg acaaccttgg 900
ttcttagaag ctggattata ttgttctttt gcccccaaaa gtgtgatttc ttctatggcg 960
aaagcagccg gaatccctct aaatgattta ttaataaccg ctattaatga aacattgggt 1020
agtaataaaa aggtgttaca aaattga 1047
<210> 104
<211> 348
<212> PRT
<213> 点状念珠藻ATCC29133
<400> 104
Met Pro Val Leu Asn Ile Leu His Leu Val Gly Ser Ala His Asp Lys
1 5 10 15
Phe Tyr Cys Asp Leu Ser Arg Leu Tyr Ala Gln Asp Cys Leu Ala Ala
20 25 30
Thr Ala Asp Pro Ser Leu Tyr Asn Phe Gln Ile Ala Tyr Ile Thr Pro
35 40 45
Asp Arg Gln Trp Arg Phe Pro Asp Ser Leu Ser Arg Glu Asp Ile Ala
50 55 60
Leu Thr Lys Pro Ile Pro Val Phe Asp Ala Ile Gln Phe Leu Thr Gly
65 70 75 80
Gln Asn Ile Asp Met Met Leu Pro Gln Met Phe Cys Ile Pro Gly Met
85 90 95
Thr Gln Tyr Arg Ala Leu Phe Asp Leu Leu Lys Ile Pro Tyr Ile Gly
100 105 110
Asn Thr Pro Asp Ile Met Ala Ile Ala Ala His Lys Ala Arg Ala Lys
115 120 125
Ala Ile Val Glu Ala Ala Gly Val Lys Val Pro Arg Gly Glu Leu Leu
130 135 140
Arg Gln Gly Asp Ile Pro Thr Ile Thr Pro Pro Ala Val Val Lys Pro
145 150 155 160
Val Ser Ser Asp Asn Ser Leu Gly Val Val Leu Val Lys Asp Val Thr
165 170 175
Glu Tyr Asp Ala Ala Leu Lys Lys Ala Phe Glu Tyr Ala Ser Glu Val
180 185 190
Ile Val Glu Ala Phe Ile Glu Leu Gly Arg Glu Val Arg Cys Gly Ile
195 200 205
Ile Val Lys Asp Gly Glu Leu Ile Gly Leu Pro Leu Glu Glu Tyr Leu
210 215 220
Val Asp Pro His Asp Lys Pro Ile Arg Asn Tyr Ala Asp Lys Leu Gln
225 230 235 240
Gln Thr Asp Asp Gly Asp Leu His Leu Thr Ala Lys Asp Asn Ile Lys
245 250 255
Ala Trp Ile Leu Asp Pro Asn Asp Pro Ile Thr Gln Lys Val Gln Gln
260 265 270
Val Ala Lys Arg Cys His Gln Ala Leu Gly Cys Arg His Tyr Ser Leu
275 280 285
Phe Asp Phe Arg Ile Asp Pro Lys Gly Gln Pro Trp Phe Leu Glu Ala
290 295 300
Gly Leu Tyr Cys Ser Phe Ala Pro Lys Ser Val Ile Ser Ser Met Ala
305 310 315 320
Lys Ala Ala Gly Ile Pro Leu Asn Asp Leu Leu Ile Thr Ala Ile Asn
325 330 335
Glu Thr Leu Gly Ser Asn Lys Lys Val Leu Gln Asn
340 345

Claims (8)

1.生产霉孢菌素样氨基酸的微生物，其中与非修饰的微生物相比，3-脱氢奎尼酸脱水酶的活性被失活，并且其中所述微生物包含霉孢菌素样氨基酸生物合成基因，所述霉孢菌素样氨基酸生物合成基因包含来自蓝细菌(Cyanobacterium)的2-脱甲基4-deoxygadusol合酶基因。
2.权利要求1所述的微生物，其中所述霉孢菌素样氨基酸生物合成基因进一步包含编码选自O-甲基转移酶和C-N连接酶的至少一种蛋白质的基因。
3.权利要求1所述的微生物，其中所述微生物包含编码选自非核糖体肽合成酶、非核糖体肽合成酶样酶(NRPS样酶)、和D-Ala D-Ala连接酶的至少一种蛋白质的基因。
4.权利要求1所述的微生物，其中与非修饰的微生物相比，选自2-脱氢-3-脱氧磷酸庚糖酸醛缩酶、磷酸烯醇丙酮酸合成酶、转酮醇酶I/II、和3-脱氢奎尼酸合酶的至少一种蛋白质的活性被进一步增强。
5.权利要求1所述的微生物，其中所述微生物是棒杆菌属(genus Corynebacterium)的微生物、埃希氏菌属(genus Escherichia)的微生物或酵母。
6.权利要求5所述的微生物，其中所述酵母被引入有编码3-脱氢奎尼酸合酶的基因。
7.权利要求1所述的微生物，其中所述霉孢菌素样氨基酸是选自霉孢菌素-2-甘氨酸、palythinol、palythenic acid、deoxygadusol、霉孢菌素-甲胺-苏氨酸、霉孢菌素-甘氨酸-缬氨酸、palythine、asterina-330、shinorine、porphyra-334、euhalothece-362、霉孢菌素-甘氨酸、霉孢菌素-鸟氨酸、霉孢菌素-赖氨酸、霉孢菌素-谷氨酸-甘氨酸、霉孢菌素-甲胺-丝氨酸、霉孢菌素-牛磺酸、palythene、palythine-丝氨酸、palythine-丝氨酸-硫酸盐、palythinol、和usujirene的至少一种。
8.生产霉孢菌素样氨基酸的方法，所述方法包括：
培养权利要求1至7中任一项所述的微生物；以及
从培养的所述微生物或培养基回收霉孢菌素样氨基酸。
CN201880066300.6A 2017-08-16 2018-08-13 生产霉孢菌素样氨基酸的微生物及利用其生产霉孢菌素样氨基酸的方法 Active CN111527211B (zh)

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