CN111433218A - 新型多肽及使用其生产基于鸟氨酸的产物的方法 - Google Patents
新型多肽及使用其生产基于鸟氨酸的产物的方法 Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本申请涉及具有输出基于鸟氨酸的产物的能力的新型多肽，以及使用其生产基于鸟氨酸的产物的方法。
Description
技术领域
本公开涉及具有输出基于鸟氨酸的产物的能力的新型多肽，以及使用其生产基于鸟氨酸的产物的方法。
背景技术
广泛存在于植物、动物和微生物中的物质鸟氨酸，是从谷氨酸生物合成的，并用作腐胺、瓜氨酸和脯氨酸的生物合成中的前体。进一步，在高等动物的体内代谢中，在通过鸟氨酸循环从氨基酸或氨产生的尿素的排泄途径中，鸟氨酸起着重要作用。鸟氨酸在增进肌肉生长和减少体脂肪方面有效，并且因此被用作营养补充剂，并也被用作改善肝硬化和肝功能障碍的药品。已知生产鸟氨酸的方法包括用消化酶处理乳酪蛋白(milk casein)和利用转化的大肠杆菌(E.coli)或棒杆菌属(genus Corynebacterium)微生物的方法(韩国专利号10-1372635；T.Gotoh等人，Bioprocess Biosyst.Eng.，33:773-777,2010)。
腐胺(或1,4-丁二胺)是生产聚酰胺-4和聚酰胺-6(包括尼龙-4和尼龙-6)的非常重要的原料，并可以通过丁二腈的氢化以工业规模生产，丁二腈是由丙烯腈通过氰化氢的添加生产的。这些化学物质的合成途径需要不可再生的石化产物作为原料。另外，还需要与昂贵的催化剂体系的使用相关的高温和高压，以及相对复杂的制备步骤和设备。因此，作为化学生产工艺的替代，需要从可再生生物质来源的碳源生产腐胺的工艺。最近，为了生产工业上可用的高浓度聚胺(腐胺)，已经连续进行了利用环境友好型微生物的研究(Qian ZG等人，Biotechnol Bioeng，104:651-662，2009；Schneider J等人，Appl MicrobiolBiotechnol，88:859-868，2000)。
同时，NCgl2522已被鉴定为具有输出腐胺能力的基因(韩国专利号2014-0115244)。然而，为了以更高的产率生产腐胺，仍然需要开发具有提高的输出腐胺能力的蛋白质，其能够更有效地从腐胺生产菌株输出腐胺。
L-精氨酸作为肝功能促进剂、脑功能促进剂以及作为多种氨基酸补充剂的成分，已经被广泛用于在医药中。另外，L-精氨酸作为鱼饼和保健饮料的食品添加剂、以及作为高血压患者的食盐替代品，在食品工业中得到了许多的关注。为了生产工业上可用的高浓度精氨酸，已经连续进行了利用微生物的研究，且其实例包括使用来自属于短杆菌属(genusBrevibacterium)或棒杆菌属的微生物诱导的突变菌株(其是谷氨酸生产菌株)的方法、或者使用氨基酸生产菌株(其生长通过细胞融合被改进)的方法。同时，具有输出L-赖氨酸能力的属于棒杆菌属的微生物的lysE也已显示输出相同的碱性氨基酸L-精氨酸(Bellmann A等人，Microbiology，147:1765-1774，2001)。进一步，已知通过上述基因的增强来增强L-精氨酸生产菌株的生产能力的方法(美国专利号2002-196232)。
发明内容
[技术问题]
本发明人已经进行了大量的努力开发输出蛋白的变体，该输出蛋白的变体能够通过增强输出鸟氨酸产物的能力，来进一步提高生产能力，并且结果确认，当在NCgl2522蛋白的氨基酸序列的特定位点上引入修饰时，输出基于鸟氨酸的产物的能力被增强。因此，他们已经发现，通过将蛋白变体引入腐胺或精氨酸生产菌株中，可以高产率生产作为基于鸟氨酸的产物的腐胺或精氨酸，从而完成本发明。
[技术方案]
本公开的一个目的是提供具有输出基于鸟氨酸的产物的能力的新型多肽。
本公开的另一个目的是提供编码多肽的多核苷酸、和包含多核苷酸的载体。
本公开的又一个目的是提供包含多肽或具有其增强活性的，生产基于鸟氨酸的产物的微生物。
本公开的又一个目的是提供用于生产基于鸟氨酸的产物的方法，其包括：
(i)在培养基中培养生产基于鸟氨酸的产物的棒杆菌属的微生物；以及
(ii)从上述获得的微生物或培养基中回收基于鸟氨酸的产物。
[有益效果]
具有输出本公开的基于鸟氨酸的产物的能力的多肽显示出优异的输出基于鸟氨酸的产物的活性，并且因此，当将这种活性引入到生产基于鸟氨酸的产物的微生物中时，能够进一步提高生产基于鸟氨酸的产物的能力。
具体实施方式
下面将详细描述本公开。同时，本公开中公开的说明和实施方式可分别应用于其它说明和实施方式。即，在本文公开的各种元素的所有组合都属于本公开的范围。另外，本公开的范围不应受下文描述的具体描述的限制。
为了实现上述目的，本公开的一方面提供了具有输出基于鸟氨酸的产物的能力的新型多肽，其中自基于鸟氨酸的产物输出蛋白的氨基酸序列的N端第152位丙氨酸残基被其它氨基酸取代。
如本文所用，基于鸟氨酸的产物输出蛋白是指在由鸟氨酸作为前体生物合成的产物的胞外输出中起作用的蛋白，且具体是指在腐胺或精氨酸的胞外输出中起作用的蛋白。更具体地，它可以是在韩国专利申请公开号2014-0115244中公开的NCgl2522蛋白。例如NCgl2522蛋白可以由SEQ ID NO:1或SEQ ID NO:2的氨基酸序列组成，但是可不受限制的包括具有与该蛋白相同的活性的任何序列，且其序列信息可以从已知的数据库NCBI的GenBank中获得。
本公开的具有输出基于鸟氨酸的产物的能力的新型多肽具有其中自腐胺输出蛋白的氨基酸序列的N端第152位丙氨酸残基被其它氨基酸取代的特征，并且因此与非修饰的多肽(具体地，在第152位具有丙氨酸残基的多肽)相比，具有提高的输出基于鸟氨酸的产物的能力。其它氨基酸可包括丝氨酸、天冬酰胺、组氨酸、脯氨酸、赖氨酸、谷氨酸、半胱氨酸、谷氨酰胺或蛋氨酸，但不限于此。因此，具有输出基于鸟氨酸的产物的能力的多肽可以是例如其中基于鸟氨酸的产物输出蛋白的氨基酸序列中第152位的丙氨酸被丝氨酸、天冬酰胺、组氨酸、脯氨酸、赖氨酸、谷氨酸、半胱氨酸、谷氨酰胺或蛋氨酸取代的那些多肽。具体地，多肽可以是由SEQ ID NO:3至SEQ ID NO:20中任一个的氨基酸序列，或者具有与其70％或更多、具体地80％或更多、更具体地85％或更多、甚至更具体地90％或更多、甚至更具体地95％或更多、和甚至更具体地99％或更多的同源性或同一性的氨基酸序列(只要它通过用其它氨基酸取代第152位的丙氨酸而具有输出腐胺的能力)组成的多肽，但不限于此。另外，应当解释的是，作为具有这种同源性或同一性的氨基酸序列，只要氨基酸序列具有与由SEQID NO:3至SEQ ID NO:20中任一个的氨基酸序列组成的多肽实质上相同或对应的生物活性，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。
如本文所用，术语“基于鸟氨酸的产物”是指可由鸟氨酸作为前体生物合成的物质。具体地，可以通过鸟氨酸循环生产的物质的实例包括腐胺、瓜氨酸、脯氨酸和精氨酸，但只要它可以由鸟氨酸作为前体被生物合成，则物质不限于此。例如，基于鸟氨酸的产物可以是腐胺和精氨酸。另外，可不受限制地包括可由鸟氨酸作为前体合成并由新型多肽(具有输出本公开的基于鸟氨酸的产物的能力)输出的任何物质。
本公开的另一方面提供了这样的多核苷酸，该多核苷酸编码具有输出基于鸟氨酸的产物的能力的多肽。
多核苷酸可以包括编码具有SEQ ID NO:3至SEQ ID NO:20中任一个的氨基酸序列的多肽，或者具有与其70％或更多、具体地80％或更多、更具体地85％或更多、甚至更具体地90％或更多、甚至更具体地95％或更多、和甚至更具体地99％或更多的同源性或同一性的多肽的多核苷酸，但只要其具有与多肽(具有输出基于鸟氨酸的产物的能力)相似的活性，就不限于此。另外，明显的是，由于密码子简并性，也可以包括能够被翻译成由SEQ IDNO:1的氨基酸序列组成的蛋白质或与其具有同源性或同一性的蛋白质的多核苷酸。可替代地，可不受限制地包括可从已知基因序列制备的探针，例如与核苷酸序列的全部或部分互补的序列在严格条件下杂交以编码具有由SEQ ID NO:1的氨基酸序列组成的蛋白质的活性的蛋白质的任何序列。具有同源性或同一性的序列可以排除上述范围内具有100％同源性的序列、或者可以是具有小于100％同源性的序列。
“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。严格条件可包括具有高度同源性或同一性(例如，同源性或同一性是80％或更多、具体地是85％或更多、更具体地是90％或更多、甚至更具体地是95％或更多、甚至更具体地是97％或更多、和甚至更具体地是99％或更多)的基因彼此杂交，而具有低于上述同源性或同一性的同源性或同一性的基因不能彼此杂交的条件；或可以包括作为Southern杂交的常规洗涤条件(即，对应于60℃下、1×SSC和0.1％SDS；具体地60℃下、0.1×SSC和0.1％SDS；更具体地68℃下、0.1×SSC和0.1％SDS的盐浓度和温度下，洗涤一次、具体地洗涤两次或三次)。
尽管根据杂交的严格性，碱基之间的错配是可能的，但杂交需要两个核苷酸具有互补序列。术语“互补”用于描述可彼此杂交的核苷酸碱基之间的关系。例如，关于DNA，腺苷与胸腺嘧啶互补，以及胞嘧啶与鸟嘌呤互补。因此，本公开还可包括与整个序列互补的分离的核酸片段以及实质上与其相似的核酸序列。
具体地，在上述条件下，可以使用包括在Tm值为55℃下的杂交步骤的杂交条件来检测具有同源性的多核苷酸。另外，Tm值可以是60℃、63℃、或65℃，但不限于此，并可由本领域技术人员根据其目的适当地控制。
用于杂交多核苷酸的适当严格性取决于多核苷酸的长度和互补程度，且这些变量是本领域公知的(Sambrook等人，同上，9.50至9.51，11.7至11.8)。
如本文所用，术语“同源性(homology)”是指两个给定氨基酸序列或核苷酸序列之间的一致程度，并且可以表示为百分比。在本公开中，具有与给定氨基酸序列或核苷酸序列的活性相同或类似的活性的同源序列，可以用术语“％同源性”表示。
如本文所用，术语“同一性”是指两个给定氨基酸序列或核苷酸序列之间的相关程度。在某些情况下，同一性是由这类序列的字符串之间的一致来确定的。
术语“同源性”和“同一性”经常彼此互换使用。
保守的多核苷酸或多肽的序列同源性或同一性可由标准对比算法确定，并可与通过正在使用的程序建立的默认空位罚值(default gap penalty)一起使用。实质上，同源或相同的多核苷酸或多肽通常预期在中等或高严格条件下与目标多核苷酸或多肽的全部或至少约50％、约60％、约70％、约80％、约85％、或约90％的全长杂交。含有代替密码子的简并密码子的多核苷酸也被考虑在杂交多核苷酸中。
任意两个多核苷酸或多肽序列是否彼此具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的同源性或同一性，可以通过已知计算机算法(如“FASTA”程序)(例如，Pearson等人，(1988)Proc.Natl.Acad.Sci.USA 85:2444)使用默认参数来确定。可替代地，可以通过使用EMBOSS包(EMBOSS:The EuropeanMolecular Biology Open Software Suite，Rice等人,2000,Trends Genet.16:276-277)(优选版本5.0.0或其后版本)(GCG程序包(Devereux,J.等人,Nucleic Acids Research12:38 7(1984))的Needleman程序执行的Needleman-Wunsch算法(Needleman and Wunsch,1970,J.Mol.Biol.48:443-453)、BLASTP、BLASTN、FASTA(Atschul，[S.][F.,][ET AL,JMOLEC BIOL 215]:403(1990)；Guide to Huge Computers,Martin J.Bishop,[ED.,]Academic Press,San Diego,1994,和[CARILLO ETA/.](1988)SIAM J Applied Math 48:1073)来确定。例如，可以使用美国国家生物技术信息中心(National Center forBiotechnology Information，NCBI)的BLAST或ClustalW来确定同源性或同一性。
多核苷酸或多肽的同源性或同一性可通过使用例如GAP计算机程序(如Smith andWaterman，Adv.Appl.Math(1981)2:482中公开的Needleman等人(1970),J Mol Biol.48:443)比较序列信息来确定。总之，GAP程序将同源性或同一性定义为相似的对比符号(即，核苷酸或氨基酸)的数量除以两序列中较短者的符号总数获得的数值。GAP程序的默认参数可包括：(1)一元比较矩阵(含有同一性值为1和非同一性值为0)和如Schwartz and Dayhoff,eds.,Atlas Of Protein Sequence And Structure,National Biomedical ResearchFoundation,pp.353-358(1979)中公开的Gribskov等人(1986),Nucl.Acids Res.14:6745的加权比较矩阵；(2)每个空位的罚值为3.0，且每个空位中每个符号为附加的0.10罚值(或空位开放罚值(gap open penalty)为10和空位延伸罚值(gap extension penalty)为0.5)；和(3)末端空位无罚值。因此，如本文所用，术语“同源性”或“同一性”是指多肽或多核苷酸之间的比较。
本发明的再一方面提供包含多核苷酸的载体。
如本文所用，术语“载体”是指为能够在适合的宿主中表达目标多肽，可操作地被连接至适合的调节序列的，含有编码目标多肽的多核苷酸的核苷酸序列的DNA构建体。调节序列可以包括能够起始转录的启动子、用于控制转录的任何操纵基因序列、编码适合的mRNA核糖体结合结构域的序列、和调节转录和翻译终止的序列。在转化到适合的宿主细胞中之后，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能够在宿主细胞中复制，其没有特别的限制，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。可用于本公开的载体没有特别限制，且可以使用已知的表达载体。具体地，可以使用载体pDZ、pACYC177、pACYC184、pCL、pECCG117、pUC19、pBR322、pMW118、pCC1BAC等。
在实施方式中，可以通过用于细胞内染色体插入的载体，用修饰的多核苷酸置换染色体中编码目标多肽的多核苷酸。多核苷酸向染色体中的插入可以通过本领域已知的任何方法(例如同源重组)进行，但不限于此。
如本文所用，术语“转化”是指将包含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多肽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。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。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要素的基因构建体。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。另外地，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。
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多肽的多核苷酸转录的上述基因序列与启动子序列之间的功能性连接。
本公开的又一方面提供了生产基于鸟氨酸的产物的微生物，其包含具有输出基于鸟氨酸的产物的能力或具有其增强的活性的多肽。
具体地，本公开提供了生产腐胺或精氨酸的棒杆菌属的微生物，其包括具有输出基于鸟氨酸的产物的能力或具有其增强的活性的多肽。
如本文所用，术语“微生物”包括所有野生型微生物、或自然或人工遗传修饰的微生物，并且它可以是由于外源基因的插入、或内源基因活性的增强或失活，而其中特定机制被减弱或增强的微生物。
如本文所用，术语“棒杆菌属的微生物”可具体是谷氨酸棒杆菌(Corynebacteriumglutamicum)、产氨棒杆菌(Corynebacterium ammoniagenes)、乳发酵短杆菌(Brevibacterium lactofermentum)、黄色短杆菌(Brevibacterium flavum)、热氨基棒杆菌(Corynebacterium thermoaminogenes)、有效棒杆菌(Corynebacterium efficiens)等，但不限于此。更具体地，本公开的棒杆菌属的微生物可以是即使当暴露于高浓度腐胺或精氨酸时其细胞生长和存活几乎不受影响的谷氨酸棒杆菌。
如本文所用，术语“生产基于鸟氨酸的产物的棒杆菌属的微生物”是指天然或经由修饰具有生产基于鸟氨酸的产物的能力的棒杆菌属的微生物。生产基于鸟氨酸的产物的棒杆菌属的微生物可以是——但并不特别限于——被修饰而使得选自以下的至少一种的活性与其内源活性相比增加的微生物：例如，将谷氨酸转化为N-乙酰谷氨酸的乙酰谷氨酸合酶、或将N-乙酰鸟氨酸转化为鸟氨酸的鸟氨酸乙酰转移酶(ArgJ)、将N-乙酰谷氨酸转化为N-乙酰谷氨酰磷酸的乙酰谷氨酸激酶(ArgB)、将N-乙酰谷氨酰磷酸转化为N-乙酰谷氨酸半醛的乙酰-γ-谷氨酰-磷酸还原酶(ArgC)、以及将N-乙酰谷氨酸半醛转化为N-乙酰鸟氨酸的乙酰鸟氨酸转氨酶(ArgD)，以增强从谷氨酸到鸟氨酸的生物合成途径，从而改善鸟氨酸生产性。
如本文所用，术语“生产腐胺或精氨酸的棒杆菌属的微生物”是指天然或经由修饰具有生产腐胺或精氨酸的能力的棒杆菌属的微生物。棒杆菌属的微生物不生产腐胺，能生产精氨酸，但精氨酸的生产性明显低下。因此，如本文所用，生产腐胺或精氨酸的棒杆菌属的微生物是指天然菌株本身、或棒杆菌属的微生物(其中插入了参与腐胺或精氨酸生产机制的外源基因、或内源基因的活性被增强或减弱，从而具有腐胺或精氨酸的提高的生产能力)。
另外，生产腐胺的微生物可以是进一步被修饰而使得选自以下的至少一种的活性与其的内源活性相比被减弱的微生物：参与由鸟氨酸合成精氨酸的鸟氨酸氨甲酰基转移酶(ArgF)、参与谷氨酸输出的蛋白质、和使腐胺乙酰化的乙酰转移酶；和/或可以是被修饰而使得鸟氨酸脱羧酶(ODC)的活性被引入的微生物。
进一步，生产精氨酸的微生物可以是进一步被修饰而使得选自以下的至少一种的活性与其内源活性相比被增强的微生物：参与由鸟氨酸合成精氨酸的鸟氨酸氨甲酰基转移酶(ArgF)、精氨琥珀酸合酶(argG)、精氨琥珀酸裂解酶(argH)、天冬氨酸氨裂解酶、和天冬氨酸转氨酶。
如本文所用，术语蛋白质活性的“增强”意思是蛋白质的活性被引入、或者与其内源活性相比，活性被增强。活性的“引入”意思是微生物原本不具备的特定多肽的活性被自然或人工表达。
如本文所用，术语蛋白质活性与其内源活性相比的“增加”意思是与微生物所具有的蛋白质的内源活性相比或与转化前非修饰蛋白质的活性相比，蛋白质的活性被提高。“内源活性”是指当由于自然或人工因素，通过遗传修饰改变了微生物的性状时，在其转化前，亲本菌株或非修饰的微生物原本具有的特定蛋白质的活性，且它可以与转化前的活性互换使用。
具体地，本公开中的活性增强可以通过以下方法来执行，但方法不限于此：
1)增加编码蛋白质的多核苷酸拷贝数的方法；
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蛋白质的活性增强的方法；
4)引入展示蛋白质的活性的外源多核苷酸或修饰的多核苷酸(其中上述多核苷酸的密码子已被优化)的方法；和
5)通过上述方法的组合进行修饰以增强活性的方法。
上述方法1)中的多核苷酸拷贝数的增加可以以多核苷酸可操作地被连接到载体的形式来执行，或者通过插入到宿主细胞的染色体中来执行，但不特别限于此。具体地，它可以通过将编码本公开的蛋白质的多核苷酸可操作地连接到载体(其可与宿主细胞无关地复制并起作用)，并将其引入到宿主细胞中来执行。可替代地，它可以通过将多核苷酸与能够将多核苷酸插入到宿主细胞染色体中的载体可操作地连接，并将其引入到宿主细胞中，用于增加宿主细胞的染色体中多核苷酸的拷贝数的方法来进行。
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的修饰，从而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执行，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、调节转录和翻译终止的序列等，但不特别限于此。
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(韩国专利号0620092和国际公开号WO2006/065095)、lysCP1启动子(国际公开号WO2009/096689)、EF-Tu启动子、groEL启动子、aceA或aceB启动子等，但强启动子不限于此。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的修饰，从而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执行，但不特别限于此。
另外，方法4)中的外源多核苷酸序列的引入可以通过将编码展示出与上述蛋白质相同或相似活性的蛋白质的外源多核苷酸、或其中外源多核苷酸的密码子已经被优化的修饰多核苷酸引入到宿主细胞中来执行。只要它展示出与蛋白质的活性相同或相似的活性，外源多核苷酸可以不受其来源或序列限制被使用。进一步，外源多核苷酸可在其密码子优化后被引入到宿主细胞中，从而在宿主细胞中实现优化的转录和翻译。引入可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执行，并且当引入的多核苷酸在宿主细胞中表达时可生产蛋白质，从而增加其活性。
最后，方法5)中通过方法1)至方法4)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执行：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、以及修饰展示蛋白质的活性的外源多核苷酸或其密码子优化的修饰多核苷酸。
如本文所用，术语蛋白质活性的“失活”包括与其内源活性相比，由于减弱导致的活性降低或完全没有活性。
蛋白质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：缺失染色体上编码蛋白质的基因的部分或全部的方法(包括消除蛋白质的活性的情况)；用突变的基因置换染色体上编码蛋白质的基因从而降低酶活性的方法；向在染色体上编码蛋白质的基因的表达调节序列中引入修饰的方法；用具有弱活性或无活性的序列置换编码蛋白质的基因的表达调节序列的方法(例如，用弱于内源性启动子的启动子置换基因启动子的方法)；缺失染色体上编码蛋白质的基因的部分或全部的方法；引入与染色体上的基因转录物互补结合以抑制mRNA翻译成蛋白质的反义寡核苷酸(例如反义RNA)的方法；人工添加与编码酶的基因的SD序列上游互补的序列以形成二级结构，从而使核糖体的附着不可能方法；以及在相应序列的开放阅读框(ORF)的3'端添加启动子从而被逆转录的逆转录工程(RTE)方法，或其组合，但不特别限于此。
具体地，缺失编码蛋白质的基因的部分或全部的方法可以通过用于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执行。在实施方式中，可以使用通过同源重组缺失基因的方法，但不限于此。另外，如本文用的，尽管术语“部分”可根据多核苷酸的种类而变化并且可由本领域技术人员适当地选择，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。
另外，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，从而进一步减弱表达调节序列的活性来执行；或通过用具有更弱活性的核酸序列置换序列来执行。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终止的序列，但不限于此。
进一步，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，从而进一步失活蛋白质的活性来执行；或通过用修饰以进一步失活活性的基因序列、或修饰以完全没有活性的基因序列置换序列来执行，但不限于此。
本公开的又一方面提供了用于生产基于鸟氨酸的产物的方法，其包括：
(i)在培养基中培养生产基于鸟氨酸的产物的微生物，微生物包含具有输出基于鸟氨酸的产物的能力的多肽或具有其增强的活性；以及
(ii)从上面获得的微生物或培养基中回收基于鸟氨酸的产物。
在具体实施方式中，本公开提供了用于生产腐胺的方法，其包括：
(i)在培养基中培养生产腐胺的微生物，微生物包含具有输出基于鸟氨酸的产物的能力的多肽或具有其增强的活性；以及
(ii)从上面获得的微生物或培养基中回收腐胺。
在另一个具体实施方式中，本公开提供了用于生产L-精氨酸的方法，其包括：
(i)在培养基中培养生产L-精氨酸的微生物，微生物包含具有输出基于鸟氨酸的产物的能力的多肽或具有其增强的活性；以及
(ii)从上面获得的微生物或培养基中回收L-精氨酸。
具有输出基于鸟氨酸的产物能力的多肽和/或生产基于鸟氨酸的产物的微生物如上所述。
在上面的方法中，微生物的培养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。特别地，关于培养条件，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调节到合适的pH(例如pH5至9、具体地pH6至8、和最具体地pH6.8)。另外，可以向培养物中注入氧气或含氧气体混合物，以维持好氧状态。培养温度可维持在20℃至45℃下、具体地是25℃至40℃下，且培养可进行约10小时至160小时，但培养不限于上述。培养生产的腐胺可能在培养基中分泌，也可能维持在细胞中。
另外，作为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。此外，培养基中可含有必需的促生长物质，如其它金属盐(如硫酸镁或硫酸铁)、氨基酸、维生素等。
在用于回收在本公开的培养步骤中生产的基于鸟氨酸的产物的方法中，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收集所期望的产物。例如，可以使用离心、过滤、阴离子交换色谱、结晶、HPLC等，但不限于此。另外，回收基于鸟氨酸的产物的方法可进一步包括使用本领域已知的适当方法的纯化过程。
优选实施方式
将通过示例性实施方式更详细地描述本公开。然而，给出这些示例性实施方式仅用于说明性目的，而不意图限制本公开的范围。
实施例1.腐胺输出蛋白的输出精氨酸的能力的确认
已经发现，谷氨酸棒杆菌的基因NCgl2522具有输出腐胺的能力(韩国专利申请公开号2014-0115244)。为了确认除了腐胺外，NCgl2522是否也能输出可由鸟氨酸作为起始物质生物合成的瓜氨酸、脯氨酸、和精氨酸的目的，进行了以下实验。
具体地，确认了在可由鸟氨酸作为起始物质生物合成的产物中，NCgl2522是否具有输出作为代表性实例的精氨酸的能力。
<1-1>基于精氨酸生产菌株的具有增强的NCgl2522活性的载体及菌株的构建
为了增强具有精氨酸生产能力的野生型ATCC21831菌株和KCCM10741P(韩国专利号10-0791659)中的NCgl2522活性，将CJ7启动子(WO 2006/065095A)引入染色体内NCgl2522起始密码子的上游。
获得了同源重组片段，其包括WO 2006/065095A中公开的CJ7启动子，且其中启动子的两端具有染色体上的原始NCgl2522序列。具体地，基于谷氨酸棒杆菌ATCC21831或KCCM10741P的基因组DNA作为模板，使用表1中所示的SEQ ID NO:63和64的引物对进行PCR，获得CJ7启动子的5'端区域。特别地，PCR反应通过重复30个循环(在94℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸30秒)进行。另外，使用表1中所示的SEQ ID NO:65和66的引物对在相同条件下进行PCR获得CJ7启动子区域，并且基于谷氨酸棒杆菌ATCC21831或KCCM10741P的基因组DNA作为模板，使用SEQ ID NO:67和68的引物对在相同条件下进行PCR获得CJ7启动子的3'端区域。用于启动子的取代的引物示于下表1中。
[表1]
将上述获得的PCR产物中的每个进行融合克隆到用BamHI和XbaI处理的pDZ载体中。使用
HD克隆试剂盒(Clontech Laboratories，Inc.)在50℃下进行10分钟的融合克隆，并将由此获得的质粒分别命名为pDZ-P(CJ7)-NCgl2522-21831和pDZ-P(CJ7)-NCgl2522-10741P。
将上述制备的质粒pDZ-P(CJ7)-NCgl2522-21831和pDZ-P(CJ7)-NCgl2522-10741P分别经由电穿孔引入到ATCC21831和KCCM10741P(它们是精氨酸生产菌株)中以获得转化体，并将由此获得的转化体平皿接种(plated)在含有卡那霉素(25μg/mL)和X-gal(5-溴-4-氯-3-吲哚啉-D-半乳糖苷)的BHIS平板培养基(37g/L的脑心浸液(Braine heartinfusion)、91g/L的山梨醇和2％的琼脂)上并培养以形成菌落。在由此形成的菌落中，选择引入有质粒pDZ-P(CJ7)-NCgl2522-21831或pDZ-P(CJ7)-NCgl2522-10741P的菌株。
选择的菌株在CM培养基(10g/L葡萄糖、10g/L聚蛋白胨、5g/L酵母提取物、5g/L牛肉提取物、2.5g/L NaCl、和2g/L尿素，pH 6.8)中振荡培养(30℃下，8小时)，并依次从10-4稀释到10-10，平皿接种在含有X-gal的固体培养基上，并培养以形成菌落。在由此形成的菌落中，选择以相对较低比率出现的白色菌落，从而最终选择其中通过二次交换用CJ7启动子取代NCgl2522基因的启动子的菌株。将最终选择的菌株使用表1中所示的SEQ ID NO:65和68的引物对进行PCR，并将由此获得的产物应用于测序。结果确认CJ7启动子被引入到染色体内NCgl2522起始密码子的上游中。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸1分钟)进行。
谷氨酸棒杆菌的由此选择的修饰菌株分别被命名为ATCC21831_Pcj7 Ncgl2522和KCCM10741P_Pcj7 Ncgl2522。
<1-2>基于精氨酸生产菌株的具有增强的NCgl2522活性的菌株的精氨酸生产能力的确认
为了确认NCgl2522基因对输出精氨酸(基于鸟氨酸的产物之一)能力的影响，在上述实施例1中制备的谷氨酸棒杆菌ATCC21831_Pcj7 NCgl2522和KCCM10741P_Pcj7NCgl2522的修饰菌株中比较精氨酸生产能力。
谷氨酸棒杆菌ATCC21831和KCCM10741P(它们是亲本菌株)被用作为对照组，且将一个铂环(platinum loop)的每种菌株接种到含有25mL生产培养基[6％葡萄糖、3％硫酸铵、0.1％磷酸二氢钾、0.2％七水硫酸镁、1.5％玉米浆(CSL)、1％NaCl、0.5％酵母抽提物、和100μg/L的生物素，pH7.2]的250-mL折角烧瓶(corner-baffled flask)中，并在30℃下以200rpm的速率培养48小时以生产精氨酸。培养完成后，通过HPLC测定精氨酸产量。结果示于下表2中。
[表2]
如表2中所示，当KCCM10741P和ATCC21831中的NCgl2522基因的启动子通过用CJ7启动子取代而增强时，与亲本菌株相比，谷氨酸棒杆菌的修饰菌株显示出精氨酸产量分别增加了20％和14％。另外，确认了与亲本菌株相比，修饰的菌株中鸟氨酸(转化为精氨酸之前的反应物)的浓度也增加了。
基于这些发现，确认了NCgl2522基因不仅是输出腐胺的基因，而且具有输出由鸟氨酸作为起始物质生物合成的产物(包括鸟氨酸)的能力。另外，从上述结果可以解释，NCgl2522基因和本公开的变体在使用生物质的基于鸟氨酸的产物的生产中是非常有用的。
实施例2.编码腐胺输出蛋白的基因变体的文库的构建及有效修饰的建立
为了增加基于鸟氨酸的产物输出蛋白的活性，本发明人构建了编码腐胺输出蛋白的基因NCgl2522(韩国专利号10-1607741)的变体。
具体地，为了构建NCgl2522基因变体的文库，基于谷氨酸棒杆菌ATCC13032的基因组DNA作为模板，使用排除表2中所示的NCgl2522基因的ORF的起始密码子的特异性引物对(SEQ ID NO:21和22)进行随机诱变PCR(JENA易错PCR)。
[表3]
将由此制备的突变基因片段进行融合克隆到用XbaI切割的pDZ载体中。使用
HD克隆试剂盒(Clontech Laboratories，Inc.)在50℃下进行10分钟的融合克隆，从而完成pDZ-N2522变体的质粒文库的构建。
经由高流量(throughput)筛选(HTS)对由此构建的重组质粒文库进行筛选。特别地，用于筛选的平台菌株(platform strain)是KCCM11240P，其是谷氨酸棒杆菌来源的能够生产腐胺的重组微生物(韩国专利号10-1493585)。
具体地，为了获得具有提高的腐胺输出活性的变体，经由电穿孔将由此构建的质粒文库引入到KCCM11240P中以获得转化体，并将由此获得的转化体平皿接种在含有卡那霉素(25μg/mL)和X-gal(5-溴-4-氯-3-吲哚啉-D-半乳糖苷)的BHIS平板培养基(37g/L脑心浸液、91g/L山梨醇和2％琼脂)上并培养以形成菌落。在由此形成的菌落中，选择引入有质粒pDZ-N2522变体文库的菌株。
通过在96深孔板及效价培养基(titer media)(2g/L葡萄糖、0.4g/L MgSO4·7H2O、0.8g/L MgCl2、1g/L KH2PO4、4g/L(NH4)2SO4、0.48g/L大豆蛋白水解液、0.01g/L MnSO4·7H2O、200μg/L硫胺素HCl、200μg/L生物素、0.01μg/L FeSO4·7H2O、1mM精氨酸、和25μg/mL卡那霉素，pH7.2)中震荡培养(30℃下24小时)选择的菌株，及测定各培养物中生成腐胺的浓度，及然后选择出与对照相比其中腐胺生产性增加最大的一个转化体。随后，确认在选择的转化体的NCgl2522蛋白的氨基酸序列中诱导了哪种修饰。Ncgl2522变体的序列确认如下：基于包括相应变体的转化体，通过使用SEQ ID NO:21和22的引物对进行菌落PCR获得同源重组片段，随后使用SEQ ID NO:21的引物将产物应用于基因组测序。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸1分钟)进行。
结果，确认选择的NCgl2522变体被修饰，使得丙氨酸(Ala)(其是自谷氨酸棒杆菌ATCC13032的NCgl2522氨基酸序列(SEQ ID NO:1)的N端第152位的氨基酸残基)被丝氨酸(Ser)取代，并且被命名为NCgl2522_M1(SEQ ID NO:3)。
实施例3.其中编码腐胺输出蛋白的基因的第152位的氨基酸残基被取代的多种变体的建立
基于实施例2中制备的NCgl2522_M1变体，本发明人认识到自N端第152位的氨基酸残基对于NCgl2522蛋白的活性是重要的。因此，制备了其中NCgl2522蛋白的第152位的氨基酸残基被其它氨基酸残基取代的各种变体。
具体地，基于谷氨酸棒杆菌ATCC13032的基因组DNA作为模板，使用排除NCgl2522基因的ORF的起始密码子的特异性引物对(SEQ ID NO:21和22)获得同源重组片段。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸30秒)进行。
随后，将上述获得的PCR产物进行融合克隆到用XbaI处理的pDZ载体中。使用
HD克隆试剂盒(Clontech Laboratories，Inc.)进行融合克隆，并且将由此获得的质粒被命名为pDZ-NCgL2522_A152。
然后，为了在NCgl2522的第152位的氨基酸残基上诱导随机诱变，基于上述构建的质粒pDZ-NCgl2522_A152作为模板，通过使用表4中所示的SEQ ID NO:23和24的引物对进行PCR来完成了pDZ-NCgl2522_A152变体的质粒文库。特别地，PCR反应通过重复25个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸5分钟)进行。
[表4]
经由高流量筛选(HTS)对由此构建的重组质粒文库进行筛选。特别地，用于筛选的平台菌株是KCCM11240P，其是谷氨酸棒杆菌来源的能够生产腐胺的重组微生物。
经由电穿孔将构建的质粒文库引入到KCCM11240P中以获得转化体，并且以与实施例2相同的方式选择引入有质粒pDZ-NCgl2522_A152变体的菌株。选择与对照组相比腐胺产率增加最大的8个转化体，并以与实施例2相同的方式确认每个转化体的NCgl2522蛋白的氨基酸序列中诱导了哪些修饰。
结果，确认了其中自谷氨酸棒杆菌ATCC13032的NCgl2522氨基酸序列(SEQ ID NO:1)的N端第152位的氨基酸残基丙氨酸被其它氨基酸取代的变体。在它们中间，其中第152位的氨基酸残基被天冬酰胺取代的变体被命名为NCgl2522_M4(SEQ ID NO:6)，其中第152位的氨基酸残基被脯氨酸取代的变体被命名为NCgl2522_M5(SEQ ID NO:7)，其中第152位的氨基酸残基被赖氨酸取代的变体被命名为NCgl2522_M6(SEQ ID NO:8)，其中第152位的氨基酸残基被谷氨酸取代的变体被命名为NCgl2522_M7(SEQ ID NO:9)，其中第152位的氨基酸残基被半胱氨酸取代的变体被命名为NCgl2522_M8(SEQ ID NO:10)，其中第152位的氨基酸残基被谷氨酰胺取代的变体被命名为NCgl2522_M9(SEQ ID NO:11)，和其中第152位的氨基酸残基被蛋氨酸取代的变体被命名为NCgl2522_M10(SEQ ID NO:12)。
另外，为了确认由于修饰而增加腐胺产率的效果是否可以应用于来源于不同菌株的NCgl2522蛋白，构建了来源于谷氨酸棒杆菌ATCC13869的NCgl2522蛋白的第152位的氨基酸残基被其他氨基酸残基取代的各种变体。
具体地，基于谷氨酸棒杆菌ATCC13869的基因组DNA作为模板，使用排除NCgl2522基因的ORF起始密码子的特异性引物对(SEQ ID NO:21和22)获得同源重组片段。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸30秒)进行。
将上述获得的PCR产物融合克隆到用XbaI处理的pDZ载体中。使用
HD克隆试剂盒(Clontech Laboratories，Inc.)进行融合克隆，和由此获得的质粒命名为pDZ-13869-NCgl2522_A152。
然后，为了在NCgl2522的第152位的氨基酸残基上诱导随机诱变，基于以上构建的质粒pDZ-13869-NCgl2522_A152作为模板，通过使用SEQ ID NO:23和24的引物对进行PCR，完成pDZ-13869-NCgl2522_A152变体的质粒文库。特别地，PCR反应通过重复25个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸5分钟)进行。
随后，经由高流量筛选(HTS)对由此构建的重组质粒文库进行筛选。特别地，用于筛选的平台菌株是DAB12-b，它是谷氨酸棒杆菌来源的能够生产腐胺的重组微生物。然后，经由电穿孔将构建的质粒文库引入到DAB12-b中以获得转化体，并且以与实施例2相同的方式选择引入有质粒pDZ-13869-NCgl2522_A152变体的菌株。
结果，选择其中自谷氨酸棒杆菌ATCC13869的NCgl2522氨基酸序列(SEQ ID NO:2)的N端第152位的氨基酸残基被取代的9个变体，作为谷氨酸棒杆菌ATCC13032的NCgl2522变体。在它们中间，其中第152位的氨基酸残基丙氨酸的被丝氨酸取代的变体被命名为NCgl2522_M2(SEQ ID NO:4)，其中第152位的氨基酸残基被天冬酰胺取代的变体被命名为NCgl2522_M11(SEQ ID NO:13)，其中152位的氨基酸残基被组氨酸取代的变体被命名为NCgl2522_M12(SEQ ID NO:14)，其中第152位的氨基酸残基被脯氨酸取代的变体被命名为NCgl2522_M13(SEQ ID NO:15)，其中第152位的氨基酸残基被赖氨酸取代的变体被命名为NCgl2522_M14(SEQ ID NO:16)，其中第152位的氨基酸残基被谷氨酸取代的变体被命名为NCgl2522_M15(SEQ ID NO:17)，其中第152位的氨基酸残基被半胱氨酸取代的变体被命名为NCgl2522_M16(SEQ ID NO:18)，其中第152位的氨基酸残基被谷氨酰胺取代的变体被命名为NCgl2522_M17(SEQ ID NO:19)，和其中第152位的氨基酸残基被蛋氨酸取代的变体被命名为NCgl2522_M18(SEQ ID NO:20)。
实施例4.NCgl2522变体菌株的构建及其腐胺生产能力的确认
<4-1>从基于ATCC13032的腐胺生产菌株构建NCgl2522变体菌株
为了增加腐胺生产菌株输出腐胺的能力，将作为NCgl2522基因的变体的NCgl2522_M1、NCgl2522_M3、NCgl2522_M4、NCgl2522_M5、NCgl2522_M6、NCgl2522_M7、NCgl2522_M8、NCgl2522_M9和NCgl2522_M10分别引入到谷氨酸棒杆菌基于ATCC13032的腐胺生产菌株的染色体中。
具体地，基于谷氨酸棒杆菌ATCC13032的基因组DNA作为模板，通过使用表5中示出的SEQ ID NO:25和28、SEQ ID NO:26和27、SEQ ID NO:25和30、SEQ ID NO:26和29、SEQ IDNO:25和32、SEQ ID NO:26和31、SEQ ID NO:25和34、SEQ ID NO:26和33、SEQ ID NO:25和36、SEQ ID NO:26和35、SEQ ID NO:25和38、SEQ ID NO:26和37、SEQ ID NO:25和40、SEQ IDNO:26和39、SEQ ID NO:25和42、SEQ ID NO:26和41、SEQ ID NO:25和44、以及SEQ ID NO:26和43的引物对进行PCR，分别获得具有NCgl2522_M1、NCgl2522_M3、NCgl2522_M4、NCgl2522_M5、NCgl2522_M6、NCgl2522_M7、NCgl2522_M8、NCgl2522_M9、和NCgl2522_M10的修饰序列的同源重组片段。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸30秒)进行。
[表5]
将上述获得的PCR产物中的每个进行融合克隆到用XbaI处理的pDZ载体中。使用
HD克隆试剂盒(Clontech Laboratories，Inc.)进行融合克隆，并将由此获得的质粒分别被命名为pDZ-NCgl2522_M1、pDZ-NCgl2522_M3、pDZ-NCgl2522_M4、pDZ-NCgl2522_M5、pDZ-NCgl2522_M6、pDZ-NCgl2522_M7、pDZ-NCgl2522_M8、pDZ-NCgl2522_M9和pDZ-NCgl2522_M10。
将上述制备的质粒pDZ-NCgl2522_M1、pDZ-NCgl2522_M3、pDZ-NCgl2522_M4、pDZ-NCgl2522_M5、pDZ-NCgl2522_M6、pDZ-NCgl2522_M7、pDZ-NCgl2522_M8、pDZ-NCgl2522_M9和pDZ-NCgl2522_M10经由电穿孔分别引入到KCCM11240P(其是谷氨酸棒杆菌基于ATCC13032的腐胺生产菌株)(韩国专利申请公开号2013-0082478)中，以获得转化体，并将由此获得的转化体平皿接种在含有卡那霉素(25μg/mL)和X-gal(5-溴-4-氯-3-吲哚啉-D-半乳糖苷)的BHIS平板培养基(37g/L脑心浸液、91g/L山梨醇和2％琼脂)上并培养以形成菌落。在由此形成的菌落中，选择了引入有每种质粒的菌株。
在CM培养基(10g/L葡萄糖，10g/L聚蛋白胨，5g/L酵母提取物，5g/L牛肉提取物，2.5g/L NaCl和2g/L尿素，pH 6.8)中震荡培养(30℃下，8小时)上述选择的菌株中的每一种，并依次从10-4稀释到10-10，平皿接种在含有X-gal的固体培养基上，并培养以形成菌落。在由此形成的菌落中，选择以相对较低比率出现的白色菌落，从而最终选择其中NCgl2522基因通过二次交换被变体中的每个取代的菌株。使用SEQ ID NO:25和26的引物对将最终选择的菌株进行PCR，并由此获得的产物应用于测序以确认被变体中的每个的取代。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸1分钟)进行。
将由此选择的谷氨酸棒杆菌的修饰菌株分别命名为KCCM11240P NCgl2522_M1、KCCM11240P NCgl2522_M3、KCCM11240P NCgl2522_M4、KCCM11240P NCgl2522_M5、KCCM11240P NCgl2522_M6、KCCM11240P NCgl2522_M7、KCCM11240P NCgl2522_M8、KCCM11240P NCgl2522_M9和KCCM11240P NCgl2522_M10。
<4-2>从基于ATCC13869的腐胺生产菌株构建NCgl2522变体菌株
基于DAB12-aΔNCgl1469(其是谷氨酸棒杆菌ATCC13869的腐胺生产菌株)(韩国专利申请公开号2013-0082478)被命名为DAB12-b。为了增加腐胺生产菌株输出腐胺的能力的目的，将作为NCgl2522基因的变体的NCgl2522_M2、NCgl2522_M11、NCgl2522_M12、NCgl2522_M13、NCgl2522_M14、NCgl2522_M15、NCgl2522_M16、NCgl2522_M17和NCgl2522_M18分别引入到DAB12-b菌株的染色体中。
具体地，基于谷氨酸棒杆菌ATCC13869的基因组DNA作为模板，通过使用表5和表6中示出的SEQ ID NO:25和46、SEQ ID NO:26和45、SEQ ID NO:25和48、SEQ ID NO:26和47、SEQ ID NO:25和50、SEQ ID NO:26和49、SEQ ID NO:25和52、SEQ ID NO:26和51、SEQ IDNO:25和54、SEQ ID NO:26和53、SEQ ID NO:25和56、SEQ ID NO:26和55、SEQ ID NO:25和58、SEQ ID NO:26和57、SEQ ID NO:25和50、SEQ ID NO:26和59、SEQ ID NO:25和62以及SEQID NO:26和61的引物对进行PCR，分别获得具有NCgl2522_M2、NCgl2522_M11、NCgl2522_M12、NCgl2522_M13、NCgl2522_M14、NCgl2522_M15、NCgl2522_M16、NCgl2522_M17和NCgl2522_M18的修饰序列的同源重组片段。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸30秒)进行。
[表6]
将上述获得的PCR产物中的每个进行融合克隆到用XbaI处理的pDZ载体中。使用
HD克隆试剂盒(Clontech Laboratories，Inc.)进行融合克隆，且将由此获得的质粒分别命名为pDZ-NCgl2522_M2、pDZ-NCgl2522_M11、pDZ-NCgl2522_M12、pDZ-NCgl2522_M13、pDZ-NCgl2522_M14、pDZ-NCgl2522_M15、pDZ-NCgl2522_M16、pDZ-NCgl2522_M17和pDZ-NCgl2522_M18。
将上述制备的质粒中的每个经由电穿孔引入到DAB12-b中以获得转化体，并将由此获得的转化体平皿接种在含有卡那霉素(25μg/mL)和X-gal(5-溴-4-氯-3-吲哚啉-D-半乳糖苷)的BHIS平板培养基(37g/L脑心浸液、91g/L山梨醇和2％琼脂)上并培养以形成菌落。在由此形成的菌落中，选择了引入有上述质粒的菌株。
在CM培养基(10g/L葡萄糖、10g/L聚蛋白胨、5g/L酵母提取物、5g/L牛肉提取物、2.5g/L NaCl和2g/L尿素，pH 6.8)中震荡培养(30℃下，8小时)上述选择的菌株中的每一种，并依次从10-4稀释到10-10，平皿接种在含有X-gal的固体培养基上，并培养以形成菌落。在由此形成的菌落中，选择以相对较低比率出现的白色菌落，从而最终选择其中NCgl2522基因通过二次交换被变体中的每个取代的菌株。使用SEQ ID NO:25和26的引物对将最终选择的菌株进行PCR，并将由此获得的产物应用于测序以确认用变体的取代。特别地，PCR反应通过重复30个循环(在95℃下变性30秒，在55℃下退火30秒，以及在72℃下延伸1分钟)进行。
将由此选择的谷氨酸棒杆菌的修饰菌株分别命名为DAB12-b NCgl2522_M2、DAB12-b NCgl2522_M11、DAB12-b NCgl2522_M12、DAB12-b NCgl2522_M13、DAB12-bNCgl2522_M14、DAB12-b NCgl2522_M15、DAB12-b NCgl2522_M16、DAB12-b NCgl2522_M17、和DAB12-b NCgl2522_M18。
<4-3>引入有NCgl2522变体的菌株的腐胺生产能力的评价
为了确认当将增加输出腐胺能力的NCgl2522基因的变体引入到腐胺生产菌株中时，NCgl2522变体对腐胺生产的效果，在实施例4-1和4-2中制备的谷氨酸棒杆菌的修饰菌株中比较腐胺生产能力。
具体地，谷氨酸棒杆菌的修饰菌株(KCCM11240P NCgl2522_M1；KCCM11240PNCgl2522_M3；KCCM11240P NCgl2522_M4；KCCM11240P NCgl2522_M5；KCCM11240PNCgl2522_M6；KCCM11240P NCgl2522_M7；KCCM11240P NCgl2522_M8；KCCM11240PNCgl2522_M9；KCCM11240P NCgl2522_M10；DAB12-b NCgl2522_M2；DAB12-b NCgl2522_M11；DAB12-b NCgl2522_M12；DAB12-b NCgl2522_M13；DAB12-b NCgl2522_M14；DAB12-bNCgl2522_M15；DAB12-b NCgl2522_M116；DAB12-b NCgl2522_M17；和DAB12-b NCgl2522_M18)以及两种亲本菌株(KCCM11240P和DAB12-b)分别平皿接种在含1mM精氨酸的CM平板培养基(1％葡萄糖、1％蛋白胨、0.5％酵母提取物、0.5％牛肉提取物、0.25％NaCl、0.2％尿素、100μL的50％NaOH和2％琼脂，pH6.8，基于1L)上，并在30℃下培养24h。将约一个铂环的从其培养的每种菌株接种到25mL的效价培养基(8％葡萄糖、0.25％大豆蛋白、0.50％玉米浆固体(corn steep solids)、4％(NH4)2SO4、0.1％KH2PO4、0.05％MgSO4.7H2O、0.15％尿素、100μg生物素、3mg硫胺素HCl、3mg泛酸钙、3mg烟酰胺和5％CaCO3，基于1升)中，并在30℃下以200rpm的速率振荡培养50小时。在所有菌株的培养过程中，向培养基中加入1mM精氨酸。培养完成后，测定各培养物中生产腐胺的浓度，且结果示于表7中。
[表7]
如上表7中所示，当将变体中的每个引入到KCCM11240P中时，与亲本菌株相比，谷氨酸棒杆菌的所有9种修饰菌株显示腐胺产量和生产性都增加了7％至14％。另外，当将变体中的每个引入到DAB12-b中时，与亲本菌株相比，谷氨酸棒杆菌的所有9种修饰菌株显示腐胺产量和生产性都增加了5％至12％。特别地，生产性代表每个转化体每小时的腐胺产量，并以g/L/h表示。
实施例5.将NCgl2522变体引入具有提高的腐胺输出能力的腐胺生产菌株及其腐胺生产能力的确认
<5-1>通过将NCgl2522变体引入具有提高的腐胺输出能力的菌株的菌株的构建
为了确认NCgl2522基因的变体的效果，将NCgl2522_M1、NCgl2522_M3、NCgl2522_M4、NCgl2522_M5、NCgl2522_M6、NCgl2522_M7、NCgl2522_M8、NCgl2522_M9和NCgl2522_M10分别引入到KCCM11240P P(CJ7)-NCgl2522(韩国专利申请公开号2014-0115244)的染色体中，KCCM11240P P(CJ7)-NCgl2522是具有增加的输出腐胺的能力的基于谷氨酸棒杆菌ATCC13032的腐胺生产菌株。
具体地，将实施例4-1中制备的质粒pDZ-NCgl2522_M1、pDZ-NCgl2522_M3、pDZ-NCgl2522_M4、pDZ-NCgl2522_M5、pDZ-NCgl2522_M6、pDZ-NCgl2522_M7、pDZ-NCgl2522_M8、pDZ-NCgl2522_M9、pDZ-NCgl2522_M10中的每一个以与实施例4-1中相同的方式转化到KCCM11240P P(CJ7)-NCgl2522中，并因此确认其染色体内的NCgl2522基因被变体取代。选择的谷氨酸棒杆菌的修饰菌株分别被命名为KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M1、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M3、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M4、KCCM11240P P(CJ7)-NCgl2522NCgl2522_M5、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M6、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M7、KCCM11240P P(CJ7)-NCgl2522NCgl2522_M8、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M9、和KCCM11240P P(CJ7)-NCgl2522NCgl2522_M10。
<5-2>通过将NCgl2522变体引入到具有提高的腐胺输出能力的菌株制备的菌株的腐胺生产能力的评价
为了确认NCgl2522变体对具有提高的输出腐胺能力的谷氨酸棒杆菌生产菌株的效果，在实施例5-1中制备的谷氨酸棒杆菌的修饰菌株和亲本菌株中比较腐胺生产能力。
具体地，对谷氨酸棒杆菌的修饰菌株(KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M1、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M3、KCCM11240P P(CJ7)-NCgl2522NCgl2522_M4、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M5、KCCM11240P P(CJ7)-NCgl2522NCgl2522_M6、KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M7、KCCM11240P P(CJ7)-NCgl2522NCgl2522_M8、KCCM11240P P(CJ7)-NCgl2522NCgl2522_M9、和KCCM11240P P(CJ7)-NCgl2522 NCgl2522_M10)和亲本菌株(KCCM11240P P(CJ7)-NCgl2522)分别平皿接种在含1mM精氨酸的CM平板培养基(1％葡萄糖、1％蛋白胨、0.5％酵母提取物、0.5％牛肉提取物、0.25％NaCl、0.2％尿素、100μL的50％NaOH和2％琼脂，pH6.8，基于1L)上，并在30℃下培养24h。将约一个铂环的从其培养的每种菌株接种到25mL的效价培养基(8％葡萄糖、0.25％大豆蛋白、0.50％玉米浆固体、4％(NH4)2SO4、0.1％KH2PO4、0.05％MgSO4.7H2O、0.15％尿素、100μg生物素、3mg硫胺素HCl、3mg泛酸钙、3mg烟酰胺和5％CaCO3，基于1升)中，并在30℃下以200rpm的速率振荡培养50小时。在所有菌株的培养过程中，向培养基中加入1mM精氨酸。培养完成后，测定各培养物中生产腐胺的浓度，且结果示于下表8中。
[表8]
如上表8中所示，当将9种变体分别引入到具有提高的腐胺输出能力的KCCM11240PP(CJ7)-NCgl2522中时，与已经显示出提高的腐胺输出能力的亲本菌株相比，谷氨酸棒杆菌的修饰菌株显示腐胺产量和生产性增加了4％至10％。特别地，生产性代表每个转化体每小时的腐胺产量，并以g/L/h表示。
实施例6.NCgl2522变体菌株的构建及其精氨酸生产能力的确认
<6-1>从L-精氨酸生产菌株构建NCgl2522变体菌株
为了增加L-精氨酸生产菌株的输出L-精氨酸的能力，将作为NCgl2522基因的变体的NCgl2522_M1、NCgl2522_M3、NCgl2522_M4、NCgl2522_M5、NCgl2522_M6、NCgl2522_M7、NCgl2522_M8、NCgl2522_M9和NCgl2522_M10分别引入到谷氨酸棒杆菌KCCM10741P(韩国专利号10-0791659)的染色体中。
具体地，基于谷氨酸棒杆菌KCCM10741P的基因组DNA作为模板，使用表5中示出的引物对，以与实施例4-2中相同的方式进行PCR，并最终选择其中用NCgl2522_M1、NCgl2522_M3、NCgl2522_M4、NCgl2522_M5、NCgl2522_M6、NCgl2522_M7、NCgl2522_M8、NCgl2522_M9或NCgl2522_M10取代NCgl2522基因的菌株。
从中选择的谷氨酸棒杆菌的修饰菌株分别被命名为KCCM10741P NCgl2522_M1、KCCM10741P NCgl2522_M3、KCCM10741P NCgl2522_M4、KCCM10741P NCgl2522_M5、KCCM10741P NCgl2522_M6、KCCM10741P NCgl2522_M7、KCCM10741P NCgl2522_M8、KCCM10741P NCgl2522_M9和KCCM10741P NCgl2522_M10。
<6-2>从基于ATCC21831的L-精氨酸生产菌株构建NCgl2522变体菌株
为了增加基于野生型谷氨酸棒杆菌ATCC21831的L-精氨酸生产菌株的输出L-精氨酸的能力，将作为NCgl2522基因的变体的NCgl2522_M2、NCgl2522_M11、NCgl2522_M12、NCgl2522_M13、NCgl2522_M14、NCgl2522_M15、NCgl2522_M16、NCgl2522_M17和NCgl2522_M18分别引入到ATCC21831的染色体中。
具体地，基于谷氨酸棒杆菌ATCC21831的基因组DNA作为模板，使用表5中示出的引物对，以与实施例4-2中相同的方式地进行PCR，并最终选择其中用NCgl2522_M2、NCgl2522_M11、NCgl2522_M12、NCgl2522_M13、NCgl2522_M14、NCgl2522_M15、NCgl2522_M16、NCgl2522_M17和NCgl2522_M18取代NCgl2522基因的菌株。
从中选择的谷氨酸棒杆菌的修饰菌株分别被命名为ATCC21831 NCgl2522_M2、ATCC21831 NCgl2522_M11、ATCC21831 NCgl2522_M12、ATCC21831 NCgl2522_M13、ATCC21831 NCgl2522_M14、ATCC21831 NCgl2522_M15、ATCC21831 NCgl2522_M16、ATCC21831 NCgl2522_M17和ATCC21831 NCgl2522_M18。
<6-3>引入有NCgl2522变体的菌株的L-精氨酸生产能力的评价
为了确认当增加输出L-精氨酸能力的NCgl2522基因的变体进入L-精氨酸生产菌株时，NCgl2522变体对L-精氨酸生产的效果，在实施例6-1和6-2中制备的谷氨酸棒杆菌的修饰菌株中比较L-精氨酸生产能力。
特别地，作为对照组，使用谷氨酸棒杆菌KCCM10741P和ATCC21831(它们是亲本菌株)，以及在实施例1中制备的KCCM10741P_Pcj7 Ncgl2522和ATCC21831_Pcj7Ncgl2522。将一个铂环的每种菌株的接种到含有25mL生产培养基[6％葡萄糖、3％硫酸铵、0.1％磷酸二氢钾、0.2％七水硫酸镁、1.5％玉米浆(CSL)、1％NaCl、0.5％酵母抽提物和100μg/L生物素，pH7.2]的250-mL折角烧瓶中，并在30℃下以200rpm的速率振荡培养48小时以生产L-精氨酸。培养完成后，通过HPLC测定L-精氨酸产量。结果示于下表9中。
[表9]
如表9中所示，当将每个变体引入到KCCM10741P中时，与亲本菌株相比，引入有变体的谷氨酸棒杆菌的所有修饰菌株显示L-精氨酸产量增加了30％和40％。另外，当将每个变体引入到ATCC21831中时，与亲本菌株相比，所有9个谷氨酸棒杆菌的修饰菌株显示L-精氨酸产量增加了19％和26％。
进一步，确认了当引入变体时，转化为L-精氨酸后输出的L-鸟氨酸的浓度也增加了。基于这些发现，可以解释谷氨酸棒杆菌的修饰菌株也可能输出由鸟氨酸作为起始物质生物合成的产物。
总之，本发明人已经确认自N端第152位的氨基酸残基在腐胺输出蛋白NCgl2522中的输出基于鸟氨酸的产物的能力中起关键作用。特别地，当第152位的氨基酸被其他氨基酸残基取代时，发现了在引入有变体的菌株中基于鸟氨酸的产物的产量增加。因此，本公开的变体可应用于使用微生物生产基于鸟氨酸的产物的方法，以进一步提高其产量，并且因此对使用生物质的基于鸟氨酸的产物的生产可以是非常有用的。
在本公开中，NCgl2522基因的变体NCgl2522_A152S被引入到基于谷氨酸棒杆菌ATCC13032的腐胺生产菌株的染色体中，且结果确认了在引入有变体的谷氨酸棒杆菌菌株中可以高得率和高生产性地生产腐胺。因此，菌株被命名为KCCM11240P NCgl2522_A152S，并于2016年9月1日根据布达佩斯条约保藏在国际保藏机构韩国微生物培养中心(KCCM)，登录号为KCCM11887P。
本领域的普通技术人员将认识到，在不脱离它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。
<110> CJ第一制糖株式会社
<120> 新型多肽及使用其生产基于鸟氨酸的产物的方法
<130> OPA18206
<150> KR 10-2017-0074981
<151> 2017-06-14
<160> 68
<170> KopatentIn 2.0
<210> 1
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522
<400> 1
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Ala Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 2
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522
<400> 2
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Ala Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 3
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M1
<400> 3
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Ser Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 4
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M2
<400> 4
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Ser Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 5
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M3
<400> 5
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Asn Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 6
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M4
<400> 6
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val His Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 7
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M5
<400> 7
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Pro Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 8
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M6
<400> 8
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Lys Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 9
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M7
<400> 9
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Glu Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 10
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M8
<400> 10
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Cys Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 11
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M9
<400> 11
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Gln Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 12
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13032 NCgl2522_M10
<400> 12
Met Thr Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Met Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Ile Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Met Pro Leu Leu Leu Gly Ala
225 230 235 240
Val Ile Met Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Ala Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Asp Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 13
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M11
<400> 13
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Asn Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 14
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M12
<400> 14
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val His Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 15
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M13
<400> 15
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Pro Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 16
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M14
<400> 16
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Lys Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 17
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M15
<400> 17
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Glu Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 18
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M16
<400> 18
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Cys Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 19
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M17
<400> 19
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Gln Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 20
<211> 494
<212> PRT
<213> 人工序列
<220>
<223> ATCC13869 NCgl2522_M18
<400> 20
Met Ile Ser Glu Thr Leu Gln Ala Gln Ala Pro Thr Lys Thr Gln Arg
1 5 10 15
Trp Ala Phe Leu Ala Val Ile Ser Gly Gly Leu Phe Leu Ile Gly Val
20 25 30
Asp Asn Ser Ile Leu Tyr Thr Ala Leu Pro Leu Leu Arg Glu Gln Leu
35 40 45
Ala Ala Thr Glu Thr Gln Ala Leu Trp Ile Ile Asn Ala Tyr Pro Leu
50 55 60
Leu Met Ala Gly Leu Leu Leu Gly Thr Gly Thr Leu Gly Asp Lys Ile
65 70 75 80
Gly His Arg Arg Met Phe Leu Met Gly Leu Ser Ile Phe Gly Ile Ala
85 90 95
Ser Leu Gly Ala Ala Phe Ala Pro Thr Ala Trp Ala Leu Val Ala Ala
100 105 110
Arg Ala Phe Leu Gly Ile Gly Ala Ala Thr Met Met Pro Ala Thr Leu
115 120 125
Ala Leu Ile Arg Ile Thr Phe Glu Asp Glu Arg Glu Arg Asn Thr Ala
130 135 140
Ile Gly Ile Trp Gly Ser Val Met Ile Leu Gly Ala Ala Ala Gly Pro
145 150 155 160
Ile Ile Gly Gly Ala Leu Leu Glu Phe Phe Trp Trp Gly Ser Val Phe
165 170 175
Leu Ile Asn Val Pro Val Ala Val Ile Ala Leu Ile Ala Thr Leu Phe
180 185 190
Val Ala Pro Ala Asn Ile Ala Asn Pro Ser Lys His Trp Asp Phe Leu
195 200 205
Ser Ser Phe Tyr Ala Leu Leu Thr Leu Ala Gly Leu Ile Val Thr Ile
210 215 220
Lys Glu Ser Val Asn Thr Ala Arg His Leu Pro Leu Leu Val Gly Ala
225 230 235 240
Ile Ile Leu Leu Ile Ile Gly Ala Val Leu Phe Ser Ser Arg Gln Lys
245 250 255
Lys Ile Glu Glu Pro Leu Leu Asp Leu Ser Leu Phe Arg Asn Arg Leu
260 265 270
Phe Leu Gly Gly Val Val Ala Ala Gly Met Ala Met Phe Thr Val Ser
275 280 285
Gly Leu Glu Met Thr Thr Ser Gln Arg Phe Gln Leu Ser Val Gly Phe
290 295 300
Thr Pro Leu Glu Ala Gly Leu Leu Met Ile Pro Ala Ala Leu Gly Ser
305 310 315 320
Phe Pro Met Ser Ile Ile Gly Gly Ala Asn Leu His Arg Trp Gly Phe
325 330 335
Lys Pro Leu Ile Ser Gly Gly Phe Leu Ala Thr Ala Val Gly Ile Ala
340 345 350
Leu Cys Ile Trp Gly Ala Thr His Thr Asp Gly Leu Pro Phe Phe Ile
355 360 365
Ala Gly Leu Phe Phe Met Gly Ala Gly Ala Gly Ser Val Met Ser Val
370 375 380
Ser Ser Thr Ala Ile Ile Gly Ser Ala Pro Val Arg Lys Ala Gly Met
385 390 395 400
Ala Ser Ser Ile Glu Glu Val Ser Tyr Glu Phe Gly Thr Leu Leu Ser
405 410 415
Val Ala Ile Leu Gly Ser Leu Phe Pro Phe Phe Tyr Ser Leu His Ala
420 425 430
Pro Ala Glu Val Ala Asp Asn Phe Ser Ala Gly Val His His Ala Ile
435 440 445
Tyr Gly Asp Ala Ala Arg Ala Ser Leu Asp Thr Ala Tyr Ile Asn Val
450 455 460
Leu Ile Ile Ala Leu Val Cys Ala Val Ala Ala Ala Leu Ile Ser Ser
465 470 475 480
Tyr Leu Phe Arg Gly Asn Pro Lys Gly Ala Asn Asn Ala His
485 490
<210> 21
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-EF-FX
<400> 21
ccggggatcc tctagaactt cagaaacctt acaggc 36
<210> 22
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-EF-RX
<400> 22
gcaggtcgac tctagactag tgcgcattat tggctc 36
<210> 23
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> SM_putE_A152-F
<400> 23
tggggttccg tgnnkattct tggcgct 27
<210> 24
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> SM_putE_A152-R
<400> 24
agcgccaaga atmnncacgg aacccca 27
<210> 25
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> pDC-putE-up-FX
<400> 25
ccggggatcc tctagagtgg tctctttctg atcg 34
<210> 26
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> pDC-putE-down-RX
<400> 26
gcaggtcgac tctagagcaa ttccttgtcc cagt 34
<210> 27
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152S-F
<400> 27
gggttccgtg tccattcttg gcg 23
<210> 28
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152S-R
<400> 28
cgccaagaat ggacacggaa ccc 23
<210> 29
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152N-F
<400> 29
gggttccgtg aatattcttg gcg 23
<210> 30
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152N-R
<400> 30
cgccaagaat attcacggaa ccc 23
<210> 31
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152H-F
<400> 31
gggttccgtg catattcttg gcg 23
<210> 32
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152H-R
<400> 32
cgccaagaat atgcacggaa ccc 23
<210> 33
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152P-F
<400> 33
gggttccgtg cctattcttg gcg 23
<210> 34
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152P-R
<400> 34
cgccaagaat aggcacggaa ccc 23
<210> 35
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152K-F
<400> 35
gggttccgtg aagattcttg gcg 23
<210> 36
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152K-R
<400> 36
cgccaagaat cttcacggaa ccc 23
<210> 37
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152E-F
<400> 37
gggttccgtg gagattcttg gcg 23
<210> 38
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152E-R
<400> 38
cgccaagaat ctccacggaa ccc 23
<210> 39
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152C-F
<400> 39
gggttccgtg tgtattcttg gcg 23
<210> 40
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152C-R
<400> 40
cgccaagaat acacacggaa ccc 23
<210> 41
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152Q-F
<400> 41
gggttccgtg cagattcttg gcg 23
<210> 42
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152Q-R
<400> 42
cgccaagaat ctgcacggaa ccc 23
<210> 43
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152M-F
<400> 43
gggttccgtg atgattcttg gcg 23
<210> 44
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13032-putE-A152M-R
<400> 44
cgccaagaat catcacggaa ccc 23
<210> 45
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152S-F
<400> 45
gggttctgtg tccattcttg gcg 23
<210> 46
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152S-R
<400> 46
cgccaagaat ggacacagaa ccc 23
<210> 47
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152N-F
<400> 47
gggttctgtg aatattcttg gcg 23
<210> 48
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152N-R
<400> 48
cgccaagaat attcacagaa ccc 23
<210> 49
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152H-F
<400> 49
gggttctgtg catattcttg gcg 23
<210> 50
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152H-R
<400> 50
cgccaagaat atgcacagaa ccc 23
<210> 51
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152P-F
<400> 51
gggttctgtg cctattcttg gcg 23
<210> 52
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152P-R
<400> 52
cgccaagaat aggcacagaa ccc 23
<210> 53
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152K-F
<400> 53
gggttctgtg aagattcttg gcg 23
<210> 54
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152K-R
<400> 54
cgccaagaat cttcacagaa ccc 23
<210> 55
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152E-F
<400> 55
gggttctgtg gagattcttg gcg 23
<210> 56
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152E-R
<400> 56
cgccaagaat ctccacagaa ccc 23
<210> 57
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152C-F
<400> 57
gggttctgtg tgtattcttg gcg 23
<210> 58
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152C-R
<400> 58
cgccaagaat acacacagaa ccc 23
<210> 59
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152Q-F
<400> 59
gggttctgtg cagattcttg gcg 23
<210> 60
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152Q-R
<400> 60
cgccaagaat ctgcacagaa ccc 23
<210> 61
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152M-F
<400> 61
gggttctgtg atgattcttg gcg 23
<210> 62
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 13869-putE-A152M-R
<400> 62
cgccaagaat catcacagaa ccc 23
<210> 63
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> NCgl2522-L5
<400> 63
tgcaggtcga ctctagagtt ctgcgtagct gtgtgcc 37
<210> 64
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> NCgl2522-L3
<400> 64
gatgtttctg gatcgtaact gtaacgaatg g 31
<210> 65
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> CJ7-F
<400> 65
agaaacatcc cagcgctact aata 24
<210> 66
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> CJ7-R
<400> 66
agtgtttcct ttcgttgggt acg 23
<210> 67
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> NCgl2522-R5
<400> 67
caacgaaagg aaacactatg atttcagaaa ctttgcaggc g 41
<210> 68
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> NCgl2522-R3
<400> 68
tcggtacccg gggatcccac aaaaagcgta gcgatcaacg 40
PCT/RO/134表
Claims (21)
1.具有输出基于鸟氨酸的产物的能力的多肽，其中自基于鸟氨酸的产物输出蛋白的氨基酸序列的N端第152位的丙氨酸残基被其它氨基酸取代，所述蛋白由SEQ ID NO:1或SEQID NO:2的氨基酸序列组成。
2.权利要求1所述的多肽，其中第152位的所述丙氨酸被丝氨酸、天冬酰胺、组氨酸、脯氨酸、赖氨酸、谷氨酸、半胱氨酸、谷氨酰胺或蛋氨酸取代。
3.权利要求1所述的多肽，其中所述多肽由SEQ ID NO:3至SEQ ID NO:20中任一项的氨基酸序列组成。
4.多核苷酸，其编码具有输出权利要求1至3中任一项的基于鸟氨酸的产物的能力的所述多肽。
5.载体，所述载体包含权利要求4所述的多核苷酸。
6.生产腐胺的棒杆菌属(genus Corynebacterium)的微生物，所述微生物包含权利要求1至4中任一项所述的或具有其增强的活性的多肽。
7.权利要求6所述的微生物，其中所述微生物是谷氨酸棒杆菌(Corynebacteriumglutamicum)。
8.权利要求6所述的微生物，其中鸟氨酸脱羧酶(ODC)的活性进一步被引入。
9.权利要求6所述的微生物，其中选自以下的至少一种的活性与其内源活性相比进一步被失活：鸟氨酸氨甲酰基转移酶(ArgF)和参与谷氨酸输出的蛋白质。
10.权利要求6所述的微生物，其中选自以下的至少一种的活性与其内源活性相比进一步被增强：乙酰-γ-谷氨酰-磷酸还原酶(ArgC)、乙酰谷氨酸合酶或鸟氨酸乙酰转移酶(argJ)、乙酰谷氨酸激酶(ArgB)、和乙酰鸟氨酸转氨酶(ArgD)。
11.权利要求6所述的微生物，其中腐胺乙酰转移酶的活性与其内源活性相比进一步被失活。
12.具有提高的精氨酸生产能力的棒杆菌属的微生物，所述微生物包含权利要求1至3中任一项所述的或具有其增强的活性的多肽。
13.权利要求12所述的微生物，其中选自以下的至少一种的活性与其内源活性相比进一步被增强：乙酰-γ-谷氨酰-磷酸还原酶(ArgC)、乙酰谷氨酸合酶或鸟氨酸乙酰转移酶(argJ)、乙酰谷氨酸激酶(ArgB)、和乙酰鸟氨酸转氨酶(ArgD)。
14.权利要求12所述的微生物，其中选自以下的至少一种的活性与其内源活性相比进一步增加：鸟氨酸氨甲酰基转移酶(ArgF)、精氨琥珀酸合酶(argG)、精氨琥珀酸裂解酶(argH)、天冬氨酸氨裂解酶和/或天冬氨酸转氨酶。
15.权利要求12所述的微生物，其中所述微生物是谷氨酸棒杆菌。
16.生产腐胺的方法，所述方法包括：
(i)在培养基中培养权利要求6至11中任一项所述的棒杆菌属的微生物；以及
(ii)从上面获得的所述微生物或所述培养基中回收腐胺。
17.生产精氨酸的方法，所述方法包括：
(i)在培养基中培养权利要求12至15中任一项所述的棒杆菌属的微生物；以及
(ii)从上面获得的所述微生物或所述培养基中回收精氨酸。
18.生产基于鸟氨酸的产物的微生物，所述微生物包含权利要求1至3中任一项所述的或具有其增强的活性的多肽。
19.权利要求18所述的微生物，其中选自以下的至少一种的活性与其内源活性相比进一步被增强：乙酰-γ-谷氨酰-磷酸还原酶(ArgC)、乙酰谷氨酸合酶或鸟氨酸乙酰转移酶(argJ)、乙酰谷氨酸激酶(ArgB)、和乙酰鸟氨酸转氨酶(ArgD)。
20.权利要求18所述的微生物，其中所述微生物是谷氨酸棒杆菌。
21.用于生产基于鸟氨酸的产物的方法，所述方法包括：
(i)在培养基中培养权利要求18至20中任一项所述的微生物；以及
(ii)从上面获得的所述微生物或所述培养基中回收基于鸟氨酸的产物。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2017-0074981 | 2017-06-14 | ||
| KR1020170074981A KR101941775B1 (ko) | 2017-06-14 | 2017-06-14 | 신규 폴리펩타이드 및 이를 이용한 오르니틴계 산물 생산방법 |
| PCT/KR2018/006733 WO2018230978A1 (ko) | 2017-06-14 | 2018-06-14 | 신규 폴리펩타이드 및 이를 이용한 오르니틴계 산물 생산방법 |
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| CN111433218A true CN111433218A (zh) | 2020-07-17 |
| CN111433218B CN111433218B (zh) | 2024-05-14 |
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| US (1) | US10870871B2 (zh) |
| EP (1) | EP3640257A4 (zh) |
| JP (2) | JP6936343B2 (zh) |
| KR (1) | KR101941775B1 (zh) |
| CN (1) | CN111433218B (zh) |
| BR (1) | BR112019026884A2 (zh) |
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| KR101999454B1 (ko) * | 2017-12-19 | 2019-07-11 | 씨제이제일제당 (주) | L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌 생산방법 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015132213A1 (de) * | 2014-03-03 | 2015-09-11 | Evocatal Gmbh | Verfahren zur herstellung von endständigen aminocarbonsäuren und aminoaldehyden mittels eines rekombinaten mikroorganismus |
| CN105492593A (zh) * | 2013-03-20 | 2016-04-13 | Cj第一制糖株式会社 | 用于腐胺生产的重组微生物和使用其生产腐胺的方法 |
| CN106459888A (zh) * | 2014-04-25 | 2017-02-22 | Cj第制糖株式会社 | 产生腐胺的变异菌株和使用其的腐胺产生方法 |
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| US7252978B2 (en) | 2001-07-25 | 2007-08-07 | Ajinomoto Co., Inc. | Method for producing L-arginine |
| KR100620092B1 (ko) | 2004-12-16 | 2006-09-08 | 씨제이 주식회사 | 코리네박테리움 속 세포로부터 유래된 신규한 프로모터서열, 그를 포함하는 발현 카세트 및 벡터, 상기 벡터를포함하는 숙주 세포 및 그를 이용하여 유전자를 발현하는방법 |
| KR100791659B1 (ko) | 2006-07-13 | 2008-01-03 | 씨제이 주식회사 | 알지닌을 생산하는 미생물 및 이를 이용한 엘-알지닌의제조방법 |
| KR100930203B1 (ko) | 2008-01-28 | 2009-12-07 | 씨제이제일제당 (주) | 개량된 프로모터 및 이를 이용한 ｌ-라이신의 생산 방법 |
| JP2009254323A (ja) * | 2008-04-21 | 2009-11-05 | Ajinomoto Co Inc | Ｌ−グルタミン酸系アミノ酸の製造法 |
| KR101372635B1 (ko) | 2010-12-08 | 2014-03-13 | 씨제이제일제당 (주) | 오르니틴 생산능이 향상된 미생물 및 이를 이용하여 오르니틴을 생산하는 방법 |
| ES2655764T3 (es) | 2012-01-11 | 2018-02-21 | Cj Cheiljedang Corporation | Microorganismo recombinante que tiene una capacidad mejorada de producción de putrescina y un procedimiento para producir putrescina utilizando el mismo |
| KR101813759B1 (ko) * | 2015-06-24 | 2018-01-02 | 씨제이제일제당 (주) | 퓨트레신 또는 오르니틴 생산 미생물 및 이를 이용한 퓨트레신 또는 오르니틴 생산방법 |
| KR101735935B1 (ko) * | 2015-07-20 | 2017-05-16 | 씨제이제일제당 (주) | 퓨트레신 또는 오르니틴 생산 미생물 및 이를 이용한 퓨트레신 또는 오르니틴 생산방법 |
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| CN105492593A (zh) * | 2013-03-20 | 2016-04-13 | Cj第一制糖株式会社 | 用于腐胺生产的重组微生物和使用其生产腐胺的方法 |
| WO2015132213A1 (de) * | 2014-03-03 | 2015-09-11 | Evocatal Gmbh | Verfahren zur herstellung von endständigen aminocarbonsäuren und aminoaldehyden mittels eines rekombinaten mikroorganismus |
| CN106459888A (zh) * | 2014-04-25 | 2017-02-22 | Cj第制糖株式会社 | 产生腐胺的变异菌株和使用其的腐胺产生方法 |
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| JP2022001049A (ja) | 2022-01-06 |
| JP7308891B2 (ja) | 2023-07-14 |
| BR112019026884A2 (pt) | 2020-07-14 |
| EP3640257A1 (en) | 2020-04-22 |
| EP3640257A4 (en) | 2021-04-21 |
| KR101941775B1 (ko) | 2019-01-24 |
| CN111433218B (zh) | 2024-05-14 |
| JP6936343B2 (ja) | 2021-09-15 |
| JP2020523988A (ja) | 2020-08-13 |
| US20200181661A1 (en) | 2020-06-11 |
| US10870871B2 (en) | 2020-12-22 |
| WO2018230978A1 (ko) | 2018-12-20 |
| KR20180136613A (ko) | 2018-12-26 |
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