CN114008070A - 导致大肠杆菌赖氨酸产量增加的全基因组合理设计的突变 - Google Patents

导致大肠杆菌赖氨酸产量增加的全基因组合理设计的突变 Download PDF

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CN114008070A
CN114008070A CN202080044725.4A CN202080044725A CN114008070A CN 114008070 A CN114008070 A CN 114008070A CN 202080044725 A CN202080044725 A CN 202080044725A CN 114008070 A CN114008070 A CN 114008070A
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理查德·福克斯
丹尼尔·赫尔德
埃里克·阿巴特
迈克尔·克莱
凯瑟琳·克劳斯
南迪尼·克里希纳穆尔蒂
克里希纳·耶拉姆塞提
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Abstract

本公开内容涉及大肠杆菌(E.coli)编码区和非编码区中的各种不同类型的变体，所述变体导致用于例如补充剂和营养制品(nutraceuticals)的赖氨酸产量增加。本公开内容提供了在培养物中产生增加量的赖氨酸的变体大肠杆菌基因和非编码序列，包括变体序列的双重和三重组合。

Description

导致大肠杆菌赖氨酸产量增加的全基因组合理设计的突变
相关申请
本国际PCT申请要求2019年6月21日提交的题为“Genome-Wide Rationally-Designed Mutations Leading to Enhanced Lysine Production in E.Coli”的美国临时申请第62/865,075号的优先权，该临时申请通过引用以其整体并入本文。
发明领域
本公开内容涉及大肠杆菌(E.coli)中导致赖氨酸产量增加的基因突变。
发明背景
在以下讨论中，将出于背景和简介的目的描述某些物品和方法。本文包含的任何内容不被解释为对现有技术的“承认”。本申请人明确地保留根据情况证明根据可适用的法定条文由本文引用的物品和方法不构成现有技术的权利。
氨基酸赖氨酸是一种α-氨基酸，用于蛋白质的生物合成，并且是大肠杆菌、酿酒酵母(S.cerevisiae)、植物、人类和其他哺乳动物以及藻类的代谢物。赖氨酸含有α-氨基基团、α-羧酸基团，并具有C6H14N2O2的化学式。赖氨酸是人体九种必需氨基酸之一，是生长和组织修复所必需的，并且具有作为微量营养素、营养制品(nutraceutical)、农业饲料补充剂、抗惊厥药以及产生肽的前体的作用。由于这些作用，例如作为补充剂和营养制品，已经有越来越多的努力来大规模生产赖氨酸。
因此，在本领域中需要产生增加量的赖氨酸的生物，其中这样的生物可用于大规模赖氨酸生产。本文公开的来自大肠杆菌的核酸序列满足了这一需求。
发明概述
提供本概述是为了以简化的形式介绍概念选择，所述概念在下文的详述中进一步描述。本概述既不意图确定所要求保护的主题的关键或基本特征，也不意图用于限制所要求保护的主题的范围。从以下撰写的详细描述，包括附图中阐明的和所附的权利要求书中限定的那些方面，所要求保护的主题的其他特征、细节、效用和优点将是明显的。
本公开内容提供了在培养物中产生增加量的赖氨酸的变体大肠杆菌基因和非编码序列，包括变体序列的双重和三重组合。因此，在一些实施方案中，本公开内容提供了SEQID No.2-42中的任何一个。
下面更详细地描述本发明的这些方面和其他特征和优点。
附图简述
从以下对说明性实施方案的详细描述，结合附图，将更充分地理解本发明的前述和其他特征和优点，在附图中：
图1A和图1B是大肠杆菌中赖氨酸途径的图解描绘，突出显示了靶向用于合理设计的编辑的途径中的酶。图1B是图1A的延续。
图2列举了对大肠杆菌赖氨酸途径进行的最初200,000个编辑的生物靶、编辑结果、编辑类型和规模。
图3A是用于在大肠杆菌中产生编辑的示例性引擎载体(engine vector)。图3B是用于在大肠杆菌中产生编辑的示例性编辑载体。
应当理解，附图不必按比例绘制，并且相同的附图标记是指相同的特征。
详细描述
除非明确声明或者特征或功能与另外的实施方案不兼容，否则结合本文描述的方法、装置或仪器的一种实施方案描述的所有功能意在适用于本文描述的方法、装置和仪器的另外的实施方案。例如，除非特征或功能与替代实施方案不兼容，否则在结合一种实施方案明确描述给定特征或功能但没有结合替代实施方案明确提及的情况下，应当理解，该特征或功能可以结合替代实施方案来部署、利用或实现。
除非另外指出，本文描述的技术的实践可以采用分子生物学(包含重组技术)、细胞生物学、生物化学和遗传工程技术的常规技术和描述，这些都在本领域的技术人员的技术内。这样的常规技术和描述可以见于标准实验室手册，诸如Green和Sambrook,MolecularCloning:A Laboratory Manual.4th,ed.,Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.,(2014)；Current Protocols in Molecular Biology,Ausubel等人编著,(2017)；Neumann等人,Electroporation and Electrofusion in Cell Biology,Plenum Press,New York,1989；和Chang等人,Guide to Electroporation andElectrofusion,Academic Press,California(1992)，所有文献出于所有目的通过引用以其整体并入本文。
注意，除非上下文另外清楚指示，如本文和所附的权利要求书中使用的，单数形式“一(a)”、“一(an)”和“该(the)”包括复数指代物。因此，例如，提及“细胞”是指一个或更多个细胞，并且提及“该系统”包括提及本领域技术人员已知的等同步骤、方法和装置，等等。
除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域内的普通技术人员通常理解的相同含义。本文提及的所有出版物出于描述和公开可以与本文描述的发明结合使用的装置、制剂和方法的目的通过引用并入。
在提供值的范围的情况下，应理解，在该范围的上限值和下限值之间的每一个中间值和该规定的范围内的任何其他规定的值或中间值被涵盖在本发明内。这些较小的范围的上限值和下限值可以独立地被包括在较小的范围内，并且也被涵盖在本发明内，受限于规定的范围内的任何特定地排除的限值。在规定的范围包括限值中的一个或两个的情况下，将那些所包括的限值中的任一个或两个排除的范围也被包括在本发明中。
在以下的描述中，阐述了许多具体细节，以提供对本发明的更充分理解。然而，对本领域技术人员将明显的是，可以在没有一个或更多个这些具体细节的情况下，实践本发明。在其他情况下，为了避免使本发明含混不清，尚未描述本领域技术人员熟知的特征和程序。本文使用的术语意在具有如由本领域普通技术人员理解的平白且普通的含义。
术语DNA“控制序列”统指启动子序列、多腺苷酸化信号、转录终止序列、上游调控结构域、复制起点、内部核糖体进入位点、核定位序列、增强子等，它们共同地提供编码序列在接受者细胞中的复制、转录和翻译。只要选择的编码序列能够在适当的宿主细胞中被复制、转录和(对于一些组分)翻译，则并非所有这些类型的控制序列都需要存在。
术语“CREATE盒”或“编辑盒”是指与供体DNA或HA连接的gRNA。用于设计和合成CREATE编辑盒的方法和组合物描述于USPN 10,240,167；USPN 10,266,849；USPN 9,982,278；USPN 10,351,877；USPN 10,364,442；USPN 10,435,715和USPN 10,465,207；和2019年8月23日提交的USSN 16/550,092；2019年8月26日提交的USSN 16/551,517；2020年1月27日提交的USSN 16/773,618；和2020年1月27日提交的USSN 16/773,712，所有这些通过引用以其整体并入本文。
如本文使用的，术语“供体DNA”或“供体核酸”是指被设计成通过使用核酸指导的核酸酶的同源重组将DNA序列修饰(插入、缺失、取代)引入基因座(例如，靶基因组DNA序列或细胞靶序列)的核酸。对于同源指导的修复，供体DNA必须与基因组靶序列中的“切割位点”或待编辑位点周围的区具有足够的同源性。一条或更多条同源臂的长度将取决于，例如，所做修饰的类型和大小。在许多情况下，并且优选地，供体DNA将与基因组靶基因座具有两个序列同源性区(例如，两个同源臂)。优选地，“插入物(insert)”区或“DNA序列修饰”区(期望引入细胞中的基因组靶基因座的核酸修饰)将位于两个同源区之间。DNA序列修饰可以改变一个特定位点或多于一个特定位点处的靶基因组DNA序列的一个或更多个碱基。改变可以包括改变基因组靶序列的1个、2个、3个、4个、5个、10个、15个、20个、25个、30个、35个、40个、50个、75个、100个、150个、200个、300个、400个或500个或更多个碱基对。缺失或插入可以是基因组靶序列的1个、2个、3个、4个、5个、10个、15个、20个、25个、30个、40个、50个、75个、100个、150个、200个、300个、400个或500个或更多个碱基对的缺失或插入。
术语“指导核酸”或“指导RNA”或“gRNA”是指包含以下的多核苷酸：1)能够与基因组靶基因座杂交的指导序列和2)能够与核酸指导的核酸酶相互作用或复合的支架序列。
“同源性”或“同一性”或“相似性”是指两个肽之间的序列相似性，或者在本公开内容的上下文中，更常见是指两个核酸分子之间的序列相似性。术语“同源区”或“同源臂”是指供体DNA上与靶基因组DNA序列具有一定程度同源性的区域。同源性可以通过比较每个序列中的位置来确定，所述每个序列可以出于比较的目的而被比对。当比较的序列中的一个位置被相同的碱基或氨基酸占据时，那么分子在该位置处是同源的。序列之间的同源性程度是序列共有的匹配或同源位置数的函数。
“可操作地连接”是指其中如此描述的组分被配置为执行其通常功能的元件的布置。因此，可操作地连接至编码序列的控制序列能够影响编码序列的转录，并且在一些情况下，能够影响编码序列的翻译。只要控制序列发挥指导编码序列的表达的功能，控制序列不必与编码序列邻接。因此，例如，不翻译但转录的间插序列可以存在于启动子序列和编码序列之间，且启动子序列仍可以被认为是“可操作地连接”至编码序列。事实上，这样的序列不必驻留于同一连续DNA分子(即染色体)上，并且仍可以具有引起调控改变的相互作用。
如本文使用的，术语“蛋白”和“多肽”可互换地使用。蛋白可能完全由氨基酸组成，也可能不完全由氨基酸组成。
“启动子”或“启动子序列”是能够与RNA聚合酶结合并启动多核苷酸或多肽编码序列(诸如信使RNA、核糖体RNA、小核RNA(small nuclear RNA)或核仁小RNA(smallnucleolar RNA)、指导RNA或由任何类别的任何RNA聚合酶I、II或III转录的任何种类的RNA)的转录的DNA调控区。启动子可以是组成型的或诱导型的，并且在一些实施方案中，核酸指导的核酸酶编辑系统的至少一种组分的转录是在诱导型启动子的控制下，并且通常核酸指导的核酸酶编辑系统的至少三种组分的转录是在诱导型启动子的控制下。已经开发了用于在植物、微生物和动物细胞(包括哺乳动物细胞)中控制基因表达的许多基因调控控制系统，包括pL启动子(通过CI857阻遏物的热失活诱导)、pPhIF启动子(通过添加2,4二乙酰基间苯三酚(DAPG)诱导)、pBAD启动子(通过将阿拉伯糖添加至细胞生长培养基中诱导)和鼠李糖诱导型启动子(通过将鼠李糖添加至细胞生长培养基中诱导)。其他系统包括四环素控制的转录激活系统(Tet-On/Tet-Off，Clontech,Inc.(Palo Alto,CA)；Bujard和Gossen,PNAS,89(12):5547-5551(1992))、Lac开关诱导型系统(Wyborski等人，Environ MolMutagen,28(4):447-58(1996)；DuCoeur等人，Strategies 5(3):70-72(1992)；美国专利第4,833,080号)、蜕皮素诱导型基因表达系统(No等人，PNAS,93(8):3346-3351(1996))、cumate基因开关系统(Mullick等人，BMC Biotechnology,6:43(2006))、以及他莫昔芬诱导型基因表达(Zhang等人，Nucleic Acids Research,24:543-548(1996))以及其他。
如本文使用的术语“可选择标记(selectable marker)”是指引入细胞中的、赋予适于人工选择的性状的基因。一般使用的可选择标记为本领域普通技术人员熟知的。可以采用可药物选择的标记，诸如氨苄青霉素/羧苄青霉素、卡那霉素、诺尔丝菌素、N-乙酰基转移酶、氯霉素、红霉素、四环素、庆大霉素、博莱霉素、链霉素、利福平、嘌呤霉素、潮霉素、杀稻瘟素和G418。在其他实施方案中，可选择标记包括但不限于糖类，诸如鼠李糖。如本文使用的“选择性培养基”是指向其中添加了选择可选择标记或针对可选择标记进行选择的化学化合物或生物部分的细胞生长培养基。
如本文使用的术语“特异性结合”包括两种分子例如工程化肽抗原和结合靶之间具有由以下解离常数表示的结合亲和力的相互作用：约10-7M、约10-8M、约10-9M、约10-10M、约10-11M、约10-12M、约10-13M、约10-14M或约10-15M。
术语“靶基因组DNA序列”、“细胞靶序列”或“基因组靶基因座”是指体外或体内，或者细胞或细胞群体的核酸(例如基因组)中的期望使用核酸指导的核酸酶编辑系统对至少一个核苷酸进行改变的任何基因座。细胞靶序列可以是基因组基因座或染色体外基因座。
术语“变体”可以指与参考多肽或多核苷酸不同但保留基本特性的多肽或多核苷酸。多肽的典型变体的氨基酸序列与另一参考多肽有差异。通常，差异是有限的，使得参考多肽和变体的序列总体上非常相似，并且在许多区域是相同的。变体和参考多肽的氨基酸序列可以相差一个或更多个修饰(例如，取代、添加和/或缺失)。多肽的变体可以是保守修饰的变体。取代的或插入的氨基酸残基可以是或可以不是由遗传密码编码的氨基酸残基(例如，非天然氨基酸)。多肽的变体可以是天然存在的，诸如等位基因变体，或者它可以是未知的天然存在的变体。
“载体”是包含待递送至细胞和/或在细胞中表达的期望的一种序列或更多种序列的多种核酸中的任一种。载体通常由DNA构成，但是RNA载体也是可用的。载体包括但不限于质粒、F粘粒(fosmid)、噬菌粒、病毒基因组、合成染色体等。如本文使用的，短语“引擎载体”包含用于本公开内容的核酸指导的核酸酶系统和方法中的核酸酶的编码序列。引擎载体还包含在大肠杆菌中的λ Red重组工程系统或其等同物，其修复由核酸酶切割产生的双链断裂。引擎载体通常还包含可选择标记。如本文使用的，短语“编辑载体”包含供体核酸和gRNA编码序列，所述供体核酸任选地包括对细胞靶序列的改变，所述改变在编辑发生后阻止核酸酶在细胞靶序列中的PAM或间隔区(spacer)处结合。编辑载体还可以并且优选地的确包括可选择标记和/或条形码。在一些实施方案中，可以将引擎载体和编辑载体组合；即，所有编辑和选择元件都可以在单个载体上找到。此外，引擎载体和编辑载体包含可操作地连接至例如核酸酶编码序列、重组工程系统编码序列(如果存在)、供体核酸、指导核酸和一种或更多种可选择标记的控制序列。
文库设计策略和核酸酶指导的基因组编辑
赖氨酸在大肠杆菌中沿着二氨基庚二酸(DAP)生物合成途径天然合成。参见，例如，图1。在历史上，用于增加大肠杆菌和其他工业相关生产宿主诸如谷氨酸棒状杆菌(Corynebacterium glutamicum)中赖氨酸产量的菌株工程化策略集中于DAP途径中的基因，作为诱变和过表达的明显目标。除了这个编码赖氨酸生物合成酶的基因的简短列表之外，很可能整个大肠杆菌基因组中的另外基因座也可能对工业生产环境中赖氨酸产量的提高做出可观的贡献(如果不那么直接的话)。因此，能够更广泛地查询整个基因组的靶向诱变策略对赖氨酸代谢工程师也具有重要价值。
本公开内容中呈现的变体是在含有诸如图3A所示的引擎质粒的野生型大肠杆菌菌株(这种转化的MG1655菌株在本文中称为大肠杆菌菌株EC83)的基因组各处的特定基因座处200,000个独特和精确设计的核酸指导的核酸酶编辑，并使用所得赖氨酸产生水平在MG1655的两个工程化菌株中进行另外的核酸指导的核酸酶编辑以产生双变体和三变体工程化菌株的结果。第一个工程化菌株是具有包含dapA E84T(SEQ ID No.1)的单突变的菌株MG1655，其赖氨酸产量是MG1655野生型赖氨酸产量的大约500倍。第二个工程化菌株是具有包含dapA E84T(SEQ ID No.1)和dapA J23100(大肠杆菌dapA启动子中的突变，SEQ IDNO.2)的双突变的菌株MG1655，其赖氨酸产量是野生型赖氨酸产量的大约10,000倍。关于用于生成变体的200,000个编辑载体中包括的编辑类型的总结参见例如图2。引擎质粒包含在诱导型pL启动子控制下的MAD7核酸酶的编码序列、在诱导型pBAD启动子控制下的λRed操纵子重组工程系统(可通过在细胞生长培养基中添加阿拉伯糖诱导)、在组成型启动子控制下的c1857基因、以及可选择标记和复制起点。如上文描述的，λRed重组工程系统修复了由MAD7核酸酶切割产生的双链断裂。c1857基因在30℃主动阻遏pL启动子(其驱动MAD7核酸酶和编辑载体(诸如图3B所示的示例性编辑载体)上的编辑盒或CREATE盒的表达)；然而，在42℃时，c1857阻遏物基因解折叠(unfold)或降解，并且在这种状态，c1857阻遏物蛋白不再能抑制pL启动子，导致MAD7核酸酶的编码序列和编辑(例如，CREATE)盒的活性转录。
图3B描绘了示例性编辑质粒，其包含由pL启动子驱动的编辑(例如，CREATE)盒(crRNA、间隔区和HA)、可选择标记和复制起点。
特异性靶向DAP途径中的基因以及一些其酶转化的产物供应进入DAP途径的基因的诱变文库被设计为饱和诱变。此外，为了更深入地探索基因组的其余部分以寻找参与赖氨酸生物合成的新靶，文库被设计成靶向所有注释的基因座，这些基因座具有过早终止密码子(对于敲除表型)或插入一组五种合成启动子变体(对于表达调节表型)。
本文描述的200,000个核酸突变或编辑使用MAD7以及gRNA和供体DNA产生。核酸指导的核酸酶诸如MAD7与适当的合成的指导核酸在细胞中复合并可以在期望的位置处切割细胞的基因组。指导核酸有助于核酸指导的核酸酶识别和切割特定靶序列处的DNA。通过操纵指导核酸的核苷酸序列，核酸指导的核酸酶可以被编程为靶向任何用于裂解的DNA序列，只要适当的前间区邻近基序(protospacer adjacent motif,PAM)在附近。在某些方面，核酸指导的核酸酶编辑系统可以使用组合起来发挥作为指导核酸的功能的两个分开的指导核酸分子，例如CRISPR RNA(crRNA)和反式激活CRISPR RNA(tracrRNA)。在其他方面，指导核酸可以是包括crRNA序列和tracrRNA序列二者的单个指导核酸。
同样，单个变体产生的赖氨酸产生水平用于在MG1655的两个工程化菌株中进行另外的核酸指导的核酸酶编辑，以产生双变体和三变体工程化菌株。第一个工程化菌株是具有包含dapA E84T(SEQ ID No.1)的单突变的菌株MG1655，其赖氨酸产量是MG1655野生型赖氨酸产量的大约500倍。第二个工程化菌株是具有包含dapA E84T(SEQ ID No.1)和dapAJ23100(大肠杆菌dapA启动子中的突变，SEQ ID NO.2)的双突变的菌株MG1655，其赖氨酸产量是野生型赖氨酸产量的大约10,000倍。
指导核酸包含指导序列，其中指导序列是与靶序列具有足够互补性以与靶序列杂交并且指导复合的核酸指导的核酸酶与靶序列的序列特异性结合的多核苷酸序列。指导序列和对应的靶序列之间的互补性程度在使用合适的比对算法进行最佳比对时是约或多于约50％、60％、75％、80％、85％、90％、95％、97.5％、99％或更多。最佳比对可以通过使用用于序列比对的任何合适的算法来确定。在一些实施方案中，指导序列的长度是约或多于约10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、35个、40个、45个、50个、75个或更多个核苷酸。在一些实施方案中，指导序列的长度为少于约75个、50个、45个、40个、35个、30个、25个、20个核苷酸。优选地，指导序列是10-30个或15-20个核苷酸长，或者长度是15个、16个、17个、18个、19个或20个核苷酸。
在产生200,000个成员文库的方法中，指导核酸作为待由质粒或载体表达的序列提供，该质粒或载体包含指导序列和支架序列二者作为诱导型启动子控制下的单一转录物。通过以下将指导核酸工程化成靶向期望的靶序列：改变指导序列，使得指导序列与期望的靶序列互补，从而允许指导序列和靶序列之间的杂交。通常，为了在靶序列中产生编辑，gRNA/核酸酶复合体与如由指导RNA确定的靶序列结合，并且核酸酶识别与靶序列邻近的前间区邻近基序(PAM)序列。这里全基因组诱变的靶序列包括在整个大肠杆菌基因组中基因组各处的特定基因座处的200,000个独特和精确的设计。
指导核酸可以是也编码供体核酸的编辑盒的一部分，并且在产生本文报告的变体的方法中是也编码供体核酸的编辑盒的一部分。靶序列与前间区突变(PAM)相关，前间区突变(PAM)是由gRNA/核酸酶复合体识别的短核苷酸序列。用于不同的核酸指导的核酸酶的精确优选的PAM序列和长度要求不同；然而，PAM通常是与靶序列邻近或接近的2-7个碱基对序列，并且取决于核酸酶，可以是靶序列的5’或3’。
在某些实施方案中，细胞靶序列的基因组编辑既将期望的DNA改变引入细胞靶序列，又去除细胞靶序列中的前间区突变(PAM)区，使细胞靶序列中的前间区突变(PAM)区突变或失活。使细胞靶序列处的PAM失活排除了对该细胞靶序列处细胞基因组的另外的编辑，例如，在后续的编辑轮中随后暴露于与合成的指导核酸复合的核酸指导的核酸酶时。因此，具有期望的细胞靶序列编辑和改变的PAM的细胞可以通过使用与合成的指导核酸复合的核酸指导的核酸酶来选择，所述合成的指导核酸与细胞靶序列互补。没有经历第一编辑事件的细胞会被切割，引起双链DNA断裂，并且因此会无法继续存活。包含期望的细胞靶序列编辑和PAM改变的细胞不会被切割，因为这些编辑的细胞不再包含必需的PAM位点，并且会继续生长和繁殖。
至于核酸指导的核酸酶编辑系统的核酸酶组分，编码核酸指导的核酸酶的多核苷酸序列可以被密码子优化以在特定细胞类型，诸如古细菌、原核细胞或真核细胞中表达。待采用的核酸指导的核酸酶的选择取决于许多因素，诸如在靶序列中待进行何种类型的编辑，以及适当的PAM是否位于期望的靶序列附近。本文描述的方法中使用的核酸酶包括但不限于Cas 9、Cas 12/CpfI、MAD2或MAD7或其他MAD酶(MADzyme)。与指导核酸一样，核酸酶由载体(例如，引擎载体—参见图3A)上的DNA序列编码，并且处于诱导型启动子的控制下。在一些实施方案中，诸如在本文描述的方法中，诱导型启动子可以与控制指导核酸转录的诱导型启动子分开但相同；即，分开的诱导型启动子驱动核酸酶和指导核酸序列的转录，但是这两个诱导型启动子可以是相同类型的诱导型启动子(例如，二者都是pL启动子)。可选地，控制核酸酶表达的诱导型启动子可以与控制指导核酸转录的诱导型启动子不同；即，例如，核酸酶可以处于pBAD诱导型启动子的控制下，并且指导核酸可以处于pL诱导型启动子的控制下。
核酸指导的核酸酶系统的另一个组分是包含与细胞靶序列的同源性的供体核酸。在一些实施方案中，供体核酸与指导核酸在同一多核苷酸(例如，编辑载体或编辑盒)上。供体核酸被设计成用作与细胞靶序列同源重组的模板，该细胞靶序列被作为gRNA/核酸酶复合体的一部分的核酸指导的核酸酶切口或裂解。供体核酸多核苷酸可以具有任何合适的长度，诸如约或多于约20个、25个、50个、75个、100个、150个、200个、500个或1000个核苷酸的长度。在某些优选的方面，供体核酸可以以20-300个核苷酸之间，更优选地50-250个核苷酸之间的寡核苷酸提供。供体核酸包含与细胞靶序列的一部分互补的区域(例如同源臂)。当最佳比对时，供体核酸与细胞靶序列重叠(互补)例如约20个、25个、30个、35个、40个、50个、60个、70个、80个、90个或更多个核苷酸。在许多实施方案中，供体核酸包含位于供体核酸和细胞靶序列之间的突变或差异的侧翼的两个同源臂(与细胞靶序列互补的区域)。供体核酸包含与细胞靶序列相比的至少一个突变或改变，诸如与细胞靶序列相比的插入、缺失、修饰或其任何组合。本文介绍了各种类型的编辑，包括定点诱变、饱和诱变、启动子交换和阶梯(promoter swaps and ladders)、敲入和敲除编辑、SNP或短串联重复序列交换以及起始/终止密码子交换。
除了供体核酸之外，编辑盒可以包含一个或更多个引物位点。引物位点可以用于通过使用寡核苷酸引物扩增编辑盒；例如，如果引物位点位于编辑盒的一个或更多个其他组分的侧翼。此外，编辑盒可以包含条形码。条形码是对应于供体DNA序列的独特DNA序列，使得条形码可以鉴定对对应细胞靶序列进行的编辑。条形码通常包含四个或更多个核苷酸。在一些实施方案中，编辑盒包含代表例如gRNA和供体核酸的全基因或全基因组文库的gRNA和供体核酸的集合或文库。编辑盒的文库被克隆到载体骨架中，其中，例如，每个不同的供体核酸与不同的条形码缔合。
感兴趣的变体包括下表1中列出的变体：
表1：变体
Figure BDA0003417731110000131
Figure BDA0003417731110000141
在表中，*表示氨基酸序列(例如，蛋白质编码区的变化)，**表示核酸序列(例如，蛋白质启动子区或其他非编码区的变化)，“NCBI-基因ID”是NCBI登录号，“表型FOWT”是超过基本培养基中的野生型(MG1655)的倍数；“表型FIOPC”是超过阳性对照(具有E84T单变体的MG1655)的提高倍数。J231XX是给定基因座处的启动子交换，并且****表示来自全基因组敲除文库的击中(hits)，其中在基因座中的给定位置处插入了三重终止(triple-stop)。注意到，对于所有三重编辑(SEQ ID No.3-8)，超过野生型的倍数等于或大于13,000倍，并且超过野生型的倍数在双重突变体dapA J21300+purM J23100(SEQ ID No.25)中高达1600倍。
实施例
特异性靶向DAP途径中的基因以及一些其酶的转化产物供应进入DAP途径的基因的诱变文库被设计为饱和诱变。此外，为了更深入地探索大肠杆菌基因组的其余部分以寻找参与赖氨酸生物合成的新靶，文库被设计成靶向所有注释的基因座，这些基因座具有过早终止密码子(对于敲除表型)或插入一组五种合成启动子变体(对于表达调节表型)。然后，单个变体产生的赖氨酸产生水平用于在MG1655的两个工程化菌株中进行另外的核酸指导的核酸酶编辑，以产生双变体和三变体工程化菌株。第一个工程化菌株是具有包含dapAE84T(SEQ ID No.1)的单突变的菌株MG1655，其赖氨酸产量是MG1655野生型赖氨酸产量的大约500倍。第二个工程化菌株是具有包含dapA E84T(SEQ ID No.1)和dapA J23100(大肠杆菌dapA启动子中的突变，SEQ ID NO.2)的双突变的菌株MG1655，其赖氨酸产量是野生型赖氨酸产量的大约10,000倍。如下文描述的，经由质谱以浅层取样(shallow sampling)筛选所有文库的赖氨酸产生。
编辑盒和骨架扩增与组装
编辑盒制备：在50μL体积中使用Q5聚合酶扩增芯片上合成的5nM寡核苷酸。PCR反应为95℃1分钟；8轮的95℃30秒/60℃30秒/72℃2.5分钟；最后在72℃保持5分钟。扩增后，PCR产物经历SPRI清理，其中将30μL SPRI混合物添加到50μL PCR反应中并孵育2分钟。将管置于磁场2分钟，去除液体，并且用80％乙醇洗涤珠2x，允许洗涤之间间隔1分钟。最后一次洗涤后，允许珠干燥2分钟，将50μL 0.5x TE pH 8.0添加到管中，并且然后将珠涡旋以混合。使浆料在室温孵育2分钟，然后置于磁场2分钟。取出洗脱液，并且对DNA进行定量。
在定量之后，使用来自SPRI清理的洗脱液的稀释液进行第二次扩增程序。PCR在以下条件下进行：95℃1分钟；18轮的95℃30秒/72℃2.5分钟；最后在72℃保持5分钟。在2％琼脂糖凝胶上检查扩增子，并且鉴定出具有一个或更多个最干净输出的池。不使用看起来具有异二聚体或嵌合体的扩增产物。
骨架制备：对纯化的骨架进行一系列的10倍系列稀释，并且稀释的骨架系列中的每一个在以下条件下扩增：95℃1分钟；然后30轮的95℃30秒/60℃1.5分钟/72℃2.5分钟；最后在72℃保持5分钟。扩增后，扩增的骨架经历SPRI清理，如上文关于盒描述的。将骨架洗脱到100μL的ddH2O中并定量，然后等温组装。
等温核酸组装：150ng骨架DNA与100ng盒DNA组合。添加等体积的2x Gibson主混合物，并使反应在50℃孵育45分钟。组装后，组装的骨架和盒经历SPRI清理，如上文描述的。
编辑载体文库向E
Figure BDA0003417731110000161
中的转化
转化：将20μL的制备的编辑载体Gibson组装反应与10μL E
Figure BDA0003417731110000162
(Lucigen,Middleton,WI)最高感受态细胞一起加入30μL冷水(chilled water)中。将转化的细胞的等分试样进行布点铺板(spot plated)以检查转化效率，其中需要>100x的覆盖率才能继续。将转化的E
Figure BDA0003417731110000163
细胞在25mL SOB+100μg/mL羧苄青霉素(carb)中生长过度(outgrown)。通过向1000μL饱和过夜培养物加入500μL 50％甘油，由饱和培养物产生甘油储备物。将储备物在-80℃冷冻。在有克隆的编辑文库的现成储备物的情况下，该步骤是任选的。可选地，可以在每次编辑实验之前进行编辑盒和载体骨架的Gibson组装或另一种组装。
用引擎载体转化的新细胞系的产生：
转化：将1μL引擎载体DNA(包含在pL诱导型启动子控制下的MAD7核酸酶的编码序列、氯霉素抗性基因和λRed重组工程系统)添加到50μL EC83菌株大肠杆菌细胞中。将转化的细胞铺板在具有25μg/mL氯霉素(chlor)的LB板上，并孵育过夜以积累克隆分离株。第二天，挑选一个菌落，在LB+25μg/mL chlor中过夜生长，并通过向1000μL培养物加入500μL50％甘油，由饱和过夜培养物制备甘油储备物。将包含引擎载体的EC1的储备物冷冻在-80℃。
感受态细胞的制备：
向含有100mL LB/SOB+25μg/mL chlor培养基的250mL烧瓶中加入用引擎载体转化的EC83细胞的新鲜生长过夜培养物的1mL等分试样。使细胞生长至0.4-0.7OD，并通过将培养物转移至冰上10分钟停止细胞生长。使细胞在JA-18转子中以8000x g沉淀5分钟，用50mL冰冷的ddH20或10％甘油洗涤3x，并在JA-18转子中以8000x g沉淀5分钟。将洗涤的细胞重悬于5mL冰冷的10％甘油中，并等分至200μL份。任选地，此时甘油储备物可以储存在-80℃以备后用。
用于赖氨酸生产的编辑文库的筛选：
将文库储备物稀释并用无菌玻璃珠铺板在含有100μg/mL羧苄青霉素(Teknova)和25μg/mL氯霉素(Teknova)的245x245mm LB琼脂板(Teknova)上。将文库适量稀释，以在板上产生～2000-3000个菌落。将板在30℃孵育～16小时，并且然后在4℃储存直至使用。使用QPixTM 420(Molecular Devices)挑取菌落，并将其沉积于无菌的1.2mL含有300μg过夜生长培养基(EZ Rich Defined Medium无赖氨酸(Teknova)、100μg/mL羧苄青霉素和25μg/mL氯霉素)的方形96孔板(Thomas Scientific)中。将板密封(AirPore片(sheets)(Qiagen))并在振荡培养箱(Climo-Shaker ISF1-X(Kuhner)，30℃，85％湿度，250rpm)中孵育～19h。然后将板培养物稀释20倍(在285μL培养基中加入15μL培养物)到含有赖氨酸产生培养基(20g/L硫酸铵(Teknova)、200mM MOPS缓冲液(Teknova)、3mg/L七水硫酸铁(II)(Sigma)、3mg/L一水硫酸锰(II)(Sigma)、0.5mg/L生物素(Sigma)、1mg/L盐酸硫胺素(Sigma)、0.7g/L氯化钾(Teknova)、20g/L葡萄糖(Teknova)、5g/L磷酸二氢钾(Sigma)、1mL/L微量金属混合物(Teknova)、1mM硫酸镁(Teknova)、100μg/mL羧苄青霉素和25μg/mL氯霉素)的新的96孔板中。将产生板在振荡培养箱(Climo-Shaker ISF1-X(Kuhner)，30℃，85％湿度，250rpm)中孵育24h。
将产生板以3,000g离心10分钟(离心机5920R,Eppendorf)以使细胞沉淀。将来自产生板的上清液稀释100倍到1.2mL方形96孔板中的水中(5μL上清液加495μL水)。将样品充分混合，并且然后进一步稀释10倍至乙腈和水的50:50混合物(20μL样品和180μL乙腈/水混合物)到96孔板(聚丙烯，335μL/孔，锥形底部)(Thomas Scientific)中。将板热密封并充分混合。
赖氨酸浓度使用与6470三重四级杆质谱仪(Triple Quad mass spectrometer)(Agilent)耦合的RapidFire高通量质谱系统(Agilent)来确定。RapidFire条件如下：泵1：80％乙腈(LC/MS级，Fisher)，20％水(LC/MS级，Fisher)，1.5mL/min，泵2：100％水，1.25mL/min，泵3：5％乙腈，95％水，1.25mL/min。RapidFire方法：抽吸：600ms，装载/清洗：2000ms，额外清洗：0ms，洗脱：3000ms，再平衡：500ms。10μL进样环(injection loop)。
用于赖氨酸检测的质谱条件：
前体离子：147.1m/z，产物离子(定量)：84m/z,驻留(Dwell)：20，碎裂电压(Fragmentor)：80，碰撞能量：20，碰撞池加速电压：4，极性：正
前体离子：147.1m/z，产物离子(定质)：130m/z,驻留：20，碎裂电压：80，碰撞能量：8，碰撞池加速电压：4，极性：正
源条件：气体温度：300℃，气流：10L/min，雾化器：45psi，鞘气温度：350℃，鞘气流：11L/min，毛细管电压：3000V(正)，喷嘴电压：1500V(正)
使用MassHunter定量分析软件(Agilent)分析数据，其中使用赖氨酸标准曲线定量样品中的赖氨酸。每个96孔板的样品包含野生型菌株的4个重复和dapA E84T阳性对照菌株的4个重复，以计算与对照相比的样品的相对赖氨酸产量。使用与上文描述的类似的方案，对来自起始筛选的击中以一式四份进行再测试。
虽然本发明通过许多不同形式的实施方案来满足，如结合本发明的优选实施方案详细描述的，但是应理解本公开内容应被认为是对本发明的原理的示例而不意在将本发明局限于本文说明和描述的具体实施方案。本领域技术人员可以作出不脱离本发明的精神的许多变化。本发明的范围将由所附权利要求书及其等同物判断。摘要和标题不应被解释为限制本发明的范围，因为它们的目的是使适当的机构以及一般公众能够迅速确定本发明的一般性质。在以下的权利要求书中，除非使用术语“手段(means)”，否则其中列举的特征或要素都不应被解释为根据35U.S.C.§112,
Figure BDA0003417731110000191
6的手段加功能的限定。
序列表
<110> 因思科瑞普特公司
<120> 导致大肠杆菌赖氨酸产量增加的全基因组合理设计的突变
<130> INSC046PCT
<140> PCT/US20/38345
<141> 2020-06-18
<150> 62/865,075
<151> 2019-06-21
<160> 42
<170> PatentIn version 3.5
<210> 1
<211> 292
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 1
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Ala Thr Leu Asn His Asp Glu His Ala Asp Val Val Met Met Thr Leu
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Asp Leu Ala Asp Gly Arg Ile Pro Val Ile Ala Gly Thr Gly Ala Asn
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Ala Thr Ala Thr Ala Ile Ser Leu Thr Gln Arg Phe Asn Asp Ser Gly
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Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Tyr Asn Arg Pro Ser Gln
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Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu His Thr Asp Leu
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Pro Gln Ile Leu Tyr Asn Val Pro Ser Arg Thr Gly Cys Asp Leu Leu
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Pro Glu Thr Val Gly Arg Leu Ala Lys Val Lys Asn Ile Ile Gly Ile
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Lys Glu Ala Thr Gly Asn Leu Thr Arg Val Asn Gln Ile Lys Glu Leu
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Val Ser Asp Asp Phe Val Leu Leu Ser Gly Asp Asp Ala Ser Ala Leu
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Ala Gly Leu Leu
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<210> 2
<211> 121
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 2
ttgcttttaa tgccatacca aacgtaccat tgagacactt gtttgcacag aggatggccc 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
a 121
<210> 3
<211> 449
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 3
Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp
1 5 10 15
Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn
20 25 30
Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu
35 40 45
Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu
50 55 60
Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr
65 70 75 80
Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr
85 90 95
Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp
100 105 110
Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu
115 120 125
Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys
130 135 140
Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala
145 150 155 160
Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu
165 170 175
Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg
180 185 190
Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu
195 200 205
Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro
210 215 220
Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile
225 230 235 240
Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala
245 250 255
Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile
260 265 270
Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu
275 280 285
Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu
290 295 300
Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His
305 310 315 320
Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn
325 330 335
Ile Ser Pro Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr
340 345 350
Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln
355 360 365
Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu
370 375 380
Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys
385 390 395 400
Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg
405 410 415
Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro
420 425 430
Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe
435 440 445
Glu
<210> 4
<211> 1693
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 4
ttatttttaa atgagaccag gtcctcattt taataacccc tggctggaga atattgcaca 60
tttacagcta gctcagtcct aggtattatg ctagctaagt aggtacgtaa ggaggtgata 120
aatggccaac atcgaaatca gacaagaaac gccaactgcg ttttatataa aagttcacga 180
cacagataat gtggcaatta ttgttaatga taatggcctg aaagcaggaa cgcgttttcc 240
ggatgggctg gaattaattg aacatattcc ccaggggcat aaagtcgcat tgctggacat 300
tccggctaat ggtgaaatta ttcgttatgg cgaagtgatt ggttacgccg tgcgtgcaat 360
cccacgcgga agctggatcg acgaatcaat ggttgtacta ccggaagcgc cgccgttaca 420
cacgctgcca ctggcaacca aagtcccgga acccttaccg ccgctggaag gatacacctt 480
tgagggctat cgcaatgccg atggcagcgt gggcaccaaa aacctgctcg gtatcaccac 540
cagcgtccac tgtgtggcag gcgtggtgga ctatgtagta aaaatcattg aacgcgatct 600
gctaccgaaa tacccgaacg tcgatggcgt ggtggggctg aatcatttgt acggttgtgg 660
cgtggcgatt aacgcaccgg cggcagttgt acctatccgt accattcaca atatttcgct 720
gaatcctaac tttggcggcg aagtaatggt gattggcctg ggttgtgaaa agttgcagcc 780
tgagcgcctg ctgactggaa cggatgatgt gcaagctatt ccagtagaaa gcgccagcat 840
tgtcagtttg caggatgaaa agcatgtcgg ttttcagtcc atggtcgagg atattttgca 900
gatcgccgaa cgccatctac aaaaactgaa tcaacggcag cgagaaacct gcccggcttc 960
agaactggtc gttggtatgc agtgcggtgg cagcgatgcg ttttctggtg taacggcaaa 1020
cccggcggtt ggctatgcgt ctgatctact ggtgcgctgc ggcgcaacgg tgatgttttc 1080
agaagtaacg gaagtgcgtg acgcgatcca tctgctgaca ccacgcgcag tgaacgaaga 1140
ggtcggcaaa cggctgctgg aggagatgga gtggtacgat aactatctca atatgggaaa 1200
aaccgaccgc agcgccaacc cttcgccggg caacaagaaa ggcggtctgg caaacgtggt 1260
agagaaggca ctcggctcca ttgctaaatc gggtaaaagc gcaattgttg aagtgctgtc 1320
gcccggtcaa cgcccgacta aacgcggatt aatttacgcc gcgacgccag ccagcgattt 1380
tgtctgtggc acgcaacagg tggcttcggg tatcacagtg caagtgttta cgaccggtcg 1440
tggtacgccg tacggcctga tggcggtacc cgtcattaaa atggcaaccc gcaccgagct 1500
ggcgaaccgc tggtttgatt taatggatat taatgcgggc accatcgcta ccggcgaaga 1560
aactattgaa gaggtgggct ggaagttgtt ccactttatt ctcgacgtcg ccagcgggaa 1620
gaagaaaacc ttctcggatc aatgggggct gcataaccag ctggcggtgt ttaacccggc 1680
accggtgacc tga 1693
<210> 5
<211> 1045
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 5
tcgccatttt tgctatcatg cctgcataca taaacgacaa aacagtatgc agagggaaaa 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgggttcc accagaaagg ggatgctgaa cgttctgatt gccgccgtgt tgtggggaag 180
ttcaggggtc tgcgcgcaat acatcatgga gcaaagccag atgtcgtcgc agtttttgac 240
tatgacgcgt ttgatattcg ccggtttgat tctactgacg ctgtcatttg ttcatggcga 300
taaaatcttt tctattatta acaatcataa agatgccatt agcctgctga ttttttccgt 360
ggttggcgcg ctaactgtac agctcacttt tttgctaacc atcgaaaaat cgaacgcagc 420
cacggcaacg gtgctgcaat tcctctcacc gacgattatc gtcgcctggt tctcactggt 480
gcgtaaatcg cgcccgggca ttctggtttt ctgcgctatt ttgacatcgc tggtcgggac 540
ttttttattg gtgacacacg gtaatccgac gtcattatcg atctctcctg ccgcgttgtt 600
ctggggcatt gcctcggcat ttgctgctgc attctatacc acctatccct caacgctaat 660
tgcccgctat ggcacgttac cagtcgtcgg ctggagtatg ctgattggcg gtctgattct 720
gttgcctttt tatgccagac aaggaacaaa ctttgtcgtt aacggcagtt tgattctggc 780
gtttttttat ttggtggtca ttggtacgtc cctgacattt agtctgtacc tgaaaggagc 840
acaattaatt ggcggtccaa aagccagcat tttgagctgt gcagaaccat taagtagcgc 900
gctactctct ttgctgttgc tggggatcac gttcacatta ccggactggc tgggaacgct 960
gctgattctg tcatcggtga ttttgatttc aatggattcc cgtcgccgcg ccagaaaaat 1020
aaatcgtccg gcgcggcata agtga 1045
<210> 6
<211> 14
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 6
Met Val Ser Glu Thr Lys Thr Thr Glu Ala Pro Gly Leu Arg
1 5 10
<210> 7
<211> 580
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 7
tatcttgcag cgataagtgc ttacagtaat ctgtaggaaa gttaactacg gatgtacatt 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatggaactg acgactcgca ctttacctgc gcggaaacat attgcgctgg tggcacacga 180
tcactgcaaa caaatgctga tgagctgggt ggaacggcat caaccgttac tggaacaaca 240
cgtactgtat gcaacaggca ctaccggtaa cttaatttcc cgcgcgaccg gcatgaacgt 300
caacgcgatg ttgagtggcc caatgggggg tgaccagcag gttggcgcat tgatctcaga 360
agggaaaatt gatgtattga ttttcttctg ggatccacta aatgccgtgc cgcacgatcc 420
tgacgtgaaa gccttgctgc gtctggcgac ggtatggaac attccggtcg ccaccaacgt 480
ggcaacggca gacttcataa tccagtcgcc gcatttcaac gacgcggtcg atattctgat 540
ccccgattat cagcgttatc tcgcggaccg tctgaagtaa 580
<210> 8
<211> 540
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 8
Met Arg Val Asn Asn Gly Leu Thr Pro Gln Glu Leu Glu Ala Tyr Gly
1 5 10 15
Ile Ser Asp Val His Asp Ile Val Tyr Asn Pro Ser Tyr Asp Leu Leu
20 25 30
Tyr Gln Glu Glu Leu Asp Pro Ser Leu Thr Gly Tyr Glu Arg Gly Val
35 40 45
Leu Thr Asn Leu Gly Ala Val Ala Val Asp Thr Gly Ile Phe Thr Gly
50 55 60
Arg Ser Pro Lys Asp Lys Tyr Ile Val Arg Asp Asp Thr Thr Arg Asp
65 70 75 80
Thr Phe Trp Trp Ala Asp Lys Gly Lys Gly Lys Asn Asp Asn Lys Pro
85 90 95
Leu Ser Pro Gln Thr Trp Gln His Leu Lys Gly Leu Val Thr Arg Gln
100 105 110
Leu Ser Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Asn
115 120 125
Pro Asp Thr Arg Leu Ser Val Arg Phe Ile Thr Glu Val Ala Trp Gln
130 135 140
Ala His Phe Val Lys Asn Met Phe Ile Arg Pro Ser Asp Glu Glu Leu
145 150 155 160
Ala Gly Phe Lys Pro Asp Phe Ile Val Met Asn Gly Ala Lys Cys Thr
165 170 175
Asn Pro Gln Trp Lys Glu Gln Gly Leu Asn Ser Glu Asn Phe Val Ala
180 185 190
Phe Asn Leu Thr Glu Arg Met Gln Leu Ile Gly Gly Thr Trp Tyr Gly
195 200 205
Gly Glu Met Lys Lys Gly Met Phe Ser Met Met Asn Tyr Leu Leu Pro
210 215 220
Leu Lys Gly Ile Ala Ser Met His Cys Ser Ala Asn Val Gly Glu Lys
225 230 235 240
Gly Asp Val Ala Val Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr
245 250 255
Leu Ser Thr Asp Pro Lys Arg Arg Leu Ile Gly Asp Asp Glu His Gly
260 265 270
Trp Asp Asp Asp Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys
275 280 285
Thr Ile Lys Leu Ser Lys Glu Ala Glu Pro Glu Ile Tyr Asn Ala Ile
290 295 300
Arg Arg Asp Ala Leu Leu Glu Asn Val Thr Val Arg Glu Asp Gly Thr
305 310 315 320
Ile Asp Phe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr
325 330 335
Pro Ile Tyr His Ile Asp Asn Ile Val Lys Pro Val Ser Lys Ala Gly
340 345 350
His Ala Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe Gly Val Leu
355 360 365
Pro Pro Val Ser Arg Leu Thr Ala Asp Gln Thr Gln Tyr His Phe Leu
370 375 380
Ser Gly Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Ile Thr Glu
385 390 395 400
Pro Thr Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu
405 410 415
His Pro Thr Gln Tyr Ala Glu Val Leu Val Lys Arg Met Gln Ala Ala
420 425 430
Gly Ala Gln Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys
435 440 445
Arg Ile Ser Ile Lys Asp Thr Arg Ala Ile Ile Asp Ala Ile Leu Asn
450 455 460
Gly Ser Leu Asp Asn Ala Glu Thr Phe Thr Leu Pro Met Phe Asn Leu
465 470 475 480
Ala Ile Pro Thr Glu Leu Pro Gly Val Asp Thr Lys Ile Leu Asp Pro
485 490 495
Arg Asn Thr Tyr Ala Ser Pro Glu Gln Trp Gln Glu Lys Ala Glu Thr
500 505 510
Leu Ala Lys Leu Phe Ile Asp Asn Phe Asp Lys Tyr Thr Asp Thr Pro
515 520 525
Ala Gly Ala Ala Leu Val Ala Ala Gly Pro Lys Leu
530 535 540
<210> 9
<211> 1609
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 9
gctcgcgaat aatccgatta cggctacgct tctaatgttc cccttgaatg gagtcgaaga 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgcgtaat cccacgctgt tacaatgttt tcactggtat tacccggaag gcggtaagct 180
ctggcctgaa ctggccgagc gcgccgacgg ttttaatgat attggtatca atatggtctg 240
gttgccgccc gcctataaag gcgcatcggg cgggtattcg gtcggctacg actcctatga 300
tttatttgat ttaggcgagt ttgatcagaa aggcagcatc cctactaaat atggcgataa 360
agcacaactg ctggccgcca ttgatgctct gaaacgtaat gacattgcgg tgctgttgga 420
tgtggtagtc aaccacaaaa tgggcgcgga tgaaaaagaa gctattcgcg tgcagcgtgt 480
aaatgctgat gaccgtacgc aaattgacga agaaatcatt gagtgtgaag gctggacgcg 540
ttacaccttc cccgcccgtg ccgggcaata ctcgcagttt atctgggatt tcaaatgttt 600
tagcggtatc gaccatatcg aaaaccctga cgaagatggc atttttaaaa ttgttaacga 660
ctacaccggc gaaggctgga acgatcaggt tgatgatgaa ttaggtaatt tcgattatct 720
gatgggcgag aatatcgatt ttcgcaatca tgccgtgacg gaagagatta aatactgggc 780
gcgctgggtg atggaacaaa cgcaatgcga cggttttcgt cttgatgcgg tcaaacatat 840
tccagcctgg ttttataaag agtggatcga acacgtacag gaagttgcgc caaagccgct 900
gtttattgtg gcggagtact ggtcgcatga agttgataag ctgcaaacgt atattgatca 960
ggtggaaggc aaaaccatgc tgtttgatgc gccgctgcag atgaaattcc atgaagcatc 1020
gcgcatgggg cgcgactacg acatgacgca gattttcacg ggtacattag tggaagccga 1080
tcctttccac gccgtgacgc tcgttgccaa tcacgacacc caaccgttgc aagccctcga 1140
agcgccggtc gaaccgtggt ttaaaccgct ggcgtatgcc ttaattttgt tgcgggaaaa 1200
tggcgttcct tcggtattct atccggacct ctacggtgcg cattacgaag atgtcggtgg 1260
tgacgggcaa acctatccga tagatatgcc aataatcgaa cagcttgatg agttaattct 1320
cgcccgtcag cgtttcgccc acggtgtaca gacgttattt ttcgaccatc cgaactgcat 1380
tgcctttagc cgcagtggca ccgacgaatt tcccggctgc gtggtggtca tgtcgaacgg 1440
ggatgatggc gaaaaaacca ttcatctggg agagaattac ggcaataaaa cctggcgtga 1500
ttttctgggg aaccggcaag agagagtagt gaccgacgaa aacggcgaag caaccttctt 1560
ttgcaacggc ggcagcgtca gcgtgtgggt tatcgaagag gtgatttaa 1609
<210> 10
<211> 14
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 10
Met Arg Asn Pro Thr Leu Leu Gln Cys Phe His Trp Tyr Tyr
1 5 10
<210> 11
<211> 4
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 11
Met Asn Thr Ala
1
<210> 12
<211> 2521
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 12
agaaaccgtg ttgaactctg gaaagccagt ctttagatgc gccaggatgc agaggtaatc 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgaagcta accgatgcgg ataatgccgc cgatggcatt tttttccccg cccttgagca 180
aaatatgatg ggtgcggtgt taattaacga aaatgatgaa gtgatgtttt tcaaccccgc 240
cgcagagaag ctctggggat acaaacgtga agaagtcatt ggcaataaca ttgatatgct 300
gattccgcgg gatttgcgtc ctgcgcatcc tgaatacatt cgtcacaacc gtgaaggcgg 360
taaagcgcgt gttgagggga tgagtcggga gctgcagctg gagaaaaaag acggcagtaa 420
aatctggacc cgttttgcgc tatcgaaagt gagcgccgag gggaaagttt attacctggc 480
gctggtacgg gatgccagcg tagaaatggc gcaaaaagaa cagacccgac aattgattat 540
tgccgttgac catctcgacc gaccggtgat tgtcctcgat ccggaacgcc atattgtgca 600
gtgcaatcgc gcatttaccg aaatgtttgg ttactgcatt agcgaagcca gcggtatgca 660
gcccgataca ctcctgaaca ttcctgaatt ccctgccgat aaccgcattc gtttacaaca 720
gttgctatgg aaaaccgccc gcgatcagga cgaatttctg ctgttgacgc gcaccggtga 780
aaaaatctgg attaaagcct ctatcagccc ggtttatgac gtgctcgcgc atctgcagaa 840
cctggtaatg actttctcgg atatcaccga agaacggcag attcgccagc ttgaaggcaa 900
tattctcgcc gccatgtgca gcagcccgcc atttcatgaa atgggggaaa tcatttgtcg 960
taacatcgaa tctgtactca acgaatcgca tgtttcgctg ttcgcactgc gcaacgggat 1020
gccgatacac tgggcgtcat cttcccacgg tgcagaaatt caaaatgcgc aaagctggtc 1080
agcgaccatt cgtcagcgtg atggcgcgcc tgcggggatc ctgcaaatta aaacctcgtc 1140
aggagcagaa accagcgcct ttatcgaacg cgtggcagat atcagccagc atatggccgc 1200
gctggcgctg gaacaggaaa aaagccgtca gcatattgaa caactcatcc aatttgatcc 1260
gatgaccggt ctgccaaatc gcaataacct gcacaattac ctcgatgacc tggtcgacaa 1320
agccgtctct cccgtggtgt atctcatcgg tgttgaccat attcaggatg tgattgatag 1380
ccttggctat gcgtgggccg atcaggcatt gctggaagtg gtcaatcgct ttcgtgaaaa 1440
actcaaaccg gatcagtatc tctgtcgtat cgaaggtacg cagtttgtcc tcgtgagcct 1500
cgaaaacgac gtcagtaaca ttacccaaat cgccgatgag ctacggaatg tggtcagcaa 1560
gccgataatg attgacgata aacccttccc gcttaccttg agtattggca tcagctacga 1620
cctgggtaaa aaccgcgatt acttgctctc cactgctcac aatgcaatgg attatattcg 1680
caagaatggc ggtaacggct ggcagttctt cagcccggcg atgaacgaaa tggtaaaaga 1740
gcgtttggtt ttaggcgcag cgctgaaaga agcgattagc aataaccaac tgaaactggt 1800
ttaccagccg caaatcttcg cagaaacggg tgaactgtac ggcatcgaag cccttgctcg 1860
ctggcacgat cccctgcatg gtcatgtgcc cccttcacgg tttattcctc tcgcagaaga 1920
gattggtgaa atcgaaaata ttgggcgctg ggtcatcgcg gaagcttgcc gtcagttagc 1980
agaatggcgt agccagaata ttcatatccc ggcgttatcc gtgaacttgt cggcgctgca 2040
ctttcgcagt aatcaactgc ctaatcaggt gtctgatgca atgcacgcct ggggtattga 2100
cggccaccag ctgacggtag aaatcacgga aagcatgatg atggaacacg ataccgaaat 2160
ctttaagcgc attcagatcc tgcgtgatat gggcgtgggc ttatcggtag atgattttgg 2220
tacgggcttt tccggattat cccgcttagt cagtcttccg gtaacggaaa tcaaaattga 2280
caaaagtttt gtcgatcgtt gtctgaccga aaaacgcatc cttgccttac ttgaagccat 2340
taccagcatt gggcaaagcc tcaatttaac cgtcgtggcg gaaggcgtcg aaaccaaaga 2400
gcaatttgag atgctacgca agatccactg tcgcgttatt cagggatatt tcttttcccg 2460
ccccctaccc gccgaagaaa ttccaggctg gatgagcagc gtgttaccgc tgaaaatctg 2520
a 2521
<210> 13
<211> 454
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 13
ggtgagttga gttcaaactg tagtacaatt ctctccagtt tgaacaggaa agaatatgct 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgaaccct tatatttatc ttggtggtgc aatacttgca gaggtcattg gtacaacctt 180
aatgaagttt tcagaaggtt ttacacggtt atggccatct gttggtacaa ttatttgtta 240
ttgtgcatca ttctggttat tagctcagac gctggcttat attcctacag ggattgctta 300
tgctatctgg tcaggagtcg gtattgtcct gattagctta ctgtcatggg gatttttcgg 360
ccaacggctg gacctgccag ccattatagg catgatgttg atttgtgccg gtgtgttgat 420
tattaattta ttgtcacgaa gcacaccaca ttaa 454
<210> 14
<211> 970
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 14
atgcagcggc tggggatctc ggttcgcgag gtgttgtaat ctgcttttgc aggagtatgc 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgagaaac aaactctctt tcgacttgca gttgagcgcc agaaaagcgg caatcgctga 180
acggattgcc gcccataaaa ttgcccgcag taaagtgtcg gtctttttaa tggcgatgtc 240
cgctggcgtg tttatggcga tcggatttac tttttacctt tccgttatcg ccgatgcccc 300
gtcttcacag gcattaaccc atctggtggg cggcctttgc tttacactcg gctttatttt 360
gctggcggtt tgcggcacca gcctgttcac ctcgtcggta atgacggtga tggcaaaaag 420
tcggggcgtt attagttggc gaacttggct gattaacgca cttctggtgg cctgcggtaa 480
tctggcaggt attgcctgtt tcagtttgtt aatctggttt tccgggctgg tgatgagtga 540
aaacgcgatg tggggagtcg cggttttaca ctgcgccgag ggcaaaatgc atcatacatt 600
tactgaatct gtcagcctcg gcattatgtg caatctgatg gtttgcctgg cgctgtggat 660
gagttattgc gggcgttcgt tatgcgacaa aatcgtcgcc atgattttgc ccatcaccct 720
gtttgtcgcc agtggctttg agcactgtat cgccaatttg tttgtgattc cgttcgccat 780
tgccattcgc catttcgccc ctcccccctt ctggcagctg gcgcacagta gcgcagacaa 840
ttttccggca ctgacggtca gccattttat taccgccaat ctgctcccgg tgatgctggg 900
taatattatc ggcggtgcgg tgctggtgag tatgtgttat cgggctattt atttacgtca 960
ggaaccctga 970
<210> 15
<211> 2794
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 15
atgaagccgg cgaatgccgg cttttttaat gcgataattt aatcttatgg gtggcgcaca 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgaatacc cttccagaac agtacgcaaa caccgctctc cccaccctgc ccggtcaacc 180
gcaaaatcca tgcgtctggc cccgtgatga attaaccgtc ggtgggataa aagcccatat 240
cgatactttc cagcgttggc tgggtgatgc ctttgacaat gggatctctg cagaacagtt 300
gattgaggcg cgcaccgagt ttatcgacca gctcctgcaa cgattatgga ttgaagcggg 360
attcagccag attgccgacc tggcattggt cgccgtcggt ggctacggtc gtggcgagct 420
gcatccactt tcagacgtcg atttactgat tttaagccgt aaaaagctcc cggacgatca 480
ggcgcaaaaa gtgggcgagc tgttaacgct gctctgggat gtaaagctgg aagtcggtca 540
tagcgtgcgc acgcttgaag agtgcatgct ggaagggtta tcggatttaa ccgtcgccac 600
caatttaatc gaatcccgct tattaattgg cgatgttgcg ctgttcctcg aactgcaaaa 660
acatattttc agcgaaggat tctggccttc cgacaagttc tacgcggcga aagttgaaga 720
acagaaccag cgccatcagc gttaccatgg caccagctac aaccttgaac cagacatcaa 780
aagcagccct ggcggcttgc gcgatatcca cactctgcaa tgggtggccc gccgtcattt 840
tggcgcaaca tcgctggatg aaatggtcgg gtttggcttc ttaacctcag cggagcgggc 900
ggaattaaac gaatgtctgc atatattgtg gcgtattcgc tttgccctgc atctggtcgt 960
cagccgttac gataatcgcc tgttattcga tcgccagctt agcgtcgccc agcgtctgaa 1020
ttacagtggt gaaggtaacg aaccggtcga gcggatgatg aaggattact tccgcgttac 1080
acgccgcgtc agtgaactca accagatgct gctgcaactg ttcgatgaag ccatcctcgc 1140
ccttcccgcc gacgaaaaac cacgtccaat cgacgatgag tttcagctac gcggtacgct 1200
aatcgacctg cgtgatgaaa cactatttat gcgccagccg gaagccatct tgcgtatgtt 1260
ctacaccatg gtgcacaaca gtgcgatcac cggcatttac tccaccacgc tgcgccagtt 1320
acgccatgcc cgtcgccatc tgcaacaacc gctgtgtaat attccggaag cacgaaaact 1380
gtttttgagc attctgcgtc accccggagc ggtgcggcgc gggctattgc caatgcatcg 1440
ccatagcgtg ctcggcgcgt atatgccgca atggtcgcat atcgtcgggc agatgcagtt 1500
tgatctgttc cacgcctaca cggtggatga acatactatc cgcgtgatgc tgaaactgga 1560
gagttttgcc agtgaagaaa cgcgccagcg ccatccgttg tgtgtggacg tctggccgcg 1620
cctgccgtca actgagctga ttttcatcgc cgcgctgttt cacgatatcg ccaaaggacg 1680
cggcggcgac cactccattc tcggtgctca ggatgtagtg cattttgccg aactccacgg 1740
gctgaactca cgcgaaacac agctggtcgc ctggctggtt cgccagcacc tgttgatgtc 1800
ggtgaccgcc caacgccgcg atattcagga cccggaagtc atcaagcagt ttgccgaaga 1860
agtgcaaacg gaaaatcgtc tgcgctatct ggtatgcctg actgtggctg acatttgcgc 1920
caccaacgaa acgctgtgga atagctggaa gcaaagtctg ttgcgtgagc tctactttgc 1980
caccgaaaag cagctacgac gcggaatgca aaacacgccg gatatgcgcg aacgggttcg 2040
ccatcaccaa ctccaggcac tggcactact gcgcatggat aacatcgacg aagaggcgct 2100
gcaccaaatt tggtcacgct gtcgtgctaa ctattttgtc cgccatagcc caaatcaact 2160
ggcctggcat gcccgccatt tattacagca tgatttaagc aaaccgctgg tattgcttag 2220
cccgcaggct acgcgtggag gcaccgagat ttttatctgg agcccggacc gcccttatct 2280
gtttgccgcc gtctgtgccg aattagaccg ccgcaattta agtgttcacg acgcacaaat 2340
tttcaccact cgcgacggta tggcgatgga tacctttatc gtgctggaac ccgatggcaa 2400
cccgctgtcc gcagatcgtc atgaggttat tcggtttggt ctggagcaag tactgacgca 2460
aagtagctgg cagccaccgc agccccgtcg ccaacccgcc aaattacgcc attttactgt 2520
tgaaaccgaa gtaacgtttt tgccgaccca taccgaccgc aaatcgttcc tcgaactgat 2580
cgccctcgac caacctggac tgctggcgcg agtcgggaaa atttttgccg atctgggaat 2640
ttcgcttcat ggtgcccgaa ttacaaccat tggcgagcga gtagaagatt tattcataat 2700
tgccaccgct gaccggcgtg cgcttaataa cgagttgcag caggaagtgc atcagcggtt 2760
gacagaggcc ctcaatccaa acgataaagg gtga 2794
<210> 16
<211> 14
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 16
Met Lys Pro Leu Ser Ser Pro Leu Gln Gln Tyr Trp Gln Thr
1 5 10
<210> 17
<211> 538
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 17
ctcggtttga gttaatcgcc aattaaaaag gttaatgaca tgcgagagac agtcgaaatt 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgcgttat cccgtcactc ttacacccgc gccggaaggc ggttatatgg tttcttttgt 180
ggatatccct gaagcgttga cccagggcga aactgtcgct gaagcgatgg aagcggcaaa 240
agatgcttta ctgaccgcat ttgattttta ttttgaagat aacgagctta tccctttacc 300
ttcgccatta aatagtcacg atcactttat tgaagtacct ttgagcgtcg cctctaaggt 360
attgctgtta aatgcttttt tacagtcaga aatcactcag caagagttag ccaggcgaat 420
tggcaaacct aaacaggaga ttactcgcct atttaacttg catcatgcga caaaaatcga 480
cgccgtccag ctcgcggcaa aggcgcttgg caaagagtta tcgctggtga tggtttaa 538
<210> 18
<211> 2401
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 18
tgggcagggg ctgttgccca cacactttat ttgtgaacgt tacgtgaaag gaacaaccaa 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatggatgac cagttaaaac aaagtgcact tgatttccat gaatttccag ttccagggaa 180
aatccaggtt tctccaacca agcctctggc aacacagcgc gatctggcgc tggcctactc 240
accaggcgtt gccgcacctt gtcttgaaat cgaaaaagac ccgttaaaag cctacaaata 300
taccgcccga ggtaacctgg tggcggtgat ctctaacggt acggcggtgc tggggttagg 360
caacattggc gcgctggcag gcaaaccggt gatggaaggc aagggcgttc tgtttaagaa 420
attcgccggg attgatgtat ttgacattga agttgacgaa ctcgacccgg acaaatttat 480
tgaagttgtc gccgcgctcg aaccaacctt cggcggcatc aacctcgaag acattaaagc 540
gccagaatgt ttctatattg aacagaaact gcgcgagcgg atgaatattc cggtattcca 600
cgacgatcag cacggcacgg caattatcag cactgccgcc atcctcaacg gcttgcgcgt 660
ggtggagaaa aacatctccg acgtgcggat ggtggtttcc ggcgcgggtg ccgcagcaat 720
cgcctgtatg aacctgctgg tagcgctggg tctgcaaaaa cataacatcg tggtttgcga 780
ttcaaaaggc gttatctatc agggccgtga gccaaacatg gcggaaacca aagccgcata 840
tgcggtggtg gatgacggca aacgtaccct cgatgatgtg attgaaggcg cggatatttt 900
cctgggctgt tccggcccga aagtgctgac ccaggaaatg gtgaagaaaa tggctcgtgc 960
gccaatgatc ctggcgctgg cgaacccgga accggaaatt ctgccgccgc tggcgaaaga 1020
agtgcgtccg gatgccatca tttgcaccgg tcgttctgac tatccgaacc aggtgaacaa 1080
cgtcctgtgc ttcccgttca tcttccgtgg cgcgctggac gttggcgcaa ccgccatcaa 1140
cgaagagatg aaactggcgg cggtacgtgc gattgcagaa ctcgcccatg cggaacagag 1200
cgaagtggtg gcttcagcgt atggcgatca ggatctgagc tttggtccgg aatacatcat 1260
tccaaaaccg tttgatccgc gcttgatcgt taagatcgct cctgcggtcg ctaaagccgc 1320
gatggagtcg ggcgtggcga ctcgtccgat tgctgatttc gacgtctaca tcgacaagct 1380
gactgagttc gtttacaaaa ccaacctgtt tatgaagccg attttctccc aggctcgcaa 1440
agcgccgaag cgcgttgttc tgccggaagg ggaagaggcg cgcgttctgc atgccactca 1500
ggaactggta acgctgggac tggcgaaacc gatccttatc ggtcgtccga acgtgatcga 1560
aatgcgcatt cagaaactgg gcttgcagat caaagcgggc gttgattttg agatcgtcaa 1620
taacgaatcc gatccgcgct ttaaagagta ctggaccgaa tacttccaga tcatgaagcg 1680
tcgcggcgtc actcaggaac aggcgcagcg ggcgctgatc agtaacccga cagtgatcgg 1740
cgcgatcatg gttcagcgtg gggaagccga tgcaatgatt tgcggtacgg tgggtgatta 1800
tcatgaacat tttagcgtgg tgaaaaatgt ctttggttat cgcgatggcg ttcacaccgc 1860
aggtgccatg aacgcgctgc tgctgccgag tggtaacacc tttattgccg atacatatgt 1920
taatgatgaa ccggatgcag aagagctggc ggagatcacc ttgatggcgg cagaaactgt 1980
ccgtcgtttt ggtattgagc cgcgcgttgc tttgttgtcg cactccaact ttggttcttc 2040
tgactgcccg tcgtcgagca aaatgcgtca ggcgctggaa ctggtcaggg aacgtgcacc 2100
agaactgatg attgatggtg aaatgcacgg cgatgcagcg ctggtggaag cgattcgcaa 2160
cgaccgtatg ccggacagct ctttgaaagg ttccgccaat attctggtga tgccgaacat 2220
ggaagctgcc cgcattagtt acaacttact gcgtgtttcc agctcggaag gtgtgactgt 2280
cggcccggtg ctgatgggtg tggcgaaacc ggttcacgtg ttaacgccga tcgcatcggt 2340
gcgtcgtatc gtcaacatgg tggcgctggc cgtggtagaa gcgcaaaccc aaccgctgta 2400
a 2401
<210> 19
<211> 127
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 19
Met Ser Arg Arg Asn Thr Asp Ala Ile Thr Ile His Ser Ile Leu Asp
1 5 10 15
Trp Ile Glu Asp Asn Leu Glu Ser Pro Leu Ser Leu Glu Lys Val Ser
20 25 30
Glu Arg Ser Gly Tyr Ser Lys Trp His Leu Gln Arg Met Phe Lys Lys
35 40 45
Glu Thr Gly His Ser Leu Gly Gln Tyr Ile Arg Ser Arg Lys Met Thr
50 55 60
Glu Ile Ala Gln Lys Leu Lys Glu Ser Asn Glu Pro Ile Leu Tyr Leu
65 70 75 80
Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln Thr Leu Thr Arg Thr Phe
85 90 95
Lys Asn Tyr Phe Asp Val Pro Pro His Lys Asp Arg Met Thr Asn Met
100 105 110
Gln Gly Glu Ser Arg Phe Leu His Pro Leu Asn His Tyr Asn Ser
115 120 125
<210> 20
<211> 810
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 20
Met Ser Val Ile Ala Gln Ala Gly Ala Lys Gly Arg Gln Leu His Lys
1 5 10 15
Phe Gly Gly Ser Ser Leu Ala Asp Val Lys Cys Tyr Leu Arg Val Ala
20 25 30
Gly Ile Met Ala Glu Tyr Ser Gln Pro Asp Asp Met Met Val Val Ser
35 40 45
Ala Ala Gly Ser Thr Thr Asn Gln Leu Ile Asn Trp Leu Lys Leu Ser
50 55 60
Gln Thr Asp Arg Leu Ser Ala His Gln Val Gln Gln Thr Leu Arg Arg
65 70 75 80
Tyr Gln Cys Asp Leu Ile Ser Gly Leu Leu Pro Ala Glu Glu Ala Asp
85 90 95
Ser Leu Ile Ser Ala Phe Val Ser Asp Leu Glu Arg Leu Ala Ala Leu
100 105 110
Leu Asp Ser Gly Ile Asn Asp Ala Val Tyr Ala Glu Val Val Gly His
115 120 125
Gly Glu Val Trp Ser Ala Arg Leu Met Ser Ala Val Leu Asn Gln Gln
130 135 140
Gly Leu Pro Ala Ala Trp Leu Asp Ala Arg Glu Phe Leu Arg Ala Glu
145 150 155 160
Arg Ala Ala Gln Pro Gln Val Asp Glu Gly Leu Ser Tyr Pro Leu Leu
165 170 175
Gln Gln Leu Leu Val Gln His Pro Gly Lys Arg Leu Val Val Thr Gly
180 185 190
Phe Ile Ser Arg Asn Asn Ala Gly Glu Thr Val Leu Leu Gly Arg Asn
195 200 205
Gly Ser Asp Tyr Ser Ala Thr Gln Ile Gly Ala Leu Ala Gly Val Ser
210 215 220
Arg Val Thr Ile Trp Ser Asp Val Ala Gly Val Tyr Ser Ala Asp Pro
225 230 235 240
Glu Lys Val Lys Asp Ala Cys Leu Leu Pro Leu Leu Arg Leu Asp Glu
245 250 255
Ala Ser Glu Leu Ala Arg Leu Ala Ala Pro Val Leu His Ala Arg Thr
260 265 270
Leu Gln Pro Val Ser Gly Ser Glu Ile Asp Leu Gln Leu Arg Cys Ser
275 280 285
Tyr Thr Pro Asp Gln Gly Ser Thr Arg Ile Glu Arg Val Leu Ala Ser
290 295 300
Gly Thr Gly Ala Arg Ile Val Thr Ser His Asp Asp Val Cys Leu Ile
305 310 315 320
Glu Phe Gln Val Pro Ala Ser Gln Asp Phe Lys Leu Ala His Lys Glu
325 330 335
Ile Asp Gln Ile Leu Lys Arg Ala Gln Val Arg Pro Leu Ala Val Gly
340 345 350
Val His Asn Asp Arg Gln Leu Leu Gln Phe Cys Tyr Thr Ser Glu Val
355 360 365
Ala Asp Ser Ala Leu Lys Ile Leu Asp Glu Ala Gly Leu Pro Gly Glu
370 375 380
Leu Arg Leu Arg Gln Gly Leu Ala Leu Val Ala Met Val Gly Ala Gly
385 390 395 400
Val Thr Arg Asn Pro Leu His Cys His Arg Phe Trp Gln Gln Leu Lys
405 410 415
Gly Gln Pro Val Glu Phe Thr Trp Gln Ser Asp Asp Gly Ile Ser Leu
420 425 430
Val Ala Val Leu Arg Thr Gly Pro Thr Glu Ser Leu Ile Gln Gly Leu
435 440 445
His Gln Ser Val Phe Arg Ala Glu Lys Arg Ile Gly Leu Val Leu Phe
450 455 460
Gly Lys Gly Asn Ile Gly Ser Arg Trp Leu Glu Leu Phe Ala Arg Glu
465 470 475 480
Gln Ser Thr Leu Ser Ala Arg Thr Gly Phe Glu Phe Val Leu Ala Gly
485 490 495
Val Val Asp Ser Arg Arg Ser Leu Leu Ser Tyr Asp Gly Leu Asp Ala
500 505 510
Ser Arg Ala Leu Ala Phe Phe Asn Asp Glu Ala Val Glu Gln Asp Glu
515 520 525
Glu Ser Leu Phe Leu Trp Met Arg Ala His Pro Tyr Asp Asp Leu Val
530 535 540
Val Leu Asp Val Thr Ala Ser Gln Gln Leu Ala Asp Gln Tyr Leu Asp
545 550 555 560
Phe Ala Ser His Gly Phe His Val Ile Ser Ala Asn Lys Leu Ala Gly
565 570 575
Ala Ser Asp Ser Asn Lys Tyr Arg Gln Ile His Asp Ala Phe Glu Lys
580 585 590
Thr Gly Arg His Trp Leu Tyr Asn Ala Thr Val Gly Ala Gly Leu Pro
595 600 605
Ile Asn His Thr Val Arg Asp Leu Ile Asp Ser Gly Asp Thr Ile Leu
610 615 620
Ser Ile Ser Gly Ile Phe Ser Gly Thr Leu Ser Trp Leu Phe Leu Gln
625 630 635 640
Phe Asp Gly Ser Val Pro Phe Thr Glu Leu Val Asp Gln Ala Trp Gln
645 650 655
Gln Gly Leu Thr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly Lys Asp
660 665 670
Val Met Arg Lys Leu Val Ile Leu Ala Arg Glu Ala Gly Tyr Asn Ile
675 680 685
Glu Pro Asp Gln Val Arg Val Glu Ser Leu Val Pro Ala His Cys Glu
690 695 700
Gly Gly Ser Ile Asp His Phe Phe Glu Asn Gly Asp Glu Leu Asn Glu
705 710 715 720
Gln Met Val Gln Arg Leu Glu Ala Ala Arg Glu Met Gly Leu Val Leu
725 730 735
Arg Tyr Val Ala Arg Phe Asp Ala Asn Gly Lys Ala Arg Val Gly Val
740 745 750
Glu Ala Val Arg Glu Asp His Pro Leu Ala Ser Leu Leu Pro Cys Asp
755 760 765
Asn Val Phe Ala Ile Glu Ser Arg Trp Tyr Arg Asp Asn Pro Leu Val
770 775 780
Ile Arg Gly Pro Gly Ala Gly Arg Asp Val Thr Ala Gly Ala Ile Gln
785 790 795 800
Ser Asp Ile Asn Arg Leu Ala Gln Leu Leu
805 810
<210> 21
<211> 4
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 21
Met Pro Glu Gln
1
<210> 22
<211> 4
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 22
Met Asp Val Met
1
<210> 23
<211> 540
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 23
Met Arg Val Asn Asn Gly Leu Thr Pro Gln Glu Leu Glu Ala Tyr Gly
1 5 10 15
Ile Ser Asp Val His Asp Ile Val Tyr Asn Pro Ser Tyr Asp Leu Leu
20 25 30
Tyr Gln Glu Glu Leu Asp Pro Ser Leu Thr Gly Tyr Glu Arg Gly Val
35 40 45
Leu Thr Asn Leu Gly Ala Val Ala Val Asp Thr Gly Ile Phe Thr Gly
50 55 60
Arg Ser Pro Lys Asp Lys Tyr Ile Val Arg Asp Asp Thr Thr Arg Asp
65 70 75 80
Thr Phe Trp Trp Ala Asp Lys Gly Lys Gly Lys Asn Asp Asn Lys Pro
85 90 95
Leu Ser Pro Glu Thr Trp Gln His Leu Lys Gly Leu Val Thr Arg Gln
100 105 110
Leu Ser Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Asn
115 120 125
Pro Asp Thr Arg Leu Ser Val Arg Phe Ile Thr Glu Val Ala Trp Gln
130 135 140
Ala His Phe Val Lys Asn Met Phe Ile Arg Pro Ser Asp Glu Glu Leu
145 150 155 160
Ala Gly Phe Lys Pro Asp Phe Ile Val Met Asn Gly Ala Lys Cys Thr
165 170 175
Asn Pro Gln Trp Lys Glu Gln Gly Leu Asn Ser Glu Asn Phe Val Ala
180 185 190
Phe Asn Leu Thr Glu Arg Met Gln Leu Ile Gly Gly Thr Trp Tyr Gly
195 200 205
Gly Glu Met Lys Lys Gly Met Phe Ser Met Met Asn Tyr Leu Leu Pro
210 215 220
Leu Lys Gly Ile Ala Ser Met Gly Cys Ser Ala Asn Val Gly Glu Lys
225 230 235 240
Gly Asp Val Ala Val Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr
245 250 255
Leu Ser Thr Asp Pro Lys Arg Arg Leu Ile Gly Asp Asp Glu His Gly
260 265 270
Trp Asp Asp Asp Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys
275 280 285
Thr Ile Lys Leu Ser Lys Glu Ala Glu Pro Glu Ile Tyr Asn Ala Ile
290 295 300
Arg Arg Asp Ala Leu Leu Glu Asn Val Thr Val Arg Glu Asp Gly Thr
305 310 315 320
Ile Asp Phe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr
325 330 335
Pro Ile Tyr His Ile Asp Asn Ile Val Lys Pro Val Ser Lys Ala Gly
340 345 350
His Ala Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe Gly Val Leu
355 360 365
Pro Pro Val Ser Arg Leu Thr Ala Asp Gln Thr Gln Tyr His Phe Leu
370 375 380
Ser Gly Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Ile Thr Glu
385 390 395 400
Pro Thr Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu
405 410 415
His Pro Thr Gln Tyr Ala Glu Val Leu Val Lys Arg Met Gln Ala Ala
420 425 430
Gly Ala Gln Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys
435 440 445
Arg Ile Ser Ile Lys Asp Thr Arg Ala Ile Ile Asp Ala Ile Leu Asn
450 455 460
Gly Ser Leu Asp Asn Ala Glu Thr Phe Thr Leu Pro Met Phe Asn Leu
465 470 475 480
Ala Ile Pro Thr Glu Leu Pro Gly Val Asp Thr Lys Ile Leu Asp Pro
485 490 495
Arg Asn Thr Tyr Ala Ser Pro Glu Gln Trp Gln Glu Lys Ala Glu Thr
500 505 510
Leu Ala Lys Leu Phe Ile Asp Asn Phe Asp Lys Tyr Thr Asp Thr Pro
515 520 525
Ala Gly Ala Ala Leu Val Ala Ala Gly Pro Lys Leu
530 535 540
<210> 24
<211> 811
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 24
tctgacgcat aatgacgtcg cattaatgat cgcaacctat ttattacaac agggcaaatc 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatggcgaga cgtattctgg tcgtagaaga tgaagctcca attcgcgaaa tggtctgctt 180
cgtgctcgaa caaaatggct ttcagccggt cgaagcggaa gattatgaca gtgctgtgaa 240
tcaactgaat gaaccctggc cggatttaat tctcctcgac tggatgttac ctggcggctc 300
cggtatccag ttcatcaaac acctcaagcg cgagtcgatg acccgggata ttccagtggt 360
gatgttgacc gccagagggg aagaagaaga tcgcgtgcgc ggccttgaaa ccggcgcgga 420
tgactatatc accaagccgt tttcgccgaa ggagctggtg gcgcgaatca aagcggtaat 480
gcgccgtatt tcgccaatgg cggtggaaga ggtgattgag atgcagggat taagtctcga 540
cccgacatct caccgagtga tggcgggcga agagccgctg gagatggggc cgacagaatt 600
taaactgctg cactttttta tgacgcatcc tgagcgcgtg tacagccgcg agcagctgtt 660
aaaccacgtc tggggaacta acgtgtatgt ggaagaccgc acggtcgatg tccacattcg 720
tcgcctgcgt aaagcactgg agcccggcgg gcatgaccgc atggtgcaga ccgtgcgcgg 780
tacaggatat cgtttttcaa cccgctttta a 811
<210> 25
<211> 1159
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 25
ctgttagaat tgcgccgaat tttatttttc taccgcaagt aacgcgtggg gacccaagca 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
agtgaccgat aaaacctctc ttagctacaa agatgccggt gttgatattg acgcgggtaa 180
tgctctggtt ggaagaatca aaggcgtagt gaagaaaacg cgtcgtccgg aagtgatggg 240
cggtctgggc ggcttcggtg cgctgtgtgc attgccgcaa aaatatcgtg aacccgtgct 300
ggtttctggc actgacggcg taggtaccaa gctgcgtctg gcaatggact taaaacgtca 360
cgacaccatt ggtattgatc tggtcgccat gtgcgttaat gacctggtgg tgcaaggtgc 420
agagccgctg tttttcctcg actattacgc aaccggaaaa ctggatgttg ataccgcttc 480
agcggtgatc agcggcattg cggaaggttg tctgcaatca ggctgttcac tggtgggtgg 540
cgaaacggca gaaatgccgg ggatgtatca cggtgaggat tacgatgtcg cgggtttctg 600
cgttggcgtg gtagaaaaat cagaaatcat cgacggctct aaagtcagcg acggcgatgt 660
gctgattgca ctcggttcca gcggtccaca ctcgaacggc tattcgctgg tgcgcaaaat 720
tcttgaagtc agcggttgtg atccgcaaac caccgaactt gatggtaagc cattagccga 780
tcatctgctg gcaccgaccc gcatttacgt gaagtcagtg ctggagttga ttgaaaaggt 840
cgatgtgcat gccattgcgc acctgaccgg cggcggcttc tgggaaaaca ttccgcgcgt 900
attgccagat aatactcagg cagtgattga tgaatcttcc tggcagtggc cggaagtgtt 960
caactggctg caaacggcag gtaacgttga gcaccatgaa atgtatcgca ccttcaactg 1020
cggcgtcggg atgattattg ccctgcctgc tccggaagtg gacaaagccc tcgccctgct 1080
caatgccaac ggtgaaaacg cgtggaaaat cggtatcatc aaagcctctg attccgaaca 1140
acgcgtggtt atcgaataa 1159
<210> 26
<211> 4
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 26
Met Asn Ser Leu
1
<210> 27
<211> 4
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 27
Met Asn Thr Asn
1
<210> 28
<211> 703
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 28
ctgttgtcgc ctgctctgga ttaacggata ataggcggct tttttatttc aggccgaaaa 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgactgac tacctgttac tgtttgtcgg aactgtactg gtcaataact ttgtactggt 180
caagtttctc ggtctctgtc cgtttatggg ggtttccaaa aaactggaaa ccgcgatggg 240
catggggctg gcaacaacgt ttgtgatgac gctggcgtct atttgcgcct ggcttatcga 300
tacgtggatt ttgatcccac ttaatctgat ttacctgcgc accctggcat ttattctggt 360
gattgctgtg gtcgtgcagt tcaccgagat ggtggtgcgc aaaaccagcc cggtgcttta 420
ccgtctgctg gggatttttt tgccgcttat caccaccaac tgtgcagtgc tcggcgtggc 480
gttgctgaat atcaatctcg ggcacaattt cttgcagtcg gcgctgtacg gtttttccgc 540
cgctgtcggt ttctcgctgg tgatggtgct cttcgccgcc atccgcgaac gccttgctgt 600
ggctgatgtc ccggcacctt ttcgcggtaa tgccattgcg ttaattaccg caggtcttat 660
gtctctggcc tttatgggct ttagtggttt ggtgaagttg taa 703
<210> 29
<211> 1480
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 29
aaagatgcgg caagtgaatt tgccgcaatt gctacctttt tagcaaatcc gaggcaccta 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatggaacaa tatgatcaaa ttggcgcaag actggaccgc ttgcctttgg cccggtttca 180
ttatcgtata tttggtatta taagctttag tctgttatta acagggtttt tgagttactc 240
cggtaatgtc gtcttagcaa agctggtaag caatggatgg tcaaataatt tcctcaatgc 300
cgcctttacc tcggcattaa tgtttggtta tttcatcggc tcacttactg gtgggtttat 360
tggtgactac tttgggcggc gcagggcgtt tcgcataaat cttctcatcg tcggtattgc 420
tgcaacaggg gccgcttttg tccctgatat gtactggctc atcttctttc gcttcctgat 480
gggaacagga atgggggcgc tgattatggt tggctatgcc tcatttacgg agtttatccc 540
cgcgacggtg cgtggaaaat ggtccgcgcg gctctcattt gttggtaact ggtcgcccat 600
gctgtctgcg gcgataggcg tggtggttat cgcttttttt agttggcgaa taatgtttct 660
gctgggtggt attggcatac tgttagcctg gtttctctca ggtaaatact ttatcgagtc 720
gccacgatgg ctggcaggga aagggcaaat cgcaggtgca gaatgccaac ttcgtgaagt 780
agagcagcaa attgaaagag agaagagtat tcgtttaccc ccgcttactt cgtatcagag 840
caacagcaag gttaaagtaa tcaagggtac tttctggctc ctgtttaaag gtgaaatgtt 900
acgacgtaca ttagtcgcga ttactgtttt aattgcaatg aacatttcgc tttataccat 960
caccgtatgg ataccgacca tatttgttaa ctccggcatt gatgtcgata aatcaatatt 1020
aatgaccgct gttattatga ttggcgctcc ggtaggaata tttattgcgg cattaattat 1080
tgatcatttt cctcgtcggt tatttggctc caccttactt attattattg ccgtgttagg 1140
ctatatctat tcaattcaga ctacagagtg ggcgatttta atctatggac tggtgatgat 1200
cttcttttta tacatgtatg tttgcttcgc gtcggcggtt tatatcccgg agctttggcc 1260
aacgcattta cgcctgcgcg gttcgggttt cgttaatgcc gtcggacgga tcgtcgcagt 1320
cttcacgccc tatggcgttg cggcattatt aacacattat gggtcgatca cggtgtttat 1380
ggtacttggt gttatgttat tgctctgtgc gctggttctc tccatttttg gcatcgaaac 1440
gcggaaggtg tcgttggaag agatttctga ggtgaattaa 1480
<210> 30
<211> 1045
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 30
tcgccatttt tgctatcatg cctgcataca taaacgacaa aacagtatgc agagggaaaa 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgggttcc accagaaagg ggatgctgaa cgttctgatt gccgccgtgt tgtggggaag 180
ttcaggggtc tgcgcgcaat acatcatgga gcaaagccag atgtcgtcgc agtttttgac 240
tatgacgcgt ttgatattcg ccggtttgat tctactgacg ctgtcatttg ttcatggcga 300
taaaatcttt tctattatta acaatcataa agatgccatt agcctgctga ttttttccgt 360
ggttggcgcg ctaactgtac agctcacttt tttgctaacc atcgaaaaat cgaacgcagc 420
cacggcaacg gtgctgcaat tcctctcacc gacgattatc gtcgcctggt tctcactggt 480
gcgtaaatcg cgcccgggca ttctggtttt ctgcgctatt ttgacatcgc tggtcgggac 540
ttttttattg gtgacacacg gtaatccgac gtcattatcg atctctcctg ccgcgttgtt 600
ctggggcatt gcctcggcat ttgctgctgc attctatacc acctatccct caacgctaat 660
tgcccgctat ggcacgttac cagtcgtcgg ctggagtatg ctgattggcg gtctgattct 720
gttgcctttt tatgccagac aaggaacaaa ctttgtcgtt aacggcagtt tgattctggc 780
gtttttttat ttggtggtca ttggtacgtc cctgacattt agtctgtacc tgaaaggagc 840
acaattaatt ggcggtccaa aagccagcat tttgagctgt gcagaaccat taagtagcgc 900
gctactctct ttgctgttgc tggggatcac gttcacatta ccggactggc tgggaacgct 960
gctgattctg tcatcggtga ttttgatttc aatggattcc cgtcgccgcg ccagaaaaat 1020
aaatcgtccg gcgcggcata agtga 1045
<210> 31
<211> 2470
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 31
caagggacaa tttttgcagc ggcacagcgt tcagatagtt attttgttaa atgtattaac 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgctgagt ttatacgaaa agataaagat aaggctgata attttatttt tattggcagc 180
actgtcattt attggtcttt ttttcatcat taactatcaa ctggtatcgg agcgcgcggt 240
aaaacgtgcc gatagccgct ttgaacttat tcagaaaaac gttggctatt tctttaaaga 300
tattgaacgt tcggccctga cattaaagga ctcactgtat ttattaaaaa atacagagga 360
gattcaacgc gccgtgattc ttaaaatgga aatgatgcca tttttagact cggtgggact 420
ggtacttgat gataataaat attatctttt ttcgcggagg gcgaatgata aaatcgttgt 480
ttatcatcag gaacaagtaa atggaccgct tgtcgacgag tcagggcggg ttatttttgc 540
cgattttaac ccatcgaaac gaccgtggtc ggtggcttca gatgactcta acaacagctg 600
gaatccggca tacaattgct ttgatcgtcc gggtaaaaaa tgtatctctt ttacgctaca 660
catcaacggc aaagatcacg atttgttagc ggtggataaa attcatgtcg atttaaactg 720
gcgatatctg aacgagtatc ttgatcaaat cagcgctaat gatgaagttc tatttttgaa 780
acaaggccat gagatcattg ccaagaatca actcgctcgt gaaaaactga ttatttataa 840
tagcgaaggt aattataata ttattgattc tgtcgatact gaatatatcg aaaaaacatc 900
agcggtgcca aacaacgcat tattcgaaat ctatttttat tatcctggcg gtaatttatt 960
gaacgcatca gataaacttt tttatctgcc gtttgcgttc attattatcg tattgctggt 1020
ggtttattta atgaccactc gtgtgttccg tcggcaattt tctgaaatga cagagctggt 1080
taatacgctg gcgtttttgc ctgactcaac ggatcaaatc gaggctctga aaattcgtga 1140
aggcgatgcg aaagagatta tcagcatcaa aaattcgatc gcggaaatga aagatgccga 1200
aattgaacgg tcaaataaat tgctctcact gatctcttac gatcaggaaa gtggttttat 1260
taaaaatatg gcgattattg agtctaacaa taatcagtat ctggctgtgg ggatcatcaa 1320
actgtgtggt ctggaagccg tggaagcggt gtttggtgtt gatgagcgca ataaaatcgt 1380
caggaaattg tgtcagcgaa ttgccgagaa atatgcgcaa tgctgcgata tcgtgacatt 1440
caatgccgat ctctatttac ttctgtgtcg ggaaaatgta cagacattta cccgtaaaat 1500
agcgatggta aacgattttg acagcagctt tggctaccgc aatctgcgca tccataagtc 1560
tgccatttgt gaacctttgc agggggaaaa cgcctggagt tacgcagaaa aactgaaact 1620
ggcgatttcc agtatccgtg accatatgtt ctcagagttt attttctgtg atgacgcgaa 1680
actcaacgaa atagaagaga atatctggat tgcgcgtaat attcgccatg caatggaaat 1740
tggcgaacta ttcctcgtct atcaaccgat cgttgatatt aacacccgcg ccattctggg 1800
cgcggaggcg ttgtgccgtt gggtgtctgc ggagcggggg atcatttcac cgctgaagtt 1860
cattaccatt gctgaagata tcgggtttat caatgagctg ggttatcaga ttattaaaac 1920
ggcaatgggt gaattcagac attttagtca gcgtgcgtcg ctgaaggatg atttcttact 1980
gcatattaat gtttcgccct ggcagttaaa cgaaccacac tttcatgagc gttttaccac 2040
catcatgaaa gaaaatggcc tgaaggcgaa cagcctctgt gttgagatca ctgaaaccgt 2100
gatcgagcga attaatgaac atttttatct caatattgaa caactgcgta aacaaggggt 2160
acggatatcg attgatgact ttggcaccgg tttgtcaaac ctgaaacgtt tttatgaaat 2220
taatccagac agcataaagg tggactcgca attcaccggc gatattttcg gtactgcggg 2280
aaaaattgtg cgcattattt tcgacctggc acgctataac cggatcccgg tgattgcgga 2340
aggcgtagag agcgaagacg ttgcgcgcga attaatcaaa ttaggatgtg ttcaggctca 2400
ggggtatctg taccagaaac ccatgccatt ctccgcctgg gataaaagtg gaaaattagt 2460
aaaagagtag 2470
<210> 32
<211> 883
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 32
aacgctgatg atatgcgcct tctatactta acgtttattc agcgttaagt ggagaactcg 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatggcacag gttgcgatta ttaccgcctc cgattcgggg atcggcaaag agtgcgcgtt 180
attactggcg cagcaggggt ttgatattgg tattacctgg cactcagatg aagaaggggc 240
aaaagatacc gcgcgtgagg tagttagcca cggcgtacgt gcggagatcg tgcagctgga 300
tctcggcaat ctaccagaag gggcactggc gctggagaaa ctcattcaac ggctggggcg 360
cattgatgtg ctggtgaata atgcgggtgc aatgaccaaa gcgccgtttc ttgatatggc 420
ttttgatgag tggcgcaaga tttttaccgt tgatgtcgat ggtgcattct tatgctcgca 480
aattgcggct cgtcagatgg tgaaacaagg gcagggcggt cgcatcatca acattacgtc 540
ggtacatgaa catacgccgc tgccggatgc cagcgcctac acagccgcta aacatgcgct 600
cggtgggtta accaaagcga tggcgctgga gctggtcagg cataagattt tggtgaacgc 660
agtcgcgcct ggggcgatcg ccacgccaat gaatggcatg gatgacagcg acgtgaagcc 720
cgacgcggag ccttcgattc ccttgcggcg ttttggcgca acgcatgaga ttgccagcct 780
ggtggtgtgg ctttgttcgg agggcgcaaa ttacaccacc gggcagtcgt tgatagtgga 840
tggcggcttt atgttggcga atccacagtt caacccagaa tag 883
<210> 33
<211> 14
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 33
Met Arg Ile Phe Val Tyr Gly Ser Leu Arg His Lys Gln Gly
1 5 10
<210> 34
<211> 93
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 34
Met Ser Arg Arg Asn Thr Asp Ala Ile Thr Ile His Ser Ile Leu Asp
1 5 10 15
Trp Ile Glu Asp Asn Leu Glu Ser Pro Leu Ser Leu Glu Lys Val Ser
20 25 30
Glu Arg Ser Gly Tyr Ser Lys Trp His Leu Gln Arg Met Phe Lys Lys
35 40 45
Glu Thr Gly His Ser Leu Gly Gln Tyr Ile Arg Ser Arg Lys Met Thr
50 55 60
Glu Ile Ala Gln Lys Leu Lys Glu Ser Asn Glu Pro Ile Leu Tyr Leu
65 70 75 80
Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln Thr Leu Thr
85 90
<210> 35
<211> 127
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 35
Met Ser Arg Arg Asn Thr Asp Ala Ile Thr Ile His Ser Ile Leu Asp
1 5 10 15
Trp Ile Glu Asp Asn Leu Glu Ser Pro Leu Ser Leu Glu Lys Val Ser
20 25 30
Glu Arg Ser Gly Tyr Ser Lys Trp His Leu Gln Arg Met Phe Lys Lys
35 40 45
Glu Thr Gly His Ser Leu Gly Gln Tyr Ile Arg Ser Arg Lys Met Thr
50 55 60
Glu Ile Ala Gln Lys Leu Lys Glu Ser Asn Glu Pro Ile Leu Tyr Leu
65 70 75 80
Ala Glu Arg Tyr Gly Phe Glu Ser Gln Gln Thr Leu Thr Arg Thr Phe
85 90 95
Lys Asn Tyr Phe Asp Val Pro Pro His Lys Lys Arg Met Thr Asn Met
100 105 110
Gln Gly Glu Ser Arg Phe Leu His Pro Leu Asn His Tyr Asn Ser
115 120 125
<210> 36
<211> 810
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 36
Met Ser Val Ile Ala Gln Ala Gly Ala Lys Gly Arg Gln Leu His Lys
1 5 10 15
Phe Gly Gly Ser Ser Leu Ala Asp Val Lys Cys Tyr Leu Arg Val Ala
20 25 30
Gly Ile Met Ala Glu Tyr Ser Gln Pro Asp Asp Met Met Val Val Ser
35 40 45
Ala Ala Gly Ser Thr Thr Asn Gln Leu Ile Asn Trp Leu Lys Leu Ser
50 55 60
Gln Thr Asp Arg Leu Ser Ala His Gln Val Gln Gln Thr Leu Arg Arg
65 70 75 80
Tyr Gln Cys Asp Leu Ile Ser Gly Leu Leu Pro Ala Glu Glu Ala Asp
85 90 95
Ser Leu Ile Ser Ala Phe Val Ser Asp Leu Glu Arg Leu Ala Ala Leu
100 105 110
Leu Asp Ser Gly Ile Asn Asp Ala Val Tyr Ala Glu Val Val Gly His
115 120 125
Gly Glu Val Trp Ser Ala Arg Leu Met Ser Ala Val Leu Asn Gln Gln
130 135 140
Gly Leu Pro Ala Ala Trp Leu Asp Ala Arg Glu Phe Leu Arg Ala Glu
145 150 155 160
Arg Ala Ala Gln Pro Gln Val Asp Glu Gly Leu Ser Tyr Pro Leu Leu
165 170 175
Gln Gln Leu Leu Val Gln His Pro Gly Lys Arg Leu Val Val Thr Gly
180 185 190
Phe Ile Ser Arg Asn Asn Ala Gly Glu Thr Val Leu Leu Gly Arg Asn
195 200 205
Gly Ser Asp Tyr Ser Ala Thr Gln Ile Gly Ala Leu Ala Gly Val Ser
210 215 220
Arg Val Thr Ile Trp Ser Asp Val Ala Gly Val Tyr Ser Ala Asp Asp
225 230 235 240
Arg Lys Val Lys Asp Ala Cys Leu Leu Pro Leu Leu Arg Leu Asp Glu
245 250 255
Ala Ser Glu Leu Ala Arg Leu Ala Ala Pro Val Leu His Ala Arg Thr
260 265 270
Leu Gln Pro Val Ser Gly Ser Glu Ile Asp Leu Gln Leu Arg Cys Ser
275 280 285
Tyr Thr Pro Asp Gln Gly Ser Thr Arg Ile Glu Arg Val Leu Ala Ser
290 295 300
Gly Thr Gly Ala Arg Ile Val Thr Ser His Asp Asp Val Cys Leu Ile
305 310 315 320
Glu Phe Gln Val Pro Ala Ser Gln Asp Phe Lys Leu Ala His Lys Glu
325 330 335
Ile Asp Gln Ile Leu Lys Arg Ala Gln Val Arg Pro Leu Ala Val Gly
340 345 350
Val His Asn Asp Arg Gln Leu Leu Gln Phe Cys Tyr Thr Ser Glu Val
355 360 365
Ala Asp Ser Ala Leu Lys Ile Leu Asp Glu Ala Gly Leu Pro Gly Glu
370 375 380
Leu Arg Leu Arg Gln Gly Leu Ala Leu Val Ala Met Val Gly Ala Gly
385 390 395 400
Val Thr Arg Asn Pro Leu His Cys His Arg Phe Trp Gln Gln Leu Lys
405 410 415
Gly Gln Pro Val Glu Phe Thr Trp Gln Ser Asp Asp Gly Ile Ser Leu
420 425 430
Val Ala Val Leu Arg Thr Gly Pro Thr Glu Ser Leu Ile Gln Gly Leu
435 440 445
His Gln Ser Val Phe Arg Ala Glu Lys Arg Ile Gly Leu Val Leu Phe
450 455 460
Gly Lys Gly Asn Ile Gly Ser Arg Trp Leu Glu Leu Phe Ala Arg Glu
465 470 475 480
Gln Ser Thr Leu Ser Ala Arg Thr Gly Phe Glu Phe Val Leu Ala Gly
485 490 495
Val Val Asp Ser Arg Arg Ser Leu Leu Ser Tyr Asp Gly Leu Asp Ala
500 505 510
Ser Arg Ala Leu Ala Phe Phe Asn Asp Glu Ala Val Glu Gln Asp Glu
515 520 525
Glu Ser Leu Phe Leu Trp Met Arg Ala His Pro Tyr Asp Asp Leu Val
530 535 540
Val Leu Asp Val Thr Ala Ser Gln Gln Leu Ala Asp Gln Tyr Leu Asp
545 550 555 560
Phe Ala Ser His Gly Phe His Val Ile Ser Ala Asn Lys Leu Ala Gly
565 570 575
Ala Ser Asp Ser Asn Lys Tyr Arg Gln Ile His Asp Ala Phe Glu Lys
580 585 590
Thr Gly Arg His Trp Leu Tyr Asn Ala Thr Val Gly Ala Gly Leu Pro
595 600 605
Ile Asn His Thr Val Arg Asp Leu Ile Asp Ser Gly Asp Thr Ile Leu
610 615 620
Ser Ile Ser Gly Ile Phe Ser Gly Thr Leu Ser Trp Leu Phe Leu Gln
625 630 635 640
Phe Asp Gly Ser Val Pro Phe Thr Glu Leu Val Asp Gln Ala Trp Gln
645 650 655
Gln Gly Leu Thr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly Lys Asp
660 665 670
Val Met Arg Lys Leu Val Ile Leu Ala Arg Glu Ala Gly Tyr Asn Ile
675 680 685
Glu Pro Asp Gln Val Arg Val Glu Ser Leu Val Pro Ala His Cys Glu
690 695 700
Gly Gly Ser Ile Asp His Phe Phe Glu Asn Gly Asp Glu Leu Asn Glu
705 710 715 720
Gln Met Val Gln Arg Leu Glu Ala Ala Arg Glu Met Gly Leu Val Leu
725 730 735
Arg Tyr Val Ala Arg Phe Asp Ala Asn Gly Lys Ala Arg Val Gly Val
740 745 750
Glu Ala Val Arg Glu Asp His Pro Leu Ala Ser Leu Leu Pro Cys Asp
755 760 765
Asn Val Phe Ala Ile Glu Ser Arg Trp Tyr Arg Asp Asn Pro Leu Val
770 775 780
Ile Arg Gly Pro Gly Ala Gly Arg Asp Val Thr Ala Gly Ala Ile Gln
785 790 795 800
Ser Asp Ile Asn Arg Leu Ala Gln Leu Leu
805 810
<210> 37
<211> 810
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 37
Met Ser Val Ile Ala Gln Ala Gly Ala Lys Gly Arg Gln Leu His Lys
1 5 10 15
Phe Gly Gly Ser Ser Leu Ala Asp Val Lys Cys Tyr Leu Arg Val Ala
20 25 30
Gly Ile Met Ala Glu Tyr Ser Gln Pro Asp Asp Met Met Val Val Ser
35 40 45
Ala Ala Gly Ser Thr Thr Asn Gln Leu Ile Asn Trp Leu Lys Leu Ser
50 55 60
Gln Thr Asp Arg Leu Ser Ala His Gln Val Gln Gln Thr Leu Arg Arg
65 70 75 80
Tyr Gln Cys Asp Leu Ile Ser Gly Leu Leu Pro Ala Glu Glu Ala Asp
85 90 95
Ser Leu Ile Ser Ala Phe Val Ser Asp Leu Glu Arg Leu Ala Ala Leu
100 105 110
Leu Asp Ser Gly Ile Asn Asp Ala Val Tyr Ala Glu Val Val Gly His
115 120 125
Gly Glu Val Trp Ser Ala Arg Leu Met Ser Ala Val Leu Asn Gln Gln
130 135 140
Gly Leu Pro Ala Ala Trp Leu Asp Ala Arg Glu Phe Leu Arg Ala Glu
145 150 155 160
Arg Ala Ala Gln Pro Gln Val Asp Glu Gly Leu Ser Tyr Pro Leu Leu
165 170 175
Gln Gln Leu Leu Val Gln His Pro Gly Lys Arg Leu Val Val Thr Gly
180 185 190
Phe Ile Ser Arg Asn Asn Ala Gly Glu Thr Val Leu Leu Gly Arg Asn
195 200 205
Gly Ser Asp Tyr Ser Ala Thr Gln Ile Gly Ala Leu Ala Gly Val Ser
210 215 220
Arg Val Thr Ile Trp Ser Asp Val Ala Gly Cys Tyr Ser Ala Asp Pro
225 230 235 240
Arg Lys Val Lys Asp Ala Cys Leu Leu Pro Leu Leu Arg Leu Asp Glu
245 250 255
Ala Ser Glu Leu Ala Arg Leu Ala Ala Pro Val Leu His Ala Arg Thr
260 265 270
Leu Gln Pro Val Ser Gly Ser Glu Ile Asp Leu Gln Leu Arg Cys Ser
275 280 285
Tyr Thr Pro Asp Gln Gly Ser Thr Arg Ile Glu Arg Val Leu Ala Ser
290 295 300
Gly Thr Gly Ala Arg Ile Val Thr Ser His Asp Asp Val Cys Leu Ile
305 310 315 320
Glu Phe Gln Val Pro Ala Ser Gln Asp Phe Lys Leu Ala His Lys Glu
325 330 335
Ile Asp Gln Ile Leu Lys Arg Ala Gln Val Arg Pro Leu Ala Val Gly
340 345 350
Val His Asn Asp Arg Gln Leu Leu Gln Phe Cys Tyr Thr Ser Glu Val
355 360 365
Ala Asp Ser Ala Leu Lys Ile Leu Asp Glu Ala Gly Leu Pro Gly Glu
370 375 380
Leu Arg Leu Arg Gln Gly Leu Ala Leu Val Ala Met Val Gly Ala Gly
385 390 395 400
Val Thr Arg Asn Pro Leu His Cys His Arg Phe Trp Gln Gln Leu Lys
405 410 415
Gly Gln Pro Val Glu Phe Thr Trp Gln Ser Asp Asp Gly Ile Ser Leu
420 425 430
Val Ala Val Leu Arg Thr Gly Pro Thr Glu Ser Leu Ile Gln Gly Leu
435 440 445
His Gln Ser Val Phe Arg Ala Glu Lys Arg Ile Gly Leu Val Leu Phe
450 455 460
Gly Lys Gly Asn Ile Gly Ser Arg Trp Leu Glu Leu Phe Ala Arg Glu
465 470 475 480
Gln Ser Thr Leu Ser Ala Arg Thr Gly Phe Glu Phe Val Leu Ala Gly
485 490 495
Val Val Asp Ser Arg Arg Ser Leu Leu Ser Tyr Asp Gly Leu Asp Ala
500 505 510
Ser Arg Ala Leu Ala Phe Phe Asn Asp Glu Ala Val Glu Gln Asp Glu
515 520 525
Glu Ser Leu Phe Leu Trp Met Arg Ala His Pro Tyr Asp Asp Leu Val
530 535 540
Val Leu Asp Val Thr Ala Ser Gln Gln Leu Ala Asp Gln Tyr Leu Asp
545 550 555 560
Phe Ala Ser His Gly Phe His Val Ile Ser Ala Asn Lys Leu Ala Gly
565 570 575
Ala Ser Asp Ser Asn Lys Tyr Arg Gln Ile His Asp Ala Phe Glu Lys
580 585 590
Thr Gly Arg His Trp Leu Tyr Asn Ala Thr Val Gly Ala Gly Leu Pro
595 600 605
Ile Asn His Thr Val Arg Asp Leu Ile Asp Ser Gly Asp Thr Ile Leu
610 615 620
Ser Ile Ser Gly Ile Phe Ser Gly Thr Leu Ser Trp Leu Phe Leu Gln
625 630 635 640
Phe Asp Gly Ser Val Pro Phe Thr Glu Leu Val Asp Gln Ala Trp Gln
645 650 655
Gln Gly Leu Thr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly Lys Asp
660 665 670
Val Met Arg Lys Leu Val Ile Leu Ala Arg Glu Ala Gly Tyr Asn Ile
675 680 685
Glu Pro Asp Gln Val Arg Val Glu Ser Leu Val Pro Ala His Cys Glu
690 695 700
Gly Gly Ser Ile Asp His Phe Phe Glu Asn Gly Asp Glu Leu Asn Glu
705 710 715 720
Gln Met Val Gln Arg Leu Glu Ala Ala Arg Glu Met Gly Leu Val Leu
725 730 735
Arg Tyr Val Ala Arg Phe Asp Ala Asn Gly Lys Ala Arg Val Gly Val
740 745 750
Glu Ala Val Arg Glu Asp His Pro Leu Ala Ser Leu Leu Pro Cys Asp
755 760 765
Asn Val Phe Ala Ile Glu Ser Arg Trp Tyr Arg Asp Asn Pro Leu Val
770 775 780
Ile Arg Gly Pro Gly Ala Gly Arg Asp Val Thr Ala Gly Ala Ile Gln
785 790 795 800
Ser Asp Ile Asn Arg Leu Ala Gln Leu Leu
805 810
<210> 38
<211> 540
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 38
Met Arg Val Asn Asn Gly Leu Thr Pro Gln Glu Leu Glu Ala Tyr Gly
1 5 10 15
Ile Ser Asp Val His Asp Ile Val Tyr Asn Pro Ser Tyr Asp Leu Leu
20 25 30
Tyr Gln Glu Glu Leu Asp Pro Ser Leu Thr Gly Tyr Glu Arg Gly Val
35 40 45
Leu Thr Asn Leu Gly Ala Val Ala Val Asp Thr Gly Ile Phe Thr Asp
50 55 60
Arg Ser Pro Lys Asp Lys Tyr Ile Val Arg Asp Asp Thr Thr Arg Asp
65 70 75 80
Thr Phe Trp Trp Ala Asp Lys Gly Lys Gly Lys Asn Asp Asn Lys Pro
85 90 95
Leu Ser Pro Glu Thr Trp Gln His Leu Lys Gly Leu Val Thr Arg Gln
100 105 110
Leu Ser Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Asn
115 120 125
Pro Asp Thr Arg Leu Ser Val Arg Phe Ile Thr Glu Val Ala Trp Gln
130 135 140
Ala His Phe Val Lys Asn Met Phe Ile Arg Pro Ser Asp Glu Glu Leu
145 150 155 160
Ala Gly Phe Lys Pro Asp Phe Ile Val Met Asn Gly Ala Lys Cys Thr
165 170 175
Asn Pro Gln Trp Lys Glu Gln Gly Leu Asn Ser Glu Asn Phe Val Ala
180 185 190
Phe Asn Leu Thr Glu Arg Met Gln Leu Ile Gly Gly Thr Trp Tyr Gly
195 200 205
Gly Glu Met Lys Lys Gly Met Phe Ser Met Met Asn Tyr Leu Leu Pro
210 215 220
Leu Lys Gly Ile Ala Ser Met His Cys Ser Ala Asn Val Gly Glu Lys
225 230 235 240
Gly Asp Val Ala Val Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr
245 250 255
Leu Ser Thr Asp Pro Lys Arg Arg Leu Ile Gly Asp Asp Glu His Gly
260 265 270
Trp Asp Asp Asp Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys
275 280 285
Thr Ile Lys Leu Ser Lys Glu Ala Glu Pro Glu Ile Tyr Asn Ala Ile
290 295 300
Arg Arg Asp Ala Leu Leu Glu Asn Val Thr Val Arg Glu Asp Gly Thr
305 310 315 320
Ile Asp Phe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr
325 330 335
Pro Ile Tyr His Ile Asp Asn Ile Val Lys Pro Val Ser Lys Ala Gly
340 345 350
His Ala Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe Gly Val Leu
355 360 365
Pro Pro Val Ser Arg Leu Thr Ala Asp Gln Thr Gln Tyr His Phe Leu
370 375 380
Ser Gly Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Ile Thr Glu
385 390 395 400
Pro Thr Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu
405 410 415
His Pro Thr Gln Tyr Ala Glu Val Leu Val Lys Arg Met Gln Ala Ala
420 425 430
Gly Ala Gln Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys
435 440 445
Arg Ile Ser Ile Lys Asp Thr Arg Ala Ile Ile Asp Ala Ile Leu Asn
450 455 460
Gly Ser Leu Asp Asn Ala Glu Thr Phe Thr Leu Pro Met Phe Asn Leu
465 470 475 480
Ala Ile Pro Thr Glu Leu Pro Gly Val Asp Thr Lys Ile Leu Asp Pro
485 490 495
Arg Asn Thr Tyr Ala Ser Pro Glu Gln Trp Gln Glu Lys Ala Glu Thr
500 505 510
Leu Ala Lys Leu Phe Ile Asp Asn Phe Asp Lys Tyr Thr Asp Thr Pro
515 520 525
Ala Gly Ala Ala Leu Val Ala Ala Gly Pro Lys Leu
530 535 540
<210> 39
<211> 1303
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 39
tgtttatttt ctgacaagca gcgtaaactc cgcgtcttcc tcttccagtg atcgaccagc 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgcataac tcccccgcag tctccagcgc gaaatcgttt gacctgacct cgacggcgtt 180
tttaatcgtt gcctttctca ccggtattgc gggcgctctg caaaccccga cactcagtat 240
ttttcttacc gatgaagtac atgcccgtcc ggcgatggtg ggattcttct ttaccggcag 300
cgctgtcatt gggattctgg taagtcagtt tctcgccggg cgctctgata agcgcggcga 360
tcgcaaatcg ctgattgtct tttgctgcct gttaggcgtg ctggcctgca ccctttttgc 420
ctggaatcgc aactactttg ttttgctatt cgttggcgtc tttcttagca gctttggctc 480
gaccgctaac ccgcaaatgt ttgcccttgc ccgtgaacat gccgacaaaa ccggacgtga 540
ggcggtgatg ttcagctctt ttttacgcgc tcaggtttca ctggcatggg tcattggccc 600
accgctggct tatgccttag cgatgggttt cagctttacg gtaatgtatc tgagcgcagc 660
ggtagcgttt attgtttgcg gtgtgatggt gtggctgttt ttaccgtcga tgcgaaaaga 720
gcttccgctg gcgaccggca cgatcgaagc gccgcgccgt aaccgtcgcg atacgctgct 780
gctgtttgtc atttgtacat tgatgtgggg ctcgaacagc ctgtacatca tcaacatgcc 840
gctatttatt atcaacgaac tgcatcttcc cgagaaactg gccggtgtga tgatggggac 900
cgccgccggg ctggaaatcc cgacgatgtt gattgccgga tatttcgcca aacgtctggg 960
taagcgtttc ttaatgcgcg ttgctgccgt gggtggcgtc tgtttttacg caggaatgct 1020
gatggcgcat tcacctgtca ttctgttggg cttgcagctg ctaaatgcta tttttattgg 1080
cattctgggc ggcatcggga tgctctattt tcaggatctg atgcccggtc aggcgggttc 1140
agccaccacg ctctatacca acacttcgcg cgtgggctgg atcatcgcag gatcagtggc 1200
gggcatcgtc gccgagatct ggaattatca cgctgtgttc tggtttgcga tggtgatgat 1260
tatcgccact ctgttttgct tactgcggat taaagatgtt taa 1303
<210> 40
<211> 532
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 40
tgaagcaatt tagataatcg tgcagactac gccccctcat atcacatgga aggtttatct 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatggatcag gtagtcattt ttaaacaaat atttgataaa gttcgaaacg atttaaacta 180
tcaatggttt tattctgagc taaaacgtca caatgtctca cattacattt actatttagc 240
cacagagaat gttcatattg tattaaaaaa tgataataca gtgttattaa agggcctaaa 300
aaacattgtg tctgtcaaat tttcaaagga taggcatctt atagaaacga cctctaataa 360
gctgaaatcc agagagatca catttcagga atacagaaga aaccttgcta aagcaggagt 420
ttttcggtgg gttacaaata tccacgaaca aaaaagatat tactatacct ttgataattc 480
attactattt actgaaagca tccagaaaac tacacagatc ttaccacgct aa 532
<210> 41
<211> 943
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 41
cgtctatagt atttatgagg gtttgctttt aataatcata attacccacc agagtgtgat 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
aatgcgtaca accattgctg tagtgttggg tgcaattagt ttgacgtctg cttttgtgtt 180
tgcagataaa ccagacgttg ccagatcggc aaacgatgag gtcagcaccc tgttttttgg 240
tcatgatgat cgtgtgccag tgaatgacac gacccaatca ccgtgggatg cggttgggca 300
actggaaacg gccagcggca atttatgtac ggcgacgctg attgcaccca atctggcatt 360
aacggcagga cactgtttat tgacacctcc aaagggtaaa gccgataaag cagtggcgct 420
gcgttttgtg tcaaataaag gtctttggcg ctatgagatc cacgacatag aaggccgcgt 480
tgatccgaca ctgggaaagc ggttaaaagc agatggggat ggttggattg tacctcccgc 540
agccgcgccg tgggacttcg gattgattgt gctacgtaat cccccttctg gcattacgcc 600
gttgccgtta tttgagggag ataaagccgc gcttaccgcc gcattaaaag cggcaggtcg 660
taaagtgact caggcaggct accctgaaga tcatctcgat acgttgtaca gtcatcaaaa 720
ctgtgaagtg actggctggg cgcaaacgtc ggtgatgtca catcagtgcg ataccttgcc 780
gggtgacagc ggttcgcctc tgatgttgca taccgatgac ggctggcaat taattggggt 840
gcaaagttcg gctcctgccg cgaaagatcg ctggcgcgcc gataaccggg ccatttctgt 900
taccggtttt cgcgacaagc tggatcaact gtcgcaaaaa taa 943
<210> 42
<211> 466
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 42
atgatgtgtt aaaattgatg taaacaaatt gtgaagtgaa tgtgcttccg gggaaaataa 60
ttgacggcta gctcagtcct aggtacagtg ctagctaagt aggtacgtaa ggaggtgata 120
agtgacttca ttacaactct caatcgtcca tcgactgccg cagaactatc gctggtctgc 180
tggtttcgca ggttcgaagg ttgaaccgat tccgcaaaat ggaccgtgcg gtgacaacag 240
cctggtggcg cttaaattgc ttagcccgga tggtgataat gcatggtcgg tgatgtataa 300
actaagccag gcgttaagcg acatcgaagt tccatgttcg gtgctggagt gtgaaggtga 360
gccgtgcctg tttgtaaatc gccaggacga gtttgctgca acatgccgat tgaaaaattt 420
tggtgtggca attgccgaac ccttttcaaa ctacaatcct ttttaa 466

Claims (20)

1.一种大肠杆菌(E.coli)细胞，所述大肠杆菌细胞具有以下：具有SEQ ID No.1的氨基酸序列的dapA蛋白、核酸SEQ ID No.2的dapA基因启动子序列和选自以下蛋白的另外蛋白：具有SEQ ID No.3的氨基酸序列的lysC蛋白、具有SEQ ID No.4的核酸序列的garD蛋白编码序列、具有SEQ ID No.5的核酸序列的yicL蛋白编码序列、具有SEQ ID No.6的氨基酸序列的lysP蛋白、具有SEQ ID No.7的核酸序列的mgSA蛋白编码序列或具有SEQ ID No.8的氨基酸序列的pckE蛋白。
2.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有SEQ IDNo.1的氨基酸序列的dapA蛋白、核酸SEQ ID No.2的dapA基因启动子序列和具有SEQ IDNo.3的氨基酸序列的lysC蛋白。
3.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有SEQ IDNo.1的氨基酸序列的dapA蛋白、核酸SEQ ID No.2的dapA基因启动子序列和具有SEQ IDNo.4的核酸序列的garD蛋白编码序列。
4.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有SEQ IDNo.1的氨基酸序列的dapA蛋白、核酸SEQ ID No.2的dapA基因启动子序列和具有SEQ IDNo.5的核酸序列的yicL蛋白编码序列。
5.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有SEQ IDNo.1的氨基酸序列的dapA蛋白、核酸SEQ ID No.2的dapA基因启动子序列和具有SEQ IDNo.6的氨基酸序列的lysP蛋白。
6.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有SEQ IDNo.1的氨基酸序列的dapA蛋白、具有核酸SEQ ID No.2的dapA基因启动子序列和具有核酸序列SEQ ID No.7的mgSA蛋白编码序列。
7.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有SEQ IDNo.1的氨基酸序列的dapA蛋白、核酸SEQ ID No.2的dapA基因启动子序列和具有SEQ IDNo.8的氨基酸序列的pckE蛋白。
8.一种大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQ ID No.2的启动子序列的dapA基因和以下蛋白之一：具有SEQ ID No.9的核酸序列的amyA蛋白编码序列、具有SEQ ID No.10的氨基酸序列的amyA蛋白、具有SEQ ID No.11的氨基酸序列的cysN蛋白、具有SEQ ID No.12的核酸序列的dosP蛋白编码序列、具有核酸序列SEQ ID No.13的emrE蛋白编码序列、具有核酸序列SEQ ID No.14的focB蛋白编码序列、具有核酸序列SEQ ID No.15的glnD蛋白编码序列、具有SEQ ID No.16的氨基酸序列的glnE蛋白、具有SEQ ID No.17的核酸序列的hicB蛋白编码序列、具有SEQ ID No.18的核酸序列的maeB蛋白编码序列、具有SEQ ID No.19的氨基酸序列的marA蛋白或具有SEQ ID No.20的氨基酸序列的metL蛋白。
9.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQ IDNo.2的启动子序列的dapA基因和具有SEQ ID No.9的核酸序列的amyA蛋白编码序列。
10.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.10的氨基酸序列的amyA蛋白。
11.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.11的氨基酸序列的cysN蛋白。
12.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.12的核酸序列的dosP蛋白编码序列。
13.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.13的核酸序列的蛋白编码序列。
14.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.14的核酸序列的focB蛋白编码序列。
15.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.15的核酸序列的glnD蛋白编码序列。
16.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.16的氨基酸序列的glnE蛋白。
17.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.17的核酸序列的hicB蛋白编码序列。
18.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.18的核酸序列的maeB蛋白编码序列。
19.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.19的氨基酸序列的marA蛋白。
20.根据权利要求1所述的大肠杆菌细胞，所述大肠杆菌细胞具有以下：具有核酸SEQID No.2的启动子序列的dapA基因和具有SEQ ID No.20的氨基酸序列的metL蛋白。
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