CN110114469A - 唾液酸化化合物的体内合成 - Google Patents

唾液酸化化合物的体内合成 Download PDF

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CN110114469A
CN110114469A CN201780080949.9A CN201780080949A CN110114469A CN 110114469 A CN110114469 A CN 110114469A CN 201780080949 A CN201780080949 A CN 201780080949A CN 110114469 A CN110114469 A CN 110114469A
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J·博普雷兹
P·库西蒙特
D·范赫普
G·彼得斯
A·瓦考特兰
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Abstract

本发明属于合成生物学和代谢工程技术领域。具体而言，本发明属于代谢工程微生物的发酵技术领域。本发明描述了能够通过细胞内生物合成途径合成唾液酸化化合物的工程微生物。这些微生物可使N‑乙酰葡糖胺‑6‑磷酸去磷酸化为N‑乙酰葡糖胺，并将N‑乙酰葡糖胺转化为N‑乙酰甘露糖胺。这些微生物还能将N‑乙酰甘露糖胺转化为N‑乙酰神经氨酸。此外，本发明提供了通过在培养基中培养微生物来大规模体内合成唾液酸化化合物的方法，所述培养基任选地包含外源性前体，例如但不限于乳糖、乳N二糖、N‑乙酰乳糖胺和/或苷元，其中所述微生物在细胞内使N‑乙酰葡糖胺‑6‑磷酸去磷酸化为N‑乙酰葡糖胺，将N‑乙酰葡糖胺转化为N‑乙酰甘露糖胺，并将后者进一步转化为N‑乙酰神经氨酸。

Description

唾液酸化化合物的体内合成
技术领域
本发明属于合成生物学和代谢工程技术领域。更具体而言，本发明属于代谢工程微生物的发酵技术领域。本发明描述了能够通过细胞内生物合成途径合成唾液酸化化合物的工程微生物。这些微生物可使N-乙酰葡糖胺-6-磷酸去磷酸化成为N-乙酰葡糖胺，并将N-乙酰葡糖胺转化为N-乙酰甘露糖胺。这些微生物还能将N-乙酰甘露糖胺转化为N-乙酰神经氨酸。此外，本发明提供了通过在培养基中培养微生物大规模体内合成唾液酸化化合物的方法，所述培养基任选地包含外源性前体，例如但不限于乳糖、乳-N-二糖、N-乙酰乳糖胺和/或苷元，其中所述微生物在细胞内使N-乙酰葡糖胺-6-磷酸去磷酸化成为N-乙酰葡糖胺，将N-乙酰葡糖胺转化为N-乙酰甘露糖胺并将后者进一步转化为N-乙酰神经氨酸。
背景技术
诸如唾液酸和唾液酸化寡糖的唾液酸化化合物由于其广泛的应用范围而在近年来受到关注。例如，唾液酸被认为是抗病毒前体。唾液酸化寡糖形成人乳的重要部分，并认为具有抗粘连特性和免疫调节特性；其他人描述了它们参与大脑发育。通常，蛋白质、脂质或苷元的唾液酸化用于抗癌药物和神经疾病的治疗中。
唾液酸是用于描述主要存在于真核细胞的细胞表面上的酸性糖大家族的通用术语。最常见的唾液酸是N-乙酰神经氨酸或Neu5Ac，Neu5Ac是一种酸性九碳糖，其经过多次修饰产生唾液酸家族成员。如在例如WO2008097366的图1中所看到的，唾液酸家族的多样性由超过50种已知成员表示。唾液酸代表衍生自称为N-乙酰神经氨酸或Neu5Ac的酸性九碳母体化合物的细胞表面碳水化合物大家族。Neu5Ac通常用乙酰基、磷酸根、甲基、硫酸根和乳酰基团修饰，它们被描述为唾液酸介导的期望的细胞信号传导和细胞粘附事件所需的。
唾液酸和唾液酸化化合物在高等真核生物中是常见的，高等真核生物在保守的生物合成途径中产生它们。该途径从内源性UDP-N-乙酰葡糖胺开始，其通过UDP-N-乙酰葡糖胺2-差向异构酶(水解)(EC 3.2.1.183)、N-酰基甘露糖胺激酶(EC 2.7.1.60)、N-酰基神经氨酸-9-磷酸合酶(EC 2.5.1.57)和Neu5Ac-9-P磷酸酶(EC 3.1.3.29)的作用转化为唾液酸。随后可以通过CMP-唾液酸合酶(EC 2.7.7.43)和例如唾液酸转移酶将该唾液酸活化并转移至所需的接纳体。
已经努力在其他真核生物中表达这种生物合成途径，然而没有报道原核系统。该途径在酵母(巴斯德毕赤酵母(Pichia pastoris))和植物(拟南芥(Arabidopsisthaliana))中功能性表达，产生唾液酸化的N-聚糖。然而，从未报道大规模生产唾液酸化寡糖。原核系统中真核基因的功能性过表达仍然是一项艰巨的任务，由于缺乏特定的分子伴侣、错误的酶折叠和缺失细胞器，因此没有确定的结果。除此以外，该途径的巨大能量需要和细胞间UDP-GlcNAc(UDP-N-乙酰葡糖胺)的消耗仍然是细胞生长所需要的。
存在基于酶促、化学以及发酵方式产生唾液酸化化合物的方法。但是，它们都有明显的缺点。例如，化学合成需要许多连续的化学步骤，酶促合成需要昂贵的前体，而发酵过程仍在尽力开发中。尽管如此，后者具有最高的工业生产潜力。
一种类型的所述发酵生产方法使用源自原核生物如天然产生唾液酸或唾液酸化化合物的空肠弯曲杆菌(Campylobacter jejuni)的生物合成途径。该生物合成途径从细胞用于其细胞壁的内源性UDP-N-乙酰葡糖胺开始。通过UDP-N-乙酰葡糖胺差向异构酶(通常称为neuC)和唾液酸合酶(通常称为neuB)的作用将其转化为N-乙酰甘露糖胺和N-乙酰神经氨酸。
仅使用该原核生物合成途径的一部分，Priem等人(Glycobiology 12,2002,235-240)描述了使用活细菌细胞来产生唾液酸寡糖。在该方法中，通过生长代谢工程大肠杆菌(E.coli)菌株的细胞直接产生唾液酸乳糖，所述大肠杆菌菌株过表达脑膜炎奈瑟氏球菌(Neisseria meningitidis)的α-2,3-唾液酸转移酶和CMP-Neu5Ac合酶的基因，这些菌株进一步缺乏β-半乳糖苷酶和N-乙酰神经氨酸(Neu5Ac)醛缩酶活性。这些微生物以甘油作为碳和能量源进行高细胞密度生长，而外源乳糖和Neu5Ac作为唾液酸乳糖合成的前体提供。在生长期间，通过诱导大肠杆菌半乳糖苷和外源性Neu5Ac通透酶表达使乳糖和Neu5Ac内在化。乳糖和Neu5Ac在细胞质中积累，然后Neu5Ac转化为CMP-Neu5Ac，进一步转移到乳糖上形成唾液酸乳糖。通过这种微生物方法大规模生产唾液酸寡糖需要大量Neu5Ac作为前体。
开发了另一种不需要外源性唾液酸供应而产生唾液酸寡糖的微生物系统。WO2007101862描述了用微生物产生唾液酸化寡糖的这样的方法，所述微生物包含编码CMP-Neu5Ac合成酶、唾液酸合酶、UDP-GlcNAc-6-磷酸2差向异构酶和唾液酸转移酶的异源基因，并且其中编码唾液酸醛缩酶(NanA)和ManNAc激酶(NanK)的内源基因已缺失或失活。这种原核生物合成途径的使用对细胞来说是非常耗能的。此外，所述用于产生唾液酸化寡糖的途径竞争对于细胞自身的肽聚糖合成是必需的UDP-GlcNAc。基于这一概念，Kang等人(Metabolic engineering 14,2012,623-629)已经创制了不使用唾液酸合酶而是具有不太有利的化学平衡的内源性唾液酸醛缩酶的生产宿主。
EP1484406描述了使用过表达N-乙酰葡糖胺2-差向异构酶和Neu5Ac合酶的大肠杆菌产生Neu5Ac，但需要N-乙酰葡糖胺(GlcNAc)作为外部前体。在所述方法中，GlcNAc需要按原样使用。因此，需要破坏EP1484406中的细胞，使得GlcNAc可以直接被GlcNAc-2-差向异构酶使用。如Lundgren等人(Org.Biomol.Chem.,2007,5,1903-1909)所述，完整细胞会将进入的GlcNAc转化为N-乙酰葡糖胺-6-磷酸(GlcNAc-6-P)，其将被细胞用于细胞生长。该GlcNAc-6-P不能在细胞间获得，因此不能用于GlcNAc-2-差向异构酶，其需要非磷酸化的GlcNAc用于差向异构化为ManNAc。这解释了为什么EP1484406中细胞的透化是必要的。如Lundgren等人所解释的，GlcNAc-6-P可用于制备Neu5Ac，但这需要另一种包括UDP-GlcNAc作为中间体的合成途径，UDP-GlcNAc在上文WO2007101862中有描述。与后一专利中描述的途径相比，所得到的途径进一步增加了能量需求，因为GlcNAc的尿苷化需要额外的ATP。
Deng等人(Metabolic Engineering 7(2005),201-214)描述了通过细胞内产生GlcNAc-6-P然后GlcNAc-6-P被有效地去磷酸化并作为GlcNAc分泌到培养基中而产生GlcNAc。根据Deng等人，这种去磷酸化发生在输出时，更具体地说是在大肠杆菌(Escherichia coli)的周质中。该方法所述的细胞外产生的GlcNAc不能用于细胞内转化。产生GlcNAc的这种方法需要两阶段补料分批过程，即细胞生长阶段，然后是GlcNAc生产阶段，仅在培养物达到高细胞密度后诱导GlcNAc产生阶段，以使磷酸化氨基糖的抑制作用最小化。
其他人通过异源表达磷酸酶尝试产生唾液酸化化合物，但是遇到了生长降低和强代谢负担的问题(Lee和Oh,Metabolic engineering,2015,143-150)。所述生长/生物质形成降低的主要原因是引入的磷酸酶的非特异性，磷酸酶使其他必需的磷酸化化合物去磷酸化。因此，这种修饰导致适应性降低和比生产率降低。它还导致在生产过程中使生产途径发生变异的选择性压力，这降低了整个过程的稳定性。
唾液酸和唾液酸化寡糖的生产途径需要形成高水平的磷酸化中间体(例如GlcNAc-6-P)和核苷酸途径中间体。通常的理解是，这种形成通过激活非特异性磷酸酶导致这些中间体的非特异性降解，这反过来导致适应性降低。为了规避代谢途径表达对生产宿主生长的影响，使用诱导型表达系统是标准的。在该方法中，首先形成生物质，然后在生产过程中，通过例如IPTG激活生产途径。其他人将其用于产生唾液酸和唾液酸化寡糖(WO2007101862；Priem等人.Glycobiology 12,2002,235-240；Kang等人,Metabolicengineering 14,2012,623-629；Yang等人,Metabolic engineering 43,2017,21-28)。除了丧失生产力和滴度之外，使用诱导型系统的另一个缺点是中间途径代谢物(诸如GlcNAc和ManNAc)的分泌。这导致纯化需要额外的下游处理步骤，因此导致在生产唾液酸、唾液酸乳糖或其他唾液酸化化合物时更高的生产成本。
上文讨论的用于产生唾液酸化化合物的方法仍不足以满足生物技术、制药和医药工业的大量需求。成功克服上述问题的代谢工程方法将代表该领域期待已久的重大进展。
发明内容
发明概述
令人惊讶的是，本发明人已经能够创建这样的生产途径：所述生成途径不需要诱导，并且不需要UDP-GlcNAc差向异构酶，但允许组成型表达，所述组成型表达还允许更好地调整代谢途径，从而在生产过程中改善生产并减少副产物形成。
根据本发明的一个实施方案，提供了用微生物产生唾液酸化化合物的不需要诱导的方法。
根据本发明的另一实施方案，提供了不需要UDP-GlcNAc差向异构酶，并且包括调节磷酸酶表达的生产途径，所述磷酸酶表达不会对细胞造成本领域先前所示的代谢负担。本发明的所述另一实施方案还通过调节磷酸酶的表达提高唾液酸化化合物的产生。
在又一个实施方案中，当与代谢途径的基因的组成型表达组合时，上述方法还允许更好地调节代谢途径，从而在生产过程中降低副产物的形成。
发明详述
本发明描述了使用微生物产生唾液酸化化合物的经济、更有效的替代的生物合成途径。
本发明提供了通过微生物的发酵生长产生唾液酸化化合物的方法。
特别是，本发明涉及产生唾液酸化化合物的方法，其中所述方法包括在培养基中培养微生物。微生物在细胞内转化以下反应：N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，N-乙酰葡糖胺转化为N-乙酰甘露糖胺，和N-乙酰甘露糖胺转化为N-乙酰神经氨酸。此外，该微生物不能：i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺。
优选地，通过细胞内表达的磷酸酶的作用获得N-乙酰葡糖胺-6-磷酸向N-乙酰葡糖胺的转化。在另一优选的实施方案中，通过细胞内表达的N-乙酰甘露糖胺差向异构酶将N-乙酰葡糖胺转化为N-乙酰甘露糖胺。在替代的优选实施方案中，通过细胞内表达的唾液酸合酶将N-乙酰甘露糖胺转化为N-乙酰神经氨酸。甚至更优选，微生物包含所有三种酶，使得所述微生物i)通过细胞内表达的磷酸酶的作用将N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，ii)通过细胞内表达的N-乙酰甘露糖胺差向异构酶将N-乙酰葡糖胺转化为N-乙酰甘露糖胺；以及iii)通过细胞内表达的唾液酸合酶将N-乙酰甘露糖胺转化为N-乙酰神经氨酸。
优选地，本发明方法所用的微生物不能产生以下酶：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶，和iii)N-乙酰神经氨酸醛缩酶。
本发明还提供了微生物，其表达i)使N-乙酰葡糖胺-6-磷酸去磷酸化为N-乙酰葡糖胺的磷酸酶(EC 3.1.3)，ii)将N-乙酰葡糖胺(GlcNAc)转化为N-乙酰甘露糖胺(manNac)的GlcNAc 2-差向异构酶(EC 5.1.3.8)，和iii)由N-乙酰甘露糖胺(ManNAc)合成N-乙酰神经氨酸(Neu5Ac)的唾液酸合成酶(EC 2.5.1.56)。此外，该微生物不能：i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺。
在一个方面，本发明提供了能够催化以下反应的微生物：N-乙酰葡糖胺-6-磷酸在细胞内转化为N-乙酰葡糖胺，N-乙酰葡糖胺在细胞内转化为N-乙酰甘露糖胺，以及N-乙酰甘露糖胺在细胞内转化为唾液酸。
通常认为N-乙酰葡糖胺-6-磷酸天然有效地分泌到细胞外，同时通过周质中的磷酸酶去磷酸化(参见Deng等人,Metabolic Engineering 7(2005),201-214，第212页，第二栏)。因此，在没有本发明的情况下，该分泌产物将不能转化为唾液酸。此外，通过磷酸化N-乙酰葡糖胺的转运蛋白发生再内在化。
细胞内N-乙酰葡糖胺-2-差向异构酶的使用确保了比经典原核途径(通过UDP-N-乙酰葡糖胺)低的能量(ATP)消耗。这使得能够以更健康和更有效的菌株更有效地产生唾液酸、唾液酸化寡糖和/或唾液酸化产物。通过优化表达水平，克服了不利的化学平衡，并且不需要大量的游离N-乙酰葡糖胺，如文献中所述。实际上，在本领域中，该酶仅用于使用高浓度的N-乙酰葡糖胺产生N-乙酰甘露糖胺的酶促反应中。因此合乎逻辑的是，差向异构酶的使用会需要已经证明释放在培养基中的大量细胞内形成的GlcNAc(参见如上所述的Deng)，然而，本发明已经证明这可以避免。本发明优于酶促方法的另一个优点是，本发明可以使用廉价的底物，例如单糖，例如葡萄糖、半乳糖或果糖，二糖，例如蔗糖或麦芽糖，或多元醇，例如但不限于甘油。这使得通过发酵实现经济生产方法。
将N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺的不同磷酸酶(EC 3.1.3.)在本领域中有记载并且可用于本发明。来自HAD超家族和HAD样家族的磷酸酶在本领域中有记载。来自这些家族的实例可以见于由来自大肠杆菌的基因yqaB、inhX、yniC、ybiV、yidA、ybjI、yigL或cof表达的酶。在埃默森小芽枝霉(Blastocladiella emersonii)中鉴定出一种催化该反应的磷酸酶。磷酸酶通常是非特异性的，并且活性通常与家族或结构无关。因此，可以在所有磷酸酶家族中找到其他实例。如Fahs等人(ACS Chem.Biol.,2016,11(11),2944-2961)所述，通过众所周知的方法可以容易地鉴定和筛选具体的磷酸酶。
优选地，本发明的磷酸酶是HAD类磷酸酶。本文定义的HAD类磷酸酶是指任何包含下文定义的下述基序中的任何一个或多个的磷酸酶多肽：
基序1：hDxDx[TV](SEQ ID NO:73)，或
基序2：[GSTDE][DSEN]x(1-2)[hP]x(1-2)[DGTS](SEQ ID NO:74、75、76、77)，
其中h表示疏水性氨基酸(A、I、L、M、F、V、P、G)，且x可以是任何不同的氨基酸。
在另一优选的实施方案中，HAD类多肽通常以优先性渐增的顺序与SEQ ID NO:43、44、45、47、48、50、51、52、54、55或57所示的任何一种多肽具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的总体序列同一性。优选地，这些多肽还包含至少一个上述鉴定的基序。更优选地，它们包含两个基序。
使用全局比对算法确定总体序列同一性，例如程序GAP(GCG Wisconsin Package，Accelrys)中的Needleman Wunsch算法，优选默认参数，并且优选成熟蛋白质的序列(即，不考虑分泌信号或转运肽)。与总体序列同一性相比，当仅考虑保守结构域或基序时，序列同一性通常会更高。
在优选的实施方案中，HAD类多肽包含SEQ ID NO:43、44、45、47、48、50、51、52、54、55或57中的任一个。
在另一个优选的实施方案中，磷酸酶选自HAD超家族或HAD类磷酸酶家族。更优选，磷酸酶选自：i)由大肠杆菌的基因yqaB、inhX、yniC、ybiV、yidA、ybjI、yigL或cof表达的酶，ii)埃默森小芽枝霉的磷酸酶和iii)其他磷酸酶家族。
N-乙酰基-D-葡糖胺-2-差向异构酶(EC 5.1.3.8)的实例可以见于原核生物和真核生物。原核生物的实例存在于蓝藻中，例如深海单细胞蓝藻(Acaryochloris marina)、多变鱼腥藻(Anabaena variabilis)、深海鱼腥藻(Anabaena marina)、点形念珠藻(Nostocpunctiforme)、单细胞蓝藻属(Acaryochloris)物种、鱼腥藻属(Anabaena)物种、念珠藻属(Nostoc)物种和集胞藻属(Synechocystis)物种。它们也存在于拟杆菌属(Bacteroides)物种中，例如卵形拟杆菌(Bacteroides ovatus)和多形拟杆菌(Bacteroidesthetaiotaomicron)以及存在于狗咬二氧化碳噬纤维菌(Capnocytophaga canimorsus)和羞怯动弯杆菌(Mobiluncus mulieris)中。在真核生物中，N-乙酰基-D-葡糖胺-2-差向异构酶存在于大豆(Glycin max)、小家鼠(Mus musculus)、智人(Homo sapiens)、褐家鼠(Rattus norvegicus)、牛(Bos Taurus)、猪(Sus scrofa)、灰狼(Canis lupus)中。优选地，在本发明的方法和微生物中，N-乙酰甘露糖胺-2-差向异构酶选自i)来自蓝藻，更特别是来自深海单细胞蓝藻、多变鱼腥藻、深海鱼腥藻、点形念珠藻、单细胞蓝藻物种、鱼腥藻物种、念珠藻物种和集胞藻物种的N-乙酰甘露糖胺-2-差向异构酶；ii)来自拟杆菌物种，更特别是来自卵形拟杆菌、多形拟杆菌、狗咬二氧化碳噬纤维菌和羞怯动弯杆菌的N-乙酰甘露糖胺-2-差向异构酶；iii)来自大豆、小家鼠、智人、褐家鼠、牛、猪或灰狼的N-乙酰基-D-葡糖胺-2-差向异构酶。
在几种原核生物中发现了N-乙酰神经氨酸合酶(本领域也称为唾液酸合酶)(EC2.5.1.56)活性，例如无乳链球菌(Streptococcus agalatiae)、枯草芽孢杆菌(Bacillussubtilis)、嗜肺军团菌(Legionella pneumophilla)、空肠弯曲杆菌(Campylobacterjejuni)、热液海源菌(Idiomarina loihiensis)、粘摩替亚氏菌(Moritella viscosa)、Aliivibrio salmonicida、大肠杆菌、詹氏甲烷球菌(Methanocaldococcus jannaschi)、索氏梭菌(Clostridium sordellii)、瘤胃溶纤维丁酸弧菌(Butyrivibrioproteoclasticus)、Micromonas commoda或脑膜炎奈瑟氏球菌。优选地，在本发明的方法和微生物中，唾液酸(或N-乙酰神经氨酸)合酶选自：来自无乳链球菌、枯草芽孢杆菌、嗜肺军团菌、空肠弯曲杆菌、热液海源菌、粘摩替亚氏菌、Aliivibrio salmonicida、大肠杆菌、詹氏甲烷球菌、索氏梭菌、瘤胃溶纤维丁酸弧菌、Micromonas commoda或脑膜炎奈瑟氏球菌的唾液酸合酶。
在一个优选的方面，在微生物中过表达磷酸酶、N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任何一种或多种。在替代优选的方面，在微生物中引入并表达磷酸酶、N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任何一种或多种。
另一方面，微生物缺乏编码下述酶的基因：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶，和iii)N-乙酰神经氨酸醛缩酶。在另一优选的方面，编码下述酶的基因活性降低：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶和iii)N-乙酰神经氨酸醛缩酶，优选所述基因在微生物中缺失或敲除。
在另一优选的方面，微生物还编码促进乳糖摄取的蛋白质并缺乏代谢乳糖的酶。WO2016/075243描述了产生抗乳糖杀伤的微生物的方法和所得的微生物，该文献通过引用并入本文。
在优选的方面，本发明的微生物和用于本发明方法的微生物也表达CMP-唾液酸合酶(EC 2.7.7.43)和唾液酸转移酶(EC 2.4.99.1)，以活化唾液酸并将其转移至期望的化合物。
在优选的方面，通过引入使用细胞内葡糖胺-6-磷酸作为底物的葡糖胺-磷酸N-乙酰转移酶(EC2.3.1.4)获得N-乙酰葡糖胺-6-磷酸。在大多数微生物中，葡萄糖胺-6-磷酸天然存在于细胞中，但可在无抑制的情况下通过表达L-谷氨酰胺:D-果糖-6-磷酸氨基转移酶提高细胞内生产，所述L-谷氨酰胺:D-果糖-6-磷酸氨基转移酶通过蛋白质工程或通过筛选例如存在于革兰氏阳性细菌中的天然酶而获得(Deng等人,Metabolic Engineering 7(2005),201-214)。
在本发明中，以能够使N-乙酰葡糖胺-6-磷酸细胞内去磷酸化、防止N-乙酰葡糖胺-6-磷酸毒性积累并防止N-乙酰葡糖胺和/或N-乙酰甘露糖胺分泌的方式，优化将N-乙酰葡糖胺-6-磷酸转化为N-乙酰神经氨酸或唾液酸的基因的表达。所述优化是使用生产途径的基因的组成型表达的结果。在优选的实施方案中，本发明防止至少10％、20％、30％、35％、40％、45％、50％或60％所形成的N-乙酰葡糖胺和/或N-乙酰甘露糖胺的分泌。在进一步优选的实施方案中，微生物产生的细胞外N-乙酰葡糖胺和/或N-乙酰甘露糖胺比唾液酸化化合物少。更优选地，微生物产生的细胞外N-乙酰葡糖胺和/或N-乙酰甘露糖胺比唾液酸化化合物少50％、40％、30％、20％、10％、5％、2％。在本发明的另一优选实施方案中，就总细胞外碳水化合物而言，微生物产生等于或多于50％、60％、70％、80％、90％、95％、98％的细胞外唾液酸化化合物。
在具体方面，本发明涉及在不向培养基中添加任何外源性唾液酸的情况下合成唾液酸化化合物的方法。
唾液酸化化合物可以是N-乙酰神经氨酸、唾液酸化寡糖、唾液酸化脂、唾液酸化糖脂(例如但不限于神经节苷脂、神经酰胺)、唾液酸化蛋白质或唾液酸化苷元。
唾液酸化寡糖是含有带电荷的唾液酸的寡糖，即具有唾液酸残基的寡糖。它具有酸性。一些实例是3-SL(3-唾液酸乳糖)、3-唾液酸乳糖胺、6-SL(6-唾液酸乳糖或n-乙酰神经氨酸α2,6半乳糖基β1,4葡萄糖)、6-唾液酸乳糖胺，寡糖，其包括6-唾液酸乳糖、SGG六糖(Neu5Acα-2,3Galβ-1,3GalNacβ-1,3Gala-1,4Galβ-1,4Gal)、唾液酸化四糖(Neu5Ac-α-2,3Galβ-1,4GlcNAcβ-14GlcNAc)、五糖LSTD(Neu5Acα-2,3Galβ-1,4GlcNAcβ-1,3Galβ-1,4Glc)、唾液酸化乳-N-三糖、唾液酸化乳-N-四糖、唾液酸乳-N-新四糖、单唾液酸乳-N-六糖、二唾液酸乳-N-六糖I、单唾液酸乳-N-新六糖I、单唾液酸乳-N-新六糖II、二唾液酸乳-N-新六糖、二唾液酸乳-N-四糖、二唾液酸乳-N-六糖II、唾液酸乳-N-四糖a、二唾液酸乳-N-六糖I、唾液酸乳-N-四糖b、3-唾液酸-3-岩藻糖基乳糖、二唾液酸单岩藻糖基乳-N-新六糖、单岩藻糖基单唾液酸乳-N-八糖(唾液酸Lea)、唾液酸乳-N-岩藻六糖II、二唾液酸乳-N-岩藻五糖II、单岩藻糖基二唾液酸乳-N-四糖，和带有一个或几个唾液酸残基的寡糖，包括但不限于：选自以下的神经节苷脂的寡糖部分：GM3(3唾液酸乳糖,Neu5Aca-2,3Galβ-4Glc)，和包括以下的寡糖：GM3基序、GD3Neu5Aca-2,8Neu5Aca-2,3Galβ-1,4Glc GT3(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca-2,3Galβ-1,4Glc)；GM2GalNAcβ-1,4(Neu5Aca-2,3)Galβ-1,4Glc,GM1Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,3)Galβ-1,4Glc，GD1a Neu5Aca-2,3Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,3)Galβ-1,4Glc GT1a Neu5Aca-2,8Neu5Aca-2,3Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,3)Galβ-1,4Glc GD2GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4GlcGT2GspalNAcβ-1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GD1b,Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GT1b Neu5Aca-2,3Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GQ1b Neu5Aca-2,8Neu5Aca-2,3Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GT1c Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GQ1c,Neu5Aca-2,3Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GP1c Neu5Aca-2,8Neu5Aca-2,3Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Galβ-1,4Glc GD1aNeu5Aca-2,3Galβ-1,3(Neu5Aca-2,6)GalNAcβ-1,4Galβ-1,4Glc岩藻糖基-GM1Fuca-1,2Galβ-1,3GalNAcβ-1,4(Neu5Aca-2,3)Galβ-1,4Glc；通过使上述寡糖部分与神经酰胺反应或在神经酰胺上合成上述寡糖，可以将所有这些都扩展到产生相应的神经节苷脂。
上文所示的术语微生物或生物或细胞是指选自包括细菌、酵母或真菌的列表中的微生物，或者是指植物细胞或动物细胞。后面的细菌优选属于变形菌门(Proteobacteria)或厚壁菌门(Firmicutes)或蓝细菌门(Cyanobacteria)或异常球菌-栖热菌门(Deinococcus-Thermus)。属于变形菌门的后面的细菌优选属于肠杆菌科(Enterobacteriaceae)，优选属于大肠杆菌物种。后面的细菌优选涉及属于大肠杆菌物种的任何菌株，例如但不限于大肠杆菌B、大肠杆菌C、大肠杆菌W、大肠杆菌K12、大肠杆菌Nissle。更具体而言，后一术语涉及培养的大肠杆菌菌株-称为大肠杆菌K12菌株-其完全适应实验室环境，并且与野生型菌株不同，它们丧失了在肠中健壮生长的能力。众所周知的大肠杆菌K12菌株的实例是K12野生型、W3110、MG1655、M182、MC1000、MC1060、MC1061、MC4100、JM101、NZN111和AA200。因此，本发明特别涉及如上所示的突变和/或转化的大肠杆菌菌株，其中所述大肠杆菌菌株是K12菌株。更具体而言，本发明涉及如上所示的突变和/或转化的大肠杆菌菌株，其中所述K12菌株是大肠杆菌MG1655。属于厚壁菌门的后面的细菌优选属于芽孢杆菌(Bacilli)，优选乳杆菌目(Lactobacilliales)，其成员如乳酸乳杆菌(Lactobacillus lactis)、肠膜明串珠菌(Leuconostoc mesenteroides)，或芽孢杆菌目(Bacillales)，其成员例如来自芽孢杆菌属(Bacillus)物种枯草芽孢杆菌(Bacillussubtilis)或解淀粉芽孢杆菌(B.amyloliquefaciens)。属于放线杆菌(Actinobacteria)门的后面的细菌优选属于棒杆菌科(Corynebacteriaceae)，其成员是谷氨酸棒杆菌(Corynebacterium glutamicum)或非发酵棒杆菌(C.afermentans)，或属于链霉菌科(Streptomycetaceae)，其成员是灰色链霉菌(Streptomyces griseus)或弗氏链霉菌(S.fradiae)。后面的酵母优选属于子囊菌门(Ascomycota)或担子菌门(Basidiomycota)或半知菌门(Deuteromycota)或接合菌(Zygomycetes)门。后面的酵母优选属于酵母属(Saccharomyces)、毕赤酵母属(Pichia)，汉逊酵母属(Hansenula)，克鲁维酵母属(Kluyveromyces)、耶氏酵母属(Yarrowia)或假丝酵母属(Starmerella)。后面的真菌优选属于根霉属(Rhizopus)、盘基网柄菌属(Dictyostelium)、青霉属(Penicillium)、毛霉属(Mucor)或曲霉属(Aspergillus)。
用于生产宿主的培养基可任选地包含外源性前体或该前体可由菌株自身产生，例如聚糖，例如乳糖、乳糖胺、乳-N-三糖、乳-N-四糖、乳-N-新四糖；寡糖；肽；脂质或苷元。在一个具体方面，本发明的方法基于外源性前体的主动摄取，所述外源性前体例如单糖、二糖或三糖，更特别是选自以下的外源性前体：乳糖、N-乙酰乳糖胺、乳-N-二糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如但不限于红细胞三糖(globotriose)(Gal-α-1,4Gal-β-1,4Glc)，而微生物在廉价的碳底物(例如二糖如蔗糖或麦芽糖)上生长。而且，这些微生物还能够在葡萄糖、果糖或甘油上生长。外源性前体的表述旨在表示参与本发明的产物的生物合成途径的被微生物内在化的化合物。
一方面，本发明提供了产生唾液酸化形式的乳-N-三糖、乳-N-四糖或乳-N-新四糖的方法。微生物通过半乳糖基转移酶(EC2.4.1.38)和/或N-乙酰葡糖胺转移酶(EC2.4.1.90)活性合成这三种分子中的任何一种，半乳糖基转移酶优选源自包括智人、牛、Musmulatta、原鸡(Gallus gallus)、斑马鱼(Danio rerio)、幽门螺杆菌(Helicobacterpylori)和杜氏嗜血菌(Haemophilus ducrey)的组，N-乙酰葡糖胺转移酶优选源自包括牛、智人和小家鼠的组。为了增强这些寡糖的形成，缺乏编码UDP糖水解酶和半乳糖-1-磷酸尿苷酰基转移酶的基因，从而在微生物中活性降低或被敲除。
在另一方面，提供了用于产生唾液酸化寡糖的方法，其中所述方法包括培养上文所述的微生物，并且其中微生物在内部产生活化的N-乙酰神经氨酸作为唾液酸转移酶的供体底物；其中所述方法还包括在包含外源性前体的培养基中培养微生物，所述外源性前体选自乳糖、N-乙酰乳糖胺、乳-N-二糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如但不限于红细胞三糖(Gal-α-1,4Gal-β-1,4Glc)半乳糖。外源性前体被主动地吸收到微生物中，外源性前体是用于产生唾液酸化寡糖的唾液酸转移酶的接纳体底物。
在另一方面，本发明的方法提供了3唾液酸乳糖(3sialyllactose)或6唾液酸乳糖的产生。在所述方法中，将微生物在碳底物(例如葡萄糖或甘油)上以高细胞密度培养，并供应乳糖。乳糖通过乳糖通透酶内在化，并使用由N-乙酰葡糖胺内源性产生的CMP-N-乙酰神经氨酸通过重组唾液酸转移酶唾液酸化。
本发明的微生物或细胞能够在作为主要碳源的单糖、二糖、寡糖、多糖、多元醇、复合培养基或其混合物上生长。术语“主要”是指生物质形成、二氧化碳和/或副产物形成(例如酸和/或醇，例如乙酸盐、乳酸盐和/或乙醇)的最重要碳源，即20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％、98％、99％的所有所需碳来自上述碳源。在本发明的一个实施方案中，所述碳源是所述生物的唯一碳源，即100％的所有所需碳来自上述碳源。
在进一步优选的实施方案中，本发明的微生物或细胞使用WO2012/007481所述的具有生产途径和生物质途径的分裂代谢，WO2012/007481通过引用并入本文。例如，通过改变选自磷酸葡萄糖异构酶基因、磷酸果糖激酶基因、果糖-6-磷酸醛缩酶基因、果糖异构酶基因和/或果糖：PEP磷酸转移酶基因的基因，可以对所述生物进行基因修饰以积累果糖-6-磷酸。
术语单糖是指不能通过水解分解成更简单的糖的糖，被归类为醛糖或酮糖，并且每分子含有一个或多个羟基。实例是葡萄糖、果糖、半乳糖、甘露糖、核糖和/或阿拉伯糖。
术语二糖是指由化学键合的两个单糖组成的糖。实例是麦芽糖、蔗糖、乳糖、海藻糖、纤维二糖和/或壳二糖。
术语寡糖是指由化学键合的三至十个单糖组成的糖。实例是麦芽三糖、低聚果糖、低聚半乳糖、低聚甘露糖(mannan oligosaccharide)、低聚异麦芽糖(isomaltooligosaccharide)、人乳寡糖和/或低聚葡萄糖(glucooligosaccharide)。
术语多元醇是指含有多个羟基的醇。例如甘油、山梨糖醇或甘露醇。
术语复合培养基是指确切组成不确定的培养基。实例是糖蜜、玉米浆、蛋白胨、胰蛋白胨或酵母提取物。
通过向培养基中添加前体，例如N-乙酰葡糖胺、N-乙酰甘露糖胺、谷氨酰胺、谷氨酸、磷酸烯醇丙酮酸和/或丙酮酸，可以增加唾液酸化化合物的产生。
可以使用本领域已知的各种方法或其组合回收在如上所述的本发明方法中产生的唾液酸化化合物。取决于产生的唾液酸化化合物，所述化合物可在细胞外部分中获得或保留在细胞中。当产生的唾液酸化化合物保留在细胞中时，唾液酸化化合物会首先通过细胞破碎从细胞中释放出来。同样取决于产生的唾液酸化化合物，可以将细胞与细胞外部分分离。在另一种情况下，在没有首先与细胞外部分分离的情况下破坏细胞，其中通过以下技术破坏细胞，例如但不限于加热、冻融和/或通过超声处理、搅拌和/或法式压滤等剪切应力。可以通过离心、过滤、微量过滤和纳米过滤将细胞外部分和/或细胞内部分与细胞和/或细胞碎片分离。絮凝剂可用于帮助产物分离。细胞外部分或细胞内部分中的唾液酸化化合物可通过离子交换、超滤或纳米过滤或电渗析、层析法如尺寸排阻、离子层析和模拟移动床提取。从液相中过滤唾液酸化化合物的另一个实例是使用带有棉花和活性炭的深床过滤器或碳过滤器进行过滤，其中在渗透物通过碳抛光器(carbon polisher)之后，接着通过例如0.2微米微量过滤膜系统，用于去除颜色、微生物和悬浮的碳颗粒。然后可将唾液酸化化合物在真空蒸发器中浓缩，得到浓缩物。可以通过加热干燥、喷雾干燥和/或冻干来沉淀和/或干燥浓缩物，以获得高纯度的唾液酸化化合物。然后可以获得无定形形式的粉末。该无定形粉末可进一步结晶以获得结晶的唾液酸化化合物。
在示例性实施方案中，可以使用本领域已知的用于发酵的方法从培养基中分离唾液酸化化合物。例如，可以通过离心、过滤、絮凝、倾析等从培养基中除去细胞。然后，可以使用诸如离子交换等方法从细胞外部分中分离唾液酸化化合物。所述唾液酸化化合物的进一步纯化可以通过例如纳米过滤或超滤或离子交换来完成，以除去任何残留的DNA、蛋白质、LPS(内毒素)或其他杂质。
在另一个示例性实施方案中，可以使用本领域已知的用于发酵的方法从培养基中分离唾液酸乳糖。例如，可以通过离心、过滤、絮凝、倾析等从培养基中除去细胞。然后，可以使用诸如离子交换等方法从细胞外部分中分离唾液酸乳糖。所述唾液酸乳糖的进一步纯化可以通过例如纳米过滤或超滤或离子交换来完成，以除去任何残留的DNA、蛋白质、LPS(内毒素)或其他杂质。另一个纯化和配制步骤通过产物的结晶或沉淀来完成。另一个配制步骤是喷雾干燥或冻干唾液酸乳糖。
唾液酸化化合物可以含有平衡离子，例如一价离子，例如质子、钠离子、钾，二价离子，例如钙、镁、铁，或三价离子，例如铁，或离子组合。
贯穿本公开的公开内容，术语唾液酸、N-乙酰神经氨酸(N-acetyl neuraminate)和N-乙酰神经氨酸(N-acetyl neuraminic acid)可互换使用。
如本文所用，术语“细胞内(intracellular)”或“在细胞内(intracellularly)”，例如细胞内转化、细胞内产生、细胞内表达、细胞内形成，必须理解为在微生物细胞内。术语细胞外必须理解为在细胞外。
贯穿本说明书所用的其他定义
同源物
蛋白质的“同源物”包括相对于所讨论的未修饰蛋白质具有氨基酸取代、缺失和/或插入并且具有与衍生它们的未修饰蛋白质相似的生物学活性和功能活性的肽、寡肽、多肽、蛋白质和酶。
缺失是指从蛋白质中去除一个或多个氨基酸。
插入是指将一个或多个氨基酸残基引入蛋白质中的预定位点。插入可以包含单个或多个氨基酸的N末端和/或C末端融合以及序列内插入。通常，氨基酸序列内的插入会比N-或C-末端融合小约1-10个残基的量级。N-或C-末端融合蛋白质或肽的实例包括酵母双杂交系统所用的转录激活剂的结合结构域或激活结构域、噬菌体外壳蛋白、(组氨酸)-6-标签、谷胱甘肽S-转移酶-标签、蛋白质A、麦芽糖结合蛋白、二氢叶酸还原酶、Tag＞＞100表位、c-myc表位、FLAG(R)-表位、lacZ、CMP(钙调蛋白结合肽)、HA表位、蛋白质C表位和VSV表位。
取代是指用具有相似性质(例如相似的疏水性、亲水性、抗原性、形成或破坏α-螺旋结构或β-片层结构的倾向)的其他氨基酸替换蛋白质的氨基酸。氨基酸取代通常由单个残基构成，但可以根据对多肽的功能限制进行聚类，并且其范围可以为1-10个氨基酸；插入通常约为1-10个氨基酸残基的量级。氨基酸取代优选为保守氨基酸取代。保守取代表是本领域熟知的(参见例如Creighton(1984)Proteins.W.H.Freeman and Company(编)和下文表1)。
表1：保守氨基酸取代的实例
残基 保守取代 残基 保守取代
Ala Ser Leu Ile；Val
Arg Lys Lys Arg；Gln
Asn Gln；His Met Leu；Ile
Asp Glu Phe Met；Leu；Tyr
Gln As, Ser Thr；Gly
Cys Ser Thr Ser；Val
Glu Asp Trp Tyr
Gly Pro Tyr Trp；Phe
His Asn；Gln Val Ile；Leu
Ile Leu；Val
使用本领域熟知的肽合成技术，例如固相肽合成等，或通过重组DNA操作，可以容易地进行氨基酸取代、缺失和/或插入。操纵DNA序列以产生蛋白质的取代、插入或缺失变体的方法是本领域熟知的。例如，用于在DNA的预定位点进行取代突变的技术是本领域技术人员公知的，包括M13诱变、17-Gen体外诱变(USB,Cleveland,OH)、快速变化定点诱变(QuickChange Site Directed mutagenesis，Stratagene,San Diego,CA)、PCR介导的定点诱变或其他定点诱变方案。
衍生物
“衍生物”包括与蛋白质例如目的蛋白质的天然存在形式的氨基酸序列相比可以包含非天然存在的氨基酸残基对氨基酸的取代或非天然存在的氨基酸残基添加的肽、寡肽、多肽。蛋白质的“衍生物”还包括，与天然存在形式的多肽的氨基酸序列相比包含天然存在的改变的(糖基化的、酰化的、异戊烯基化的(prenylated)、磷酸化的、豆蔻酰化的、硫酸化的等)或非天然改变的氨基酸残基的肽、寡肽、多肽。衍生物与衍生其的氨基酸序列相比还可以包含一个或多个非氨基酸取代基或添加，例如与氨基酸序列共价或非共价键合的报告分子或其他配体，例如键合而促进其检测的报告分子，和相对于天然存在的蛋白质的氨基酸序列的非天然存在的氨基酸残基。此外，“衍生物”还包括天然存在形式的蛋白质与标记肽如FLAG、HIS6或硫氧还蛋白的融合(有关标记肽的综述，参见Terpe,Appl.Microbiol.Biotechnol.60,523-533,2003)。
直系同源物/旁系同源物
直系同源物和旁系同源物包括用于描述基因祖先关系的进化概念。旁系同源物是同一物种中通过复制祖先基因而产生的基因；直系同源物是来自不同生物的、起源于物种形成并且也衍生自共同祖先基因的基因。
结构域、基序/共有序列/特征(Signature)
术语“结构域”是指沿着进化相关蛋白质序列的比对在特定位置保守的一组氨基酸。虽然其他位置的氨基酸在同源物之间可以变化，但在特定位置高度保守的氨基酸表示可能在蛋白质的结构、稳定性或功能中必需的氨基酸。通过它们在蛋白质同源物家族的比对序列中的高度保守性鉴定，它们可以用作确定所讨论的任何多肽是否属于先前鉴定的多肽家族的标识符。
术语“基序”或“共有序列”或"特征"是指进化相关蛋白质序列中的短保守区域。基序通常是结构域的高度保守部分，但也可仅包括结构域的一部分，或位于保守结构域之外(如果所述基序的所有氨基酸都落在限定的结构域之外)。
存在用于鉴定结构域的专家数据库，例如SMART(Schultz等人.(1998)Proc.Natl.Acad.Sci.USA 95,5857-5864；Letunic等人(2002)Nucleic Acids Res 30,242-244)，InterPro(Mulder等人.,(2003)Nucl.Acids.Res.31,315-318)，Prosite(Bucher和Bairoch(1994),A generalized profile syntax for biomolecular sequencesmotifs and its function in automatic sequence interpretation.(In)ISMB-94；Proceedings 2nd International Conference on Intelligent Systems for MolecularBiology.Altman R.,Brutlag D.,Karp P.,Lathrop R.,Searls D.,Eds.,pp53-61,AAAIPress,Menlo Park；Hulo等人.,Nucl.Acids.Res.32:D134-D137,(2004))，或Pfam(Bateman等人.,Nucleic Acids Research30(1):276-280(2002))。用于蛋白质序列计算机分析的一组工具可在ExPASy蛋白质组学服务器上获得(Swiss Institute of Bioinformatics(Gasteiger等人,ExPASy:the proteomics server for in-depth protein knowledgeand analysis,Nucleic Acids Res.31:3784-3788(2003))。也可以使用常规技术，例如通过序列比对鉴定结构域或基序。
用于比较的序列比对的方法是本领域熟知的，这些方法包括GAP、BESTFIT、BLAST、FASTA和TFASTA。GAP使用Needleman和Wunsch((1970)J Mol Biol 48:443-453)的算法来找到使匹配数最大化并使缺口数最小化的两条序列的全局(即跨越完整序列)比对。BLAST算法(Altschul等人(1990)J Mol Biol 215:403-10)计算序列同一性百分比并对两条序列之间的相似性进行统计分析。用于进行BLAST分析的软件可通过国家生物技术信息中心(National Centre for Biotechnology Information,NCBI)公开获得。使用例如ClustalW多序列比对算法(版本1.83)，使用默认的成对比对参数和百分比评分方法可以容易地鉴定同源物。全局相似性和同一性百分比也可以使用可在MatGAT软件包中找到的方法之一来确定(Campanella等人,BMC Bioinformatics.2003Jul 10；4:29.MatGAT:an applicationthat generates similarity/identity matrices using protein or DNA sequences.)。可以进行次要手动编辑以优化保守基序之间的比对，这对于本领域技术人员来说是显而易见的。此外，对于同源物鉴定，也可以使用特定结构域，代替使用全长序列。可以使用上述程序使用默认参数，在整个核酸或氨基酸序列上或在选择的结构域或保守基序上确定序列同一性值。对于局部比对，Smith-Waterman算法特别有用(Smith TF,Waterman MS(1981)J.Mol.Biol147(1)；195-7)。
互惠BLAST
通常，这涉及第一次BLAST，其涉及将查询序列(例如，使用实施例部分的表A所列的任何序列)针对任何序列数据库(例如公众可获得的NCBI数据库)进行BLAST。当从核苷酸序列开始时通常使用BLASTN或TBLASTX(使用标准默认值)，并且当从蛋白质序列开始时使用BLASTP或TBLASTN(使用标准默认值)。可任选地过滤BLAST结果。然后将过滤结果或未过滤结果的全长序列针对来自衍生查询序列的生物的序列进行返回BLAST(第二次BLAST)。然后比较第一次和第二次BLAST的结果。如果来自第一次BLAST的高等级命中(hit)来自与衍生查询序列的物种相同的物种，则鉴别出旁系同源物，然后返回BLAST理想地导致处在最高命中之中的查询序列；如果第一次BLAST中的高等级命中不是来自与衍生查询序列的物种相同的物种，则鉴别出直系同源物，并且优选地在查询序列中返回BLAST时的结果在最高命中之中。
高等级命中是具有低E值的那些命中。E值越低，则得分越显著(或者换句话说，偶然发现命中的机会越低)。E值的计算在本领域中是公知的。除了E值之外，还通过同一性百分比对比较进行评分。同一性百分比是指在特定长度上两条比较的核酸(或多肽)序列之间的相同核苷酸(或氨基酸)的数目。在大家族的情况下，可以使用ClustalW，然后使用邻接树，以帮助显现相关基因的聚类并鉴别直系同源物和旁系同源物。
构建体
其他调节元件可包括转录增强子和翻译增强子。本领域技术人员应当知道可适用于实施本发明的终止子序列和增强子序列。如定义部分所述，还可以将内含子序列添加到5非翻译区(UTR)或编码序列中以增加在胞质溶胶中积累的成熟信息(mature message)的量。其他控制序列(除了启动子序列、增强子序列、沉默子序列、内含子序列、3UTR和/或5UTR区)可以是蛋白质和/或RNA稳定元件。这些序列是已知的或可由本领域技术人员容易地获得。
本发明的基因构建体还可以包括在具体细胞类型中维持和/或复制所需的复制起点序列。一个实例是当需要基因构建体作为附加型基因元件(例如质粒或粘粒分子)保持在细菌细胞中时。
为了检测本发明方法所用的核酸序列的成功转移和/或包含这些核酸的转基因微生物的成功选择，使用标记基因(或报告基因)是有利的。因此，基因构建体可任选地包含选择标记基因。一旦不再需要标记基因，可以将其从转基因细胞中除去或切除。用于去除标记物的技术是本领域已知的，上文定义部分描述了有用的技术。
调节元件/控制序列/启动子
术语“调节元件”、“控制序列”和“启动子”在本文中都可互换使用，并且在广义上下文中理解为表示能够实现与它们连接的序列的表达的调节核酸序列。术语“启动子”通常是指位于基因转录起始上游并参与RNA聚合酶和其他蛋白质的识别和结合从而指导可操作连接的核酸的转录的核酸控制序列。上述术语包括来自经典真核基因组基因的转录调控序列(包括准确转录起始所需的TATA盒，有或没有CCAAT盒序列)和响应于发育和/或外部刺激或以组织特异性方式改变基因表达的其他调节元件(即上游激活序列、增强子和沉默子)。所述术语还包括经典原核基因的转录调控序列，在这种情况下，它可以包括-35盒序列和/或-10盒转录调控序列。术语“调节元件”还包括赋予、激活或增强细胞、组织或器官中核酸分子表达的合成融合分子或衍生物。
组成型启动子
“组成型启动子”是指在大多数但不一定是所有生长和发育阶段以及在大多数环境条件下在至少一个细胞、组织或器官中具有转录活性的启动子。
转基因的/转基因/重组
出于本发明的目的，根据本发明，“转基因的”、“转基因”或“重组”就例如核酸序列而言是指表达盒、基因构建体或包含所述核酸序列的载体或用所述核酸序列、表达盒或载体转化的生物，所有这些构建体都通过重组方法产生，其中
(a)核酸序列，其编码可用于本发明方法的蛋白质，或
(b)基因控制序列，其与本发明的核酸序列可操作地连接，例如启动子，或
(c)a)和b)不在其天然遗传环境中或已通过重组方法进行修饰，修饰可以采取例如一个或多个核苷酸残基的取代、添加、缺失、倒置或插入的形式。天然遗传环境应理解为意指原始微生物中的天然基因组或染色体基因座或基因组文库中的存在。在基因组文库的情况下，优选至少部分地保留核酸序列的天然遗传环境。环境至少在一侧位于核酸序列的侧翼，并且其序列长度为至少50bp，优选至少500bp，特别优选至少1000bp，最优选至少5000bp。通过非天然的合成(“人工”)方法(例如诱变处理)进行修饰时，天然存在的表达盒——例如，核酸序列的天然启动子与编码可用于本发明方法的多肽的相应核酸序列的天然存在的组合，如上所定义——变成转基因表达盒。合适的方法描述于例如US5,565,350或WO 00/15815中。
因此，用于本发明目的的转基因微生物应理解为如上所述表示本发明方法所用的核酸不存在于所述微生物的基因组中，或不源自所述微生物的基因组，或存在于所述微生物的基因组中，但是不是在所述微生物基因组中的天然基因座上，所述核酸可以同源或异源表达。然而，如上所述，转基因还表示，虽然本发明的核酸或本发明方法所用的核酸位于微生物基因组中的天然位置，但该序列已经相对于天然序列得以修饰，和/或已经修改了天然序列的调节序列。转基因优选理解为是指本发明的核酸在基因组中的非天然基因座处的表达，即发生所述核酸的同源表达或优选异源表达。本文提及了优选的转基因微生物。
还应注意，在本发明的上下文中，术语“分离的核酸”或“分离的多肽”在某些情况下可以分别视为是“重组核酸”或“重组多肽”的同义词，并且是指不位于其天然遗传环境中和/或已经通过重组方法得以修饰的核酸或多肽。
调节
术语“调节”是指与表达或基因表达有关的过程，其中与对照微生物相比，所述基因表达改变表达水平，表达水平可以增加或降低。原始的未调节的表达可以是结构RNA(rRNA，tRNA)或mRNA的任何种类的表达，随后进行翻译。出于本发明的目的，原始的未调节的表达也可以不存在任何表达。术语“调节活性”是指本发明的核酸序列或编码的蛋白质的任何表达变化，这导致微生物的产量增加和/或微生物生长增加。表达可以从零(不存在表达或表达不可测量)增加到一定量，或者可以从一定量减少到不可测量的少量或零。
表达
术语“表达”或“基因表达”是指一个具体基因或数个具体基因或具体基因构建体的转录。术语“表达”或“基因表达”特别是指一个基因或数个基因或基因构建体转录成结构RNA(rRNA，tRNA)或mRNA，随后将后者翻译成蛋白质，或不翻译。该过程包括DNA的转录和所得mRNA产物的加工。
表达增加/过表达
本文所用的术语“表达增加”或“过表达”是指除原始野生型表达水平之外的任何形式的表达。出于本发明的目的，原始野生型表达水平也可以是零，即不存在表达或表达不可测量。
用于增加基因表达或基因产物的方法在本领域中有很好的记载，包括例如由适当启动子驱动的过表达，转录增强子或翻译增强子的使用。可以将用作启动子元件或增强子元件的分离的核酸引入非异源形式的多核苷酸的适当位置(通常是上游)，以便上调编码目的多肽的核酸的表达。例如，可以通过突变、缺失和/或取代在体内改变内源启动子(参见Kmiec,US 5,565,350；Zarling等人,W09322443)，或者可以以适当的方向、距本发明的基因适当的距离，将分离的启动子引入微生物细胞中，以控制基因的表达。
如果需要多肽表达，通常需要在多核苷酸编码区的3端包含多腺苷酸化区域。多腺苷酸化区域可以衍生自天然基因、衍生自多种其他微生物基因或衍生自T-DNA。
此外，本发明涉及以下具体实施方案：
1.产生唾液酸化化合物的方法，所述方法包括：
-在培养基中培养微生物，所述培养基任选地包含外源性前体，
-其中所述微生物在细胞内将N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，将所述N-乙酰葡糖胺转化为N-乙酰甘露糖胺，并将所述N-乙酰甘露糖胺转化为N-乙酰神经氨酸；和
-其中所述微生物不能i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸化转化为N-乙酰甘露糖胺。
2.根据实施方案1所述的方法，其中：
i)通过细胞内表达的磷酸酶的作用获得N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，
ii)通过细胞内表达的N-乙酰甘露糖胺差向异构酶进行所述N-乙酰葡糖胺向N-乙酰甘露糖胺的转化；和
iii)细胞内表达的唾液酸合酶将所述N-乙酰甘露糖胺转化为N-乙酰神经氨酸。
3.根据实施方案1或2中任一项所述的方法，其中所述生物不能产生以下酶：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶，和iii)N-乙酰神经氨酸醛缩酶。
4.根据实施方案1-3中任一项所述的方法，其中通过组成型表达的基因所编码的酶催化所有所述转化。
5.根据实施方案2所述的方法，其中所述磷酸酶选自HAD超家族或HAD类磷酸酶家族，优选所述磷酸酶选自：i)由大肠杆菌的基因yqaB、inhX、yniC、ybiV、yidA、ybjI、yigL或cof表达的酶，ii)埃默森小芽枝霉(Blastocladiella emersonii)的磷酸酶，和iii)其他磷酸酶家族，更优选地，所述磷酸酶是权利要求所定义的HAD类磷酸酶多肽。
6.根据实施方案2、3、4或5中任一项所述的方法，其中所述N-乙酰甘露糖胺-2-差向异构酶选自：i)来自蓝藻，特别是来自深海单细胞蓝藻(Acaryochloris marina)、多变鱼腥藻(Anabaena variabilis)、深海鱼腥藻(Anabaena marina)、点形念珠藻(Nostocpunctiforme)、单细胞蓝藻属(Acaryochloris)物种、鱼腥藻属(Anabaena)物种、念珠藻属(Nostoc)物种和集胞藻属(Synechocystis)物种的N-乙酰甘露糖胺-2-差向异构酶；ii)来自拟杆菌属(Bacteroides)物种，特别是来自卵形拟杆菌(Bacteroides ovatus)、多形拟杆菌(Bacteroides thetaiotaomicron)、狗咬二氧化碳噬纤维菌(Capnocytophagacanimorsus)和羞怯动弯杆菌(Mobiluncus mulieris)的N-乙酰甘露糖胺-2-差向异构酶；iii)来自大豆(Glycin max)、小家鼠(Mus musculus)、智人(Homo sapiens)、褐家鼠(Rattus norvegicus)、牛(Bos Taurus)、猪(Sus scrofa)或灰狼(Canis lupus)的N-乙酰基-D-葡糖胺-2-差向异构酶。
7.根据实施方案2、3、4、5或6中任一项所述的方法，其中所述唾液酸合酶选自来自以下的唾液酸合酶：无乳链球菌(Streptococcus agalatiae)，枯草芽孢杆菌(Bacillussubtilis)、嗜肺军团菌(Legionella pneumophilla)、空肠弯曲杆菌(Campylobacterjejuni)、热液海源菌(Idiomarina loihiensisf)、粘摩替亚氏菌(Moritella viscosa)、Aliivibrio salmonicida、大肠杆菌、詹氏甲烷球菌(Methanocaldococcus jannaschi)、索氏梭菌(Clostridium sordellii)、瘤胃溶纤维丁酸弧菌(Butyrivibrioproteoclasticus)、Micromonas commoda或脑膜炎奈瑟氏球菌(Neisseria meningitis)。
8.根据前述实施方案中任一项所述的方法，其中所述唾液酸化化合物选自N-乙酰神经氨酸、唾液酸化寡糖、唾液酸化脂质、唾液酸化蛋白质、唾液酸化苷元。
9.根据前述实施方案所述的方法，其中所述唾液酸化化合物是唾液酸化寡糖。
10.根据实施方案9所述的方法，其中所述唾液酸化寡糖是唾液酸乳糖，优选3-SL或6-SL中的任何一种。
11.根据实施方案9所述的方法，其中所述唾液酸化寡糖是二唾液酸乳-N-四糖。
12.根据实施方案8所述的方法，其中所述唾液酸化化合物是N-乙酰神经氨酸。
13.根据实施方案1-10中任一项所述的方法，其中所述唾液酸化化合物是唾液酸化乳-N-三糖、乳-N-四糖或乳-N-新四糖，并且其中所述微生物还包含半乳糖基转移酶(EC2.4.1.38)活性，优选地，所述半乳糖基转移酶源自包括智人(Homo sapiens)、牛(Bostaurus)、Mus mulatta、原鸡(Gallus gallus)、斑马鱼(Danio rerio)、幽门螺杆菌(Helicobacter pylori)和杜氏嗜血菌(Haemophilus ducrey)的组；和/或所述微生物包含N-乙酰葡糖胺转移酶(EC 2.4.1.90)活性，优选地，所述N-乙酰葡糖胺转移酶源自包括牛、智人和小家鼠的组。
14.根据实施方案13所述的方法，其中所述微生物不能表达编码UDP糖水解酶和半乳糖-1-磷酸尿苷酰基转移酶的基因。
15.根据实施方案1-14中任一项所述的方法，其中所述微生物产生的细胞外N-乙酰葡糖胺和/或N-乙酰甘露糖胺比唾液酸化化合物少50％、40％、30％、20％、10％、5％、2％，和/或就总碳水化合物而言，所述微生物产生等于或大于50％、60％、70％、80％、90％、95％、98％的唾液酸化化合物。
16.产生唾液酸化寡糖的方法，包括：
a)根据实施方案1-7、14和15中任一项所述的方法培养微生物，并且其中所述微生物在内部产生活化的N-乙酰神经氨酸作为唾液酸转移酶的供体底物；和
b)在包含外源性前体的培养基中培养所述微生物，所述外源性前体选自乳糖、N-乙酰乳糖胺、乳-N-二糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如但不限于红细胞三糖(Gal-α-1,4Gal-β-1,4Glc)半乳糖，其中发生所述微生物对所述外源性前体的主动摄取，并且其中所述外源性前体是用于产生所述唾液酸化寡糖的所述唾液酸转移酶的接纳体底物。
17.根据实施方案2所述的方法，其中在所述微生物中过表达所述磷酸酶、N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任何一种或多种。
18.根据实施方案2所述的方法，其中在所述微生物中引入并表达所述磷酸酶、N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任何一种或多种。
19.根据实施方案3所述的方法，其中所述微生物缺乏编码以下酶的基因：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶，和iii)N-乙酰神经氨酸醛缩酶。
20.根据实施方案3所述的方法，其中在所述微生物中，编码以下酶的基因活性降低：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶和iii)N-乙酰神经氨酸醛缩酶，优选地，所述基因缺失或敲除。
21.根据实施方案1-20中任一项所述的方法，其中所述微生物还编码促进乳糖摄取的蛋白质并且缺乏代谢乳糖的酶。
22.根据实施方案1-21中任一项所述的方法，其中所述微生物是细菌，优选大肠杆菌菌株，更优选大肠杆菌菌株是K12菌株，甚至更优选大肠杆菌K12菌株是大肠杆菌MG1655。
23.根据实施方案1-21中任一项所述的方法，其中所述微生物是酵母。
24.根据实施方案1-23中任一项所述的方法，其中所述外源性前体选自乳糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如红细胞三糖(Gal-α-1,4Gal-β-1,4Glc)。
25.用于产生唾液酸化化合物的微生物，所述微生物
-在细胞内将N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，将所述N-乙酰葡糖胺转化为N-乙酰甘露糖胺，并将所述N-乙酰甘露糖胺转化为N-乙酰神经氨酸；和
-不能i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺。
26.用于产生唾液酸化化合物的微生物，所述微生物由实施方案2-24中任一项所定义。
27.细胞培养基，其包含乳糖作为前体和实施方案25或26中任一项所述的微生物。
28.根据实施方案1-24之一所述的方法，用于产生3唾液酸乳糖或6唾液酸乳糖，其中将所述微生物在碳底物如葡萄糖或甘油上以高细胞密度培养并供应乳糖，所述乳糖通过乳糖通透酶内在化，并使用由N-乙酰葡糖胺内源产生的CMP-N-乙酰神经氨酸，通过所述重组唾液酸转移酶进行唾液酸化。
29.根据实施方案1-24中任一项所述的方法，其中通过选自离心、过滤、微量过滤、超滤、纳米过滤、离子交换、电渗析、层析法、模拟移动床、蒸发、沉淀、结晶、冻干和/或喷雾干燥的单元操作从所述培养基中分离所述唾液酸化化合物。
30.根据实施方案1-24中任一项所述的方法产生的唾液酸化化合物，其中所述唾液酸化化合物通过离心和/或过滤、离子交换、通过蒸发或纳米过滤浓缩、通过结晶或喷雾干燥配制或冻干来纯化。
31.根据实施方案1-24中任一项所述的方法产生的唾液酸化化合物，其中将所述唾液酸化化合物加入食品制剂、饲料制剂、药物制剂、化妆品制剂或农业化学制剂中。
32.根据实施方案1-24中任一项所述的方法，其中所述培养基包含以下中的任何一种或多种作为主要碳源：单糖、二糖、寡糖、多糖、多元醇、复合培养基。
33.根据实施方案32所述的方法，其中所述主要碳源为所述微生物的生长提供至少20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％、98％、99％或100％所有所需碳。
34.根据实施方案32所述的方法，其中所述单糖选自葡萄糖、果糖、半乳糖、甘露糖、核糖或阿拉伯糖。
35.根据实施方案32所述的方法，其中所述二糖选自麦芽糖、蔗糖、乳糖、海藻糖、纤维二糖或壳二糖。
36.根据实施方案32所述的方法，其中所述寡糖选自麦芽三糖、低聚果糖、低聚半乳糖、低聚甘露糖、低聚异麦芽糖或低聚葡萄糖。
37.根据实施方案32所述的方法，其中所述多元醇选自甘油。
38.根据实施方案32所述的方法，其中所述复合培养基选自糖蜜、玉米浆、蛋白胨、胰蛋白胨或酵母提取物。
在优选的方面，本发明涉及以下优选的具体实施方案：
1.用于在微生物中产生唾液酸化化合物的方法，所述方法包括：
-在培养基中培养微生物，所述培养基任选地包含外源性前体，
其中所述微生物包含至少一种编码磷酸酶的核酸、至少一种编码N-乙酰甘露糖胺差向异构酶的核酸和至少一种编码唾液酸合酶的核酸，且
其中所述微生物不能i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰-葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺；和
-调节所述微生物中编码HAD类磷酸酶多肽的核酸的表达，其中所述HAD类磷酸酶多肽包含：
-至少一个下述基序：
基序1：hDxDx[TV](SEQ ID NO:73)，或
基序2：[GSTDE][DSEN]x(1-2)[hP]x(1-2)[DGTS](SEQ ID NO:74、75、76、77)，
其中h表示疏水性氨基酸(A、I、L、M、F、V、P、G)，且x可以是任何不同的氨基酸；
-或SEQ ID NO:43、44、45、47、48、50、51、52、54、55或57中任一个的与所述多肽具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的总体序列同一性的同源物或衍生物。
2.根据优选实施方案1所述的方法，其中所述HAD类多肽包含SEQ ID NO:43、44、45、47、48、50、51、52、54、55、57中的任一个。
3.根据优选实施方案1所述的方法，其中通过在微生物中引入并表达编码HAD类多肽的核酸来实现受调节的表达。
4.根据优选实施方案1所述的方法，其中通过组成型启动子的作用实现受调节的表达。
5.根据前述优选实施方案中任一项所述的方法，其中所述唾液酸化化合物选自N-乙酰神经氨酸、唾液酸化寡糖、唾液酸化脂质、唾液酸化蛋白质、唾液酸化苷元。
6.根据前述优选实施方案所述的方法，其中所述唾液酸化化合物是唾液酸化寡糖。
7.根据优选实施方案8所述的方法，其中所述唾液酸化寡糖是唾液酸乳糖。
8.根据优选实施方案8所述的方法，其中所述唾液酸化寡糖是二唾液酸乳-N-四糖。
9.根据优选实施方案7所述的方法，其中所述唾液酸化化合物是N-乙酰神经氨酸。
10.根据优选实施方案1-9中任一项所述的方法，其中所述唾液酸化化合物是唾液酸化乳-N-三糖、乳-N-四糖或乳-N-新四糖，并且其中所述微生物还包含半乳糖基转移酶(EC 2.4.1.38)活性，优选地，所述半乳糖基转移酶源自包括智人(Homo sapiens)、牛(Bostaurus)、Mus mulatta、原鸡(Gallus gallus)、斑马鱼(Danio rerio)、幽门螺杆菌(Helicobacter pylori)和杜氏嗜血菌(Haemophilus ducrey)的组；和/或所述微生物包含N-乙酰葡糖胺转移酶(EC 2.4.1.90)活性，所述N-乙酰葡糖胺转移酶源自包含牛、智人和小家鼠的组。
11.根据优选实施方案12所述的方法，其中所述微生物不能表达编码UDP糖水解酶和半乳糖-1-磷酸尿苷酰基转移酶的基因。
12.根据优选实施方案1-13中任一项所述的方法，其中所述微生物产生的细胞外N-乙酰葡糖胺和/或N-乙酰甘露糖胺比唾液酸化化合物少50％、40％、30％、20％、10％、5％、2％，和/或就总碳水化合物而言，所述微生物产生等于或大于50％、60％、70％、80％、90％、95％、98％的唾液酸化化合物。
13.产生唾液酸化寡糖的方法，包括：
a)根据优选实施方案1-12中任一项所述的方法培养微生物，并且其中所述微生物在内部产生活化的N-乙酰神经氨酸作为唾液酸转移酶的供体底物；和
b)在包含外源性前体的培养基中培养所述微生物，所述外源性前体选自乳糖、N-乙酰乳糖胺、乳-N-二糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如但不限于红细胞三糖(Gal-α-1,4Gal-β-1,4Glc)半乳糖，其中发生所述微生物对所述外源性前体的主动摄取，并且其中所述外源性前体是用于产生所述唾液酸化寡糖的所述唾液酸转移酶的接纳体底物。
14.根据优选实施方案1所述的方法，其中在所述微生物中过表达所述N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任一种或两种。
15.根据优选实施方案1所述的方法，其中在所述微生物中引入并表达所述N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任一种或两种。
16.根据优选实施方案1所述的方法，其中所述微生物缺乏编码以下酶的基因：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶，和iii)N-乙酰神经氨酸醛缩酶。
17.根据优选实施方案1所述的方法，其中在所述微生物中，编码以下酶的基因活性降低：i)N-乙酰葡糖胺-6-磷酸脱乙酰酶，ii)N-乙酰葡糖胺激酶和iii)N-乙酰神经氨酸醛缩酶，优选地，所述基因缺失或敲除。
18.根据优选实施方案1-17中任一项所述的方法，其中所述微生物还编码促进乳糖摄取的蛋白质并缺乏代谢乳糖的酶。
19.根据优选实施方案1-18中任一项所述的方法，其中所述微生物是细菌，优选大肠杆菌菌株，更优选大肠杆菌菌株是K12菌株，甚至更优选大肠杆菌K12菌株是大肠杆菌MG1655。
20.根据优选实施方案1-18中任一项所述的方法，其中所述微生物是酵母。
21.根据优选实施方案1-20中任一项所述的方法，其中所述外源性前体选自乳糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如红细胞三糖(Gal-α-1,4Gal-β-1,4Glc)。
22.能够通过权利要求1-21中任一项所述的方法获得的微生物，其中所述微生物包含编码HAD类多肽的重组核酸。
23.用于产生唾液酸化化合物的微生物，其中所述微生物包含至少一种编码磷酸酶的核酸、至少一种编码N-乙酰甘露糖胺差向异构酶的核酸和至少一种编码唾液酸合酶的核酸，并且其中所述微生物不能i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺；其特征在于，所述微生物包括优选实施方案1中所定义的编码HAD类磷酸酶多肽的核酸的受调节的表达。
24.构建体，其包含：
(i)优选实施方案1或2所定义的编码HAD类多肽的核酸；
(ii)一种或多种能够驱动(i)的核酸序列表达的控制序列；和可选地
(iii)转录终止序列。
25.根据优选实施方案24所述的构建体，其中所述控制序列之一是组成型启动子。
26.优选实施方案24或25所述的构建体在产生唾液酸化化合物的方法中的用途。
27.根据优选实施方案1-21中任一项所述方法产生的唾液酸化化合物，其中将所述唾液酸化化合物加入食品制剂、饲料制剂、药物制剂、化妆品制剂或农业化学制剂中。
28.用于产生唾液酸化化合物的微生物，所述微生物由实施方案2-21中任一项所定义。
29.细胞培养基，其包含乳糖作为前体和实施方案22、23或28中任一项所述的微生物。
30.实施方案1-21之一所述的方法，其用于产生3唾液酸乳糖或6唾液酸乳糖，其中将所述微生物在诸如葡萄糖或甘油或蔗糖的碳底物中以高细胞密度培养，并供应乳糖，所述乳糖通过乳糖通透酶内在化，并使用由N-乙酰葡糖胺内源产生的CMP-N-乙酰神经氨酸，通过所述重组唾液酸转移酶进行唾液酸化。
31.根据实施方案1-21中任一项所述的方法，其中通过选自离心、过滤、微量过滤、超滤、纳米过滤、离子交换、电渗析、层析法、模拟移动床、蒸发、沉淀、结晶、冻干和/或喷雾干燥的单元操作从所述培养基中分离所述唾液酸化化合物。
32.根据实施方案1-21中任一项所述方法产生的唾液酸化化合物，其中通过离心和/或过滤、离子交换、通过蒸发或纳米过滤浓缩、通过结晶或喷雾干燥配制或冻干来纯化所述唾液酸化化合物。
33.根据实施方案1-21中任一项所述方法产生的唾液酸化化合物，其中将所述唾液酸化化合物加入食品制剂、饲料制剂、药物制剂、化妆品制剂或农业化学制剂中。
34.根据实施方案1-21中任一项所述的方法，其中所述培养基包含以下中的任何一种或多种作为主要碳源：单糖、二糖、寡糖、多糖、多元醇、复合培养基。
35.根据实施方案34所述的方法，其中所述主要碳源为所述微生物的生长提供至少20％、30％、40％、50％、60％、70％、75％、80％、85％、90％、95％、98％、99％或100％的所有所需碳。
36.根据实施方案34所述的方法，其中所述单糖选自葡萄糖、果糖、半乳糖、甘露糖、核糖或阿拉伯糖。
37.根据实施方案34所述的方法，其中所述二糖选自麦芽糖、蔗糖、乳糖、海藻糖、纤维二糖或壳二糖。
38.根据实施方案34所述的方法，其中所述寡糖选自麦芽三糖、低聚果糖、低聚半乳糖、低聚甘露糖、低聚异麦芽糖或低聚葡萄糖。
39.根据实施方案34所述的方法，其中所述多元醇选自甘油。
40.根据实施方案34所述的方法，其中所述复合培养基选自糖蜜、玉米浆、蛋白胨、胰蛋白胨或酵母提取物。
以下附图和实施例将用于进一步说明和澄清本发明，而非意图限制。
附图说明
图1显示了实施例2中用于根据本发明产生唾液酸的示例性途径。图1A显示了没有所有KO和过表达迹象的途径。图1B显示了实施例2中使用的途径，其中敲除用叉号表示，过表达用所示酶旁边的向上箭头表示。
图2显示了如实施例2中所述的能够产生唾液酸的大肠杆菌菌株的生产结果。
图3显示了可以在本发明的方法中产生的不同唾液酸化化合物的实例。
图4显示了补充有指定磷酸酶的菌株的光密度和唾液酸产量。
图5显示了补充有指定磷酸酶的菌株的生长速率。
图6显示了实施例中测试的磷酸酶的比对部分。
具体实施方式
实施例1：材料和方法
方法和材料：大肠杆菌
培养基
使用三种不同的培养基，即丰富Luria Broth(LB)，用于摇瓶的基本培养基(MMsf)和用于发酵的基本培养基(MMf)。两种基本培养基都使用微量元素混合物。
微量元素混合物由以下组成：3.6g/L FeCl2.4H2O、5g/L CaCl2.2H2O、1.3g/LMnCl2.2H2O、0.38g/L CuCl2.2H2O、0.5g/L CoCl2.6H2O、0.94g/L ZnCl2、0.0311g/LH3BO4、0.4g/L Na2EDTA.2H2O和1.01g/L盐酸硫胺素。钼酸盐溶液含有0.967g/LNa2MoO4.2H2O。硒溶液含有42g/L SeO2。
Luria Broth(LB)培养基由1％胰蛋白胨蛋白胨(Difco,Erembodegem,Belgium)、0.5％酵母提取物(Difco)和0.5％氯化钠(VWR,Leuven,Belgium)组成。
Luria Broth琼脂(LBA)平板由添加有12g/L琼脂(Difco,Erembodegem,Belgium)的LB培养基组成。
用于摇瓶实验的基本培养基(MMsf)含有2.00g/L NH4Cl、5.00g/L(NH4)2SO4、2.993g/L KH2PO4、7.315g/L K2HPO4、8.372g/L MOPS、0.5g/L NaCl、0.5g/L MgSO4.7H2O。使用选自但不限于葡萄糖、果糖、麦芽糖、甘油和麦芽三糖的碳源。浓度默认为15g/L，但这取决于实验而变化。1mL/L微量元素混合物，100μL/L钼酸盐溶液和1mL/L硒溶液。用1M KOH将培养基设定为pH7。根据实验，可以添加乳糖作为前体。
用于发酵的基本培养基含有6.75g/L NH4Cl，1.25g/L(NH4)2SO4，1.15g/L KH2PO4(低磷酸盐培养基)或2.93g/L KH2PO4和7.31g/L KH2PO4(高磷酸盐培养基)，0.5g/L NaCl，0.5g/L MgSO4.7H2O，碳源包括但不限于葡萄糖，蔗糖、果糖、麦芽糖、甘油和麦芽三糖，1mL/L微量元素混合物，100μL/L钼酸盐溶液，和1mL/L具有与上述相同组成的硒溶液。
通过高压灭菌(121℃，21)对复合培养基，例如LB进行灭菌，通过过滤(0.22μmSartorius)对基本培养基(MMsf和MMf)进行灭菌。必要时，通过添加抗生素(例如氨苄青霉素(100mg/L)、氯霉素(20mg/L)、羧苄青霉素(100mg/L)、壮观霉素(40mg/L)和/或卡那霉素(50mg/L))使培养基具有选择性。
菌株
大肠杆菌MG1655[λ-,F-,rph-1]于2007年3月从Coli Genetic Stock Center(US)获得,CGSC菌株编号为7740。使用同源重组构建突变菌株，如Datsenko和Wanner所述(PNAS97(2000),6640-6645)。
质粒
pKD46(Red辅助质粒，氨苄青霉素抗性)、pKD3(含有FRT侧翼氯霉素抗性(cat)基因)、pKD4(含有FRT侧翼卡那霉素抗性(kan)基因)和pCP20(表达FLP重组酶活性)质粒获自R.Cunin教授(Vrije Universiteit Brussel,Belgium，2007年)。
使用Gibson组装构建质粒pCX-CjneuB。使用Aerts等人(Eng.Life Sci.2011,11,No.1,10-19)描述的表达载体表达基因CjneuB1。
使用Gibson组装构建质粒pCX-CjneuB-NmneuA-Pdbst。使用Aerts等人(Eng.LifeSci.2011,11，No.1,10-19)描述的表达载体表达基因CjneuB1、NmneuA和Pdbst。
使用Golden Gate组装构建用于磷酸酶表达的质粒。使用启动子apFAB87和apFAB346以及UTRs基因10_SD2-接合_HisHA和UTR1AATTCGCCGGAGGGATATTAAAAtgaatggaaaattgAAACATCTTAATCATGCTAAGGAGGTTTTCTAATG(SEQ ID NO:41)表达磷酸酶(EcAphA、EcCof、EcHisB、EcOtsB、EcSurE、EcYaed、EcYcjU、EcYedP、EcYfbT、EcYidA、EcYigB、EcYihX、EcYniC、EcYqaB、EcYrbL和PsMupP)。Mutalik等人(Nat.Methods 2013,No.10,354-360)描述了除UTR1之外的所有启动子和UTR。还使用相同的启动子和UTR表达磷酸酶EcAppA、EcGph、EcSerB、EcNagD、EcYbhA、EcYbiV、EcYbjL、EcYfbR、EcYieH、EcYjgL、Ec YjjG、EcYrfG、EcYbiU、ScDOG1和BsAraL。
使用pBR322载体通过Golden Gate组装构建质粒pBR322-NmneuB。用于表达NmNeuB的启动子和UTR是启动子apFAB299和UTR galE_SD2-接合_BCD12。使用pSC101载体通过Golden Gate组装构建质粒pSC101-NmneuA-Pdbst。用于表达NmneuA的启动子和UTR是启动子apFAB37和UTR galE_SD2-接合_BCD18。用于表达Pdbst的启动子和UTR是启动子apFAB339和UTR galE_SD2-接合_BCD12。Mutalik等人(Nat.Methods 2013,No.10,354-360)描述了所有启动子和UTR。
将质粒维持在宿主大肠杆菌DH5α(F-,phi80dlacZδM15,δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk-,mk+),phoA,supE44,λ-,thi-1,gyrA96,relA1)中，其购自Invitrogen。
基因破坏
使用Datsenko和Wanner(PNAS 97(2000),6640-6645)公布的技术进行基因破坏以及基因导入。该技术基于由λRed重组酶进行同源重组后的抗生素选择。随后翻转酶重组酶的催化确保去除最终生产菌株中的抗生素选择盒。
在表A中列出了构建基因破坏盒所必需的引物。
表A：构建用于靶基因的破坏盒的引物列表。
基于与之前讨论的用于基因破坏的相同技术，将必需基因基因组整合到生产宿主基因组中，对破坏盒进行特定改变。整合的构建体位于同源位点和破坏盒的FRT位点之间。这允许构建体在同源位点所示的区域中巧妙整合。
使用该工作流程，直接基因破坏和基因组整合是可能的。用于在特定位点靶整合的引物列于表B。
表B：基因组整合所用的引物
使携带温度敏感性pKD46辅助质粒的克隆在10mL具有氨苄青霉素(100mg/L)和L-阿拉伯糖(10mM)的LB培养基中于30℃生长至OD600nm为0.6。通过连续洗涤，一次用50mL冰冷的去离子水，一次用1mL冰冷的去离子水，使细胞成为电感受态。接下来，将细胞重悬于50μL冰冷水中。最后，将10-100ng破坏/整合盒加入到50μL经洗涤的细胞溶液中，用于电穿孔。使用Gene Pulser(BioRad的商标)(600Ohm 25μFD，和250V)进行电穿孔。
电穿孔后，将细胞在1mL LB培养基中于37℃复苏1小时，最后铺板到含有25mg/L氯霉素或50mg/L卡那霉素的LB琼脂上，以选择抗生素抗性转化体。通过用修饰区上游和下游的引物的PCR验证所选突变体，随后使其在42℃在LB琼脂上生长，以丢失pKD46辅助质粒。最后测试突变体的氨苄青霉素敏感性。
用pCP20质粒转化选择的突变体(氯霉素或卡那霉素抗性)，pCP20质粒是表现出温度敏感性复制和FLP合成热诱导的氨苄青霉素和氯霉素抗性质粒。在30℃选择氨苄青霉素抗性转化体，然后在42℃在LB中对一些转化体进行菌落纯化，然后测试丧失所有抗生素抗性并因此丧失FLP辅助质粒的转化体。用对照引物检查基因破坏和/或基因整合，并测序。这些引物列于表C中。
表C：用于验证特定基因靶标的基因破坏和/或基因组整合的引物。
异源表达和同源表达
需要表达的基因，无论是来自质粒还是来自基因组，都用以下公司之一以合成方式合成：DNA2.0、Gen9或IDT。
从大肠杆菌K-12MG1655基因组中挑选大肠杆菌天然基因，例如磷酸酶。其他基因的来源在相关表中列出。
通过将密码子应用优化成表达宿主的密码子应用，可以进一步促进表达。使用供应商的工具优化基因。
培养条件
96孔微量滴定板实验的预培养从在175μL中的LB平板上的单个菌落开始，并在37℃在轨道振荡器上以800rpm孵育8小时。该培养物用作96孔微量滴定板的接种体，使用175μL MMsf培养基稀释300倍。再次通过稀释300倍，这些培养物进而用作96孔板中最终实验的预培养物。96孔板可以是微量滴定板，培养体积为175μL，或24孔深孔板，培养体积为3mL。
摇瓶实验的预培养从在5mL LB培养基中的LB平板上的单个菌落开始，并在37℃在轨道振荡器上以200rpm孵育8小时。从该培养物中，将1mL转移至500mL摇瓶中的100mL基本培养基(MMsf)中，并在37℃在轨道振荡器上以200rpm孵育。该设置用于摇瓶实验。
生长16小时的摇瓶实验也可用作生物反应器的接种体。用4％的该细胞溶液接种具有4L工作体积的2L Biostat Dcu-B，其由MFCS控制软件(Sartorius Stedim Biotech,Melsungen,Germany)控制。培养条件设定为37℃，800rpm搅拌，气体流速为1.5L/min。使用0.5M H2SO4和25％NH4OH将pH控制在7。将废气冷却。当在发酵过程中起泡(约106L)时加入10％的硅氧烷消泡剂溶液。不需要使用诱导剂，因为所有基因都是组成型表达的。
材料和方法：酿酒酵母(Saccharomyces cerevisae)
培养基
使菌株在具有完全补充混合物(Complete Supplement Mixture，SD CSM)或不含CSM(SD CSM-Ura)的合成的确定成分的酵母培养基上生长，所述培养基含有6.7g/L不含氨基酸的酵母氮碱(YNB，不含AA，Difco)、20g/L琼脂(Difco)(固体培养物)、22g/L葡萄糖一水合物或20g/L乳糖和0.79g/L CSM或0.77g/L CSM-Ura(MP Biomedicals)。
菌株
由Bachmann等人(Yeast(1998)14:115-32)创制的酿酒酵母(Saccharomycescerevisiae)BY4742用于Euroscarf培养物收集。使用Gietz(Yeast 11:355-360,1995)的方法通过同源重组或质粒转化创制所有突变菌株。乳酸马克思克鲁维酵母(Kluyveromycesmarxianus lactis)可在LMG培养物保藏中心(LMG culture collection,Ghent,Belgium)获得。
质粒
酵母表达质粒p2a_2μ_sia_GFA1(Chan 2013(Plasmid 70(2013)2-17))用于在酿酒酵母中表达外源基因。该质粒含有氨苄青霉素抗性基因和细菌复制起点，以允许在大肠杆菌中进行选择和维持。质粒还含有2μ酵母起点(ori)和Ura3选择标记，用于在酵母中进行选择和维持。最后，所述质粒可含有β-半乳糖苷酶表达盒。接下来，该质粒还含有N-乙酰葡糖胺-2-差向异构酶(例如来自卵形拟杆菌(BoAGE))和唾液酸合酶(例如来自空肠弯曲杆菌(CjneuB))。最后，它还含有来自酿酒酵母的果糖-6-P-氨基转移酶ScGFA1。
酵母表达质粒p2a_2μ_sia_glmS基于p2a_2μ_sia，但是以表达glmS*54(来自大肠杆菌的果糖-6-P-氨基转移酶)的方式进行了修饰。
酵母表达质粒p2a_2μ_sia_glmS_phospha基于p2a_2μ_sia_glmS，但是也以表达以下的方式进行了修饰：EcAphA(SEQ ID NO:42)、EcCof(SEQ ID NO:43)、EcHisB(SEQ ID NO:44)、EcOtsB(SEQ ID NO:45)、EcSurE(SEQ ID NO:46)、EcYaed(SEQ ID NO:47)、EcYcjU(SEQID NO:48)、EcYedP(SEQ ID NO:49)、EcYfbT(SEQ ID NO:50)、EcYidA(SEQ ID NO:51)、EcYigB(SEQ ID NO:52)、EcYihX(SEQ ID NO:53)、EcYniC(SEQ ID NO:54)、EcYqaB(SEQ IDNO:55)、EcYrbL(SEQ ID NO:56)、PsMupP(SEQ ID NO:57)、EcAppA(SEQ ID NO:58)、EcGph(SEQ ID NO:59)、EcSerB(SEQ ID NO:60)、EcNagD(SEQ ID NO:61)、EcYbhA(SEQ ID NO:62)、EcYbiV(SEQ ID NO:63)、EcYbjL(SEQ ID NO:64)、EcYfbR(SEQ ID NO:65)、EcYieH(SEQID NO:66)、EcYjgL(SEQ ID NO:67)、Ec YjjG(SEQ ID NO:68)、EcYrfG(SEQ ID NO:69)、EcYbiU(SEQ ID NO:70)、ScDOG1(SEQ ID NO:71)和BsAraL(SEQ ID NO:72)。
酵母表达质粒p2a_2μ_SL-glmS基于p2a_2μ_sia，但是也以表达KlLAC12(来自乳酸克鲁维酵母(Kluyveromyces lactis)的乳糖通透酶)、NmneuA(来自脑膜炎奈瑟氏球菌(Neisseria meningitides)的CMP-唾液酸合酶)和Pdbst(来自美人鱼发光杆菌(Photobacterium damselae)的唾液酸转移酶)的方式进行了修饰。
将质粒维持在宿主大肠杆菌DH5α(F-,phi80dlacZδM15,δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk-,mk+),phoA,supE44,λ-,thi-1,gyrA96,relA1)中。其购自Invitrogen。
基因表达启动子
如Blazeck(Biotechnology and Bioengineering,Vol.109,No.11,2012)所述，使用合成的组成型启动子表达基因。
异源表达和同源表达
需要表达的基因，无论是来自质粒还是来自基因组，都可以通过以下公司之一以合成方式合成：DNA2.0、Gen9或IDT。
通过将密码子应用优化为表达宿主的密码子应用，可以进一步促进表达。使用供应商的工具优化基因。
培养条件
通常，最初使酵母菌株在SD CSM平板上生长，以获得单个菌落。使这些平板在30℃生长2-3天。
从单个菌落开始，使预培养物在30℃以200rpm振荡以5mL生长过夜。随后在100mL培养基中用2％这种预培养物接种500mL摇瓶实验。将这些摇瓶在30℃以200rpm轨道振荡孵育。不需要使用诱导剂，因为所有基因都是组成型表达的。
材料和方法：枯草芽孢杆菌(Bacillus subtilis)
培养基
使用两种不同的培养基，即丰富Luria Broth(LB)、用于摇瓶的基本培养基(MMsf)。基本培养基使用微量元素混合物。
微量元素混合物由以下组成：0.735g/L CaCl2.2H2O、0.1g/L MnCl2.2H2)、0.033g/L CuCl2.2H2O、0.06g/L CoCl2.6H2O、0.17g/L ZnCl2、XX g/L H3BO4、XX g/LNa2EDTA.2H2O和0.06g/L Na2MoO4。柠檬酸铁溶液含有0.135g/L FeCl3.6H2O、1g/L柠檬酸钠(Hoch 1973PMC1212887)。
Luria Broth(LB)培养基由以下组成：1％胰蛋白胨蛋白胨(Difco,Erembodegem,Belgium)、0.5％酵母提取物(Difco)和0.5％氯化钠(VWR,Leuven,Belgium)。
Luria Broth琼脂(LBA)平板由添加了12g/L琼脂(Difco,Erembodegem,Belgium)的LB培养基组成。
用于摇瓶实验的基本培养基(MMsf)含有2g/L(NH4)2SO4、7.5g/L KH2PO4、17.5g/LK2HPO4、1.25g/L柠檬酸钠、0.25g/L MgS04.7H2O、0.05g/L色氨酸，10-30g/L葡萄糖或另一种碳源，包括但不限于葡萄糖、果糖、麦芽糖、甘油和麦芽三糖，10mL/L微量元素混合物和10mL/L柠檬酸铁溶液。用1M KOH将培养基设定为pH 7。
通过高压灭菌(121℃，21)对复合培养基，例如LB进行灭菌，通过过滤(0.22μmSartorius)对基本培养基(MMsf)进行灭菌。如果需要，通过加入抗生素(例如博来霉素(zeocin)(20mg/L))使培养基具有选择性。
菌株
枯草芽孢杆菌168，可从Bacillus Genetic Stock Center(Ohio,USA)获得。
质粒和基因过表达
如Yan等人(Appl&environm microbial,sept 2008,p5556-5562)所述，通过Cre/lox构建用于基因缺失的质粒。
表达载体可以见于Mobitec(Germany)或ATCC(number 87056)。在这些表达载体中克隆基因BsglmS、ScGNA1和CjneuB。用于表达的合适启动子可以衍生自部分储存库(part repository)(iGem)：序列id:BBa_K143012,BBa_K823000,BBa_K823002或BBa_K823003。可以使用Gibson组装、Golden Gate组装、Cliva组装、LCR或限制连接进行克隆。
将质粒维持在宿主大肠杆菌DH5α(F-,phi 80dlacZδM15,δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk-,mk+),phoA,supE44,λ-,thi-1,gyrA96,relA1)中。其其购自Invitrogen。
基因破坏
如Xue等人(J.microb.Meth.34(1999)183-191)所述，通过与线性DNA的同源重组和通过电穿孔的转化进行基因破坏。Liu等人(Metab.Engine.24(2014)61-69)描述了基因敲除的方法。该方法使用靶基因上游和下游的1000bp同源物。表D列出了本发明所用的同源物。修饰后，使用修饰区上游和下游的引物验证突变体。这些引物在表E中给出。接下来，通过测序确认修饰(在LGC Genomics(LGC group,Germany)进行)。
表D
表E
靶基因 正向引物 反向引物
nagA-nagB Tgtaatcgggcggttaattc(SEQ ID NO:37) Gccctttcaggcgatagag(SEQ ID NO:38)
gamA Acggcgaaaatcagcggaag(SEQ ID NO:39) Tcactctccgtcggcagctg(SEQ ID NO:40)
异源表达和同源表达
需要表达的基因，无论是来自质粒还是来自基因组，都用以下公司之一以合成方式合成：DNA2.0、Gen9或IDT。
通过将密码子应用优化成表达宿主的密码子使应，可以进一步促进表达。使用供应商的工具优化基因。
培养条件
将来自LB-平板上的单个菌落的预培养物在5mL LB培养基中在37℃在轨道振荡器上以200rpm孵育8小时。从该培养物中，将1mL转移至500mL摇瓶中的100mL基本培养基(MMsf)中，并在37℃在轨道振荡器上以200rpm孵育。该设置用于摇瓶实验。不需要使用诱导剂，因为所有基因都是组成型表达的。
分析方法
光密度
通过测量600nm处的光密度(Implen Nanophotometer NP80,Westburg,Belgium)经常监测培养物的细胞密度。通过在预先干燥并称重的Falcons离心(10min,5000g,LegendX1R Thermo Scientific,Belgium)20g反应器培养液获得细胞干重。随后用20mL生理溶液(9g/L NaCI)洗涤沉淀物一次，并在70℃干燥至恒重。为了能够将OD600nm测量值转换为生物质浓度，制作OD600nm与生物质浓度的相关性曲线。
细胞干重的测量
将4×10g从培养液样品中转移至离心管中，将细胞离心(5000g,4℃,5min)，并用0.9％NaCl溶液洗涤细胞两次。将含有细胞沉淀物的离心管在烘箱中在70℃干燥48小时直至恒重。通过重量分析获得细胞干重；在称重之前，将试管在干燥器中冷却。
液相色谱法
使用具有ELSD检测器的Waters Acquity UPLC H-class系统，使用Acquity UPLCBEH酰胺，1.7μm，在35℃加热2.1mm x 50mm，使用具有0.2％三乙胺的75/25乙腈/水溶液(0.130mL/min)作为流动相，测定诸如但不限于葡萄糖、果糖和乳糖的碳水化合物的浓度。
在同一台机器上，用相同的柱定量唾液酸乳糖。然而将洗脱液修改为具有1％甲酸的75/25乙腈/水溶液。将流量设定为0.130mL/min，将柱温设定为35℃。
使用REZEX ROA柱(300x 7.8mm ID)在同一台机器上定量唾液酸。洗脱液为0.08％乙酸水溶液。将流量设定为0.5mL/min，将柱温设定为65℃。还使用这种方法测量GlcNAc和ManNAc。
生长速率测量
使用R Package grofit基于在600nm处观察到的光密度计算最大生长速率(μMax)。
实施例2：大肠杆菌中唾液酸的产生
第一个实施例提供了能够产生N-乙酰神经氨酸(唾液酸)的大肠杆菌菌株(参见图1B)。
将能够使用蔗糖作为碳源累积葡糖胺-6-磷酸的菌株进一步改造以允许N-乙酰神经氨酸产生。基础菌株过表达来自青春双歧杆菌(Bifidobacterium adolescentis)的蔗糖磷酸化酶(BaSP)、来自运动发酵单胞菌(Zymomonas mobilis)的果糖激酶(Zmfrk)、突变果糖-6-P-氨基转移酶(EcglmS*54,如Deng等人(Biochimie 88,419-429(2006))所述)。为了允许基因唾液酸产生，破坏操纵子nagABCDE、nanATEK和manXYZ。在nagABCDE的位置引入BaSP和Zmfrk，并在nanATEK的位置引入EcglmS*54。如实施例1中所述，并且基于Datsenko&Wanner(PNAS USA 97,6640-6645(2000))的原则，进行这些修饰。
在这种菌株中，通过过表达来自酿酒酵母的葡糖胺-6-P-氨基转移酶(ScGNA1)、来自卵形拟杆菌的N-乙酰葡糖胺-2-差向异构酶(BoAGE)和来自空肠弯曲杆菌的唾液酸合成酶(CjneuB)，来实施本发明所述的用于产生唾液酸的生物合成途径。分别在位置nagABCDE和manXYZ上表达ScGNA1和BoAGE。使用高拷贝质粒pCX-CjneuB表达CjneuB。
如实施例1(材料和方法)所述培养菌株。简而言之，接种5mL LB预培养物，并使之在37℃生长过夜。该培养物在使用100mL培养基的摇瓶实验中用作接种体，所述培养基含有10g/L蔗糖，并如实施例1所述制备。如实施例1所述采集常规样品并进行分析。容易追踪生物质、蔗糖和唾液酸的浓度变化，在细胞外产生的N-乙酰神经氨酸的终浓度为0.22g/L，如图2所示。
相同生物也基于葡萄糖、麦芽糖或甘油作为碳源产生N-乙酰神经氨酸。
实施例3：大肠杆菌中6-唾液酸乳糖的产生
本发明的另一个实例是所述方法和菌株用于产生6-唾液酸乳糖的用途。
实施例3的菌株是实施例2所用的菌株的子菌株。通过过表达来自大肠杆菌的乳糖通透酶EclacY(如WO 2016/075243的实施例1所述和所证明的，还通过引用将WO 2016/075243并入本文)、来自脑膜炎奈瑟氏球菌的CMP-唾液酸合成酶(NmneuA)和来自发光杆菌的唾液酸转移酶(Pdbst)，进一步修饰所述菌株。除此之外，破坏了lacZ。
由质粒表达基因NmneuA和Pdbst以及CjneuB。该质粒是pCX-CjneuB-NmneuA-Pdbst，并如实施例1中所述制备。
如实施例1中所述，接种所述菌株，作为由5ml LB培养基组成的预培养物。在37℃在培养箱中生长过夜后，将1％的预培养物接种在含有100ml培养基(MMsf)的摇瓶中，所述培养基含有10g/l蔗糖作为碳源和10g/l乳糖作为前体。使菌株在37℃生长300小时。
该菌株产生大量6-唾液酸乳糖。
实施例4：使用异源果糖-6-P-氨基转移酶在酿酒酵母中产生唾液酸
另一个实例提供了酿酒酵母形式的真核生物在本发明中的用途。通过引入并表达N-乙酰葡糖胺-2-差向异构酶(例如来自卵形拟杆菌(BoAGE))和唾液酸合酶(例如来自空肠弯曲杆菌(CjneuB))在酿酒酵母中获得利用本发明途径的这种方法。
作为起点，需要具有增加的向N-乙酰葡糖胺-6-磷酸的代谢通量的菌株。这通过过表达来自大肠杆菌的果糖-6-P-氨基转移酶突变体(EcglmS*54)来实现。
为了创制本发明的产生N-乙酰神经氨酸的酿酒酵母，通过2-micron质粒(Chan2013(Plasmid 70(2013)2-17))引入所述基因，并且还如实施例1所述，使用合成的组成型启动子(Blazeck 2012(Biotechnology and Bioengineering,Vol.109,No.11))表达所述基因。本实施方案所用的具体质粒是p2a_2μ_sia_glmS。使用Gietz和Woods(2002,PMID12073338)描述的转化技术将该质粒引入酿酒酵母中，并获得突变菌株。
所述菌株能够将果糖-6-磷酸转化为葡糖胺-6-磷酸，然后将葡糖胺-6-磷酸转化为N-乙酰葡糖胺-6-磷酸。该N-乙酰葡糖胺-6-磷酸部分进一步转化为N-乙酰葡糖胺，所述N-乙酰葡糖胺转化为N-乙酰甘露糖胺，最后将该N-乙酰甘露糖胺转化为N-乙酰神经氨酸。
所述菌株的预培养在5mL含有22g/L葡萄糖的合成的确定成分培养基SD-CSM中进行，并如实施例1所述在30℃生长。将该预培养物接种于摇瓶中具有10g/L蔗糖作为唯一碳源的100mL培养基中，并在30℃生长。如实施例1所述采集常规样品并测量N-乙酰神经氨酸的产生。这种菌株和方法产生大量N-乙酰神经氨酸。
相同生物也基于作为碳源的葡萄糖、麦芽糖或甘油产生N-乙酰神经氨酸。
实施例5：酿酒酵母中6-唾液酸乳糖的产生
另一个实例提供了酿酒酵母形式的真核生物在本发明中的用途。通过引入并表达N-乙酰葡糖胺-2-差向异构酶(例如来自卵形拟杆菌(BoAGE))和唾液酸合酶(例如来自空肠弯曲杆菌(CjneuB))在酿酒酵母中获得利用本发明途径的这种方法。
除此之外，进行进一步的修饰以产生6唾液酸乳糖。这些修饰包括添加乳糖通透酶、CMP-唾液酸合酶和唾液酸转移酶。优选的乳糖通透酶是来自乳酸克鲁维酵母的KlLAC12基因(WO 2016/075243)。优选的CMP-唾液酸合酶和唾液酸转移酶是分别来自脑膜炎奈瑟氏球菌的NmneuA和来自发光杆菌的Pdbst，如实施例3所述。
作为起点，需要具有增加的向N-乙酰葡糖胺-6-磷酸的代谢通量的菌株。这是通过过表达来自大肠杆菌的果糖-6-P-氨基转移酶突变体(EcglmS*54)来实现的。
为了创制本发明的产生N-乙酰神经氨酸的酿酒酵母，通过2-micron质粒(Chan2013(Plasmid 70(2013)2-17))引入所述基因，并且还如实施例1所述，使用合成的组成型启动子(Blazeck 2012(Biotechnology and Bioengineering,Vol.109,No.11))表达所述基因。本实施方案中使用的具体质粒是p2a_2μ_sia_glmS。使用Gietz和Woods(2002)描述的转化技术将这种质粒引入酿酒酵母中，并获得突变菌株。
所述菌株能够将果糖-6-磷酸转化为葡糖胺-6-磷酸，能够将所述葡糖胺-6-磷酸转化为N-乙酰葡糖胺-6-磷酸，能够将所述N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，能够将所述N-乙酰葡糖胺转化为N-乙酰甘露糖胺，最后能够将N-乙酰甘露糖胺转化为N-乙酰神经氨酸。然后将所述N-乙酰甘露糖胺转化为CMP-唾液酸并转移至乳糖中以获得6唾液酸乳糖。
所述菌株的预培养在5mL含有22g/L葡萄糖的合成的确定成分培养基SD-CSM中进行，并如实施例1所述在30℃生长。将该预培养物接种于摇瓶中具有10g/L蔗糖作为唯一碳源的100mL培养基中，并在30℃生长。如实施例1所述采集常规样品并测量N-乙酰神经氨酸的产生。这种菌株和方法产生大量的6唾液酸乳糖。
相同生物也基于作为碳源的葡萄糖、麦芽糖或甘油产生N-乙酰神经氨酸。
实施例6：使用自体同源的果糖-6-P-氨基转移酶在酿酒酵母中产生唾液酸
另一个实例提供了酿酒酵母形式的真核生物在本发明中的用途。通过引入并表达N-乙酰葡糖胺-2-差向异构酶(例如来自卵形拟杆菌(BoAGE))和唾液酸合酶(例如来自空肠弯曲杆菌(CjneuB))在酿酒酵母中获得利用本发明途径的这种方法。
作为起点，需要具有增加的向N-乙酰葡糖胺-6-磷酸的代谢通量的菌株。这通过过表达天然果糖-6-P-氨基转移酶ScGFA1来实现。
为了创制本发明的产生N-乙酰神经氨酸的酿酒酵母，通过2-micron质粒(Chan2013(Plasmid 70(2013)2-17))引入所述基因，并且，还如实施例1中所述，使用合成的组成型启动子(Blazeck 2012(Biotechnology and Bioengineering,Vol.109,No.11))表达所述基因。本实施方案中使用的具体质粒是p2a_2μ_sia_GFA1。使用Gietz和Woods(2002)描述的转化技术将该质粒引入酿酒酵母中，并获得突变菌株。
所述菌株能够将果糖-6-磷酸转化为葡糖胺-6-磷酸，能够将所述葡糖胺-6-磷酸转化为N-乙酰葡糖胺-6-磷酸，能够将所述N-乙酰葡糖胺-6-磷酸转化为N-乙酰葡糖胺，能够将所述N-乙酰葡糖胺转化为N-乙酰甘露糖胺，最后能够将N-乙酰甘露糖胺转化为N-乙酰神经氨酸。
所述菌株的预培养在5mL含有22g/L葡萄糖的合成的确定成分培养基SD-CSM中进行，并如实施例1所述在30℃生长。将该预培养物接种于摇瓶中具有10g/L蔗糖作为唯一碳源的100mL培养基中，并在30℃生长。如实施例1所述采集常规样品并测量N-乙酰神经氨酸的产生。这种菌株和方法产生大量N-乙酰神经氨酸。
相同生物也基于作为碳源的葡萄糖、麦芽糖或甘油产生N-乙酰神经氨酸。
实施例7：唾液酸乳糖和其他唾液酸化化合物的产生
在实施例3的替代实施方案中，将唾液酸转移酶改变为具有不同活性的另一种唾液酸转移酶。这可以是α-2,3-唾液酸转移酶、α-2,6-唾液酸转移酶、α-2,8-唾液酸转移酶或其组合。这些唾液酸转移酶在自然界中广泛获得并且有充分的注解。
以这种方式，可以获得不同的唾液酸乳糖，例如6-唾液酸乳糖、3-唾液酸乳糖或其混合物。
如实施例1和实施例3中所述培养所述菌株。
实施例2-7中产生的途径也可以与其他途径组合，用于合成较大的寡糖，例如唾液酸-乳-N-三糖、唾液酸乳-N-四糖、二唾液酸乳糖-N-四糖、唾液酸乳-N-新四糖和二唾液酸乳糖-N-新四糖。为此，合成这些糖苷键的转移酶与途径基因共表达以形成CMP-唾液酸和转移酶(如上所述)以唾液酸化所述寡糖。
如Datta(Current Drug Targets,2009,10,483-498)和Harduin-Lepers(Biochimie 83(2001)727-737)所述，这类唾液酸转移酶的实例是ST6GalI、ST6GalII、ST3GalI至VI，ST6GalNAc I至VI和ST8Sia I至VI。Yamamoto(Mar.Drugs 2010,8,2781-2794)描述了源自海洋生物的其他实例。
实施例8：唾液酸化的乳-N-新四糖的产生
本实验的目的是，证明本发明产生其他唾液酸化寡糖的功能，在这种情况下是唾液酸化乳-N-新四糖。
按照实施例1描述的方案，开发产生乳-N-新四糖的菌株。为了产生，需要表达N-乙酰葡糖胺转移酶和半乳糖基转移酶，这通过分别引入都来自脑膜炎奈瑟氏球菌的基因NmlgtA和NmlgtB来实现。接下来，引入来自大肠杆菌的乳糖输入EclacY(如WO 2016/075243的实施例1所述和所证明的，通过引用将WO 2016/075243并入本文)。最后，敲除基因ushA和galT。以此方式，获得产生乳-N-新四糖的菌株。
为了能够在乳糖上生长并产生N-乙酰葡糖胺-6-磷酸，如实施例1所述过表达来自青春双歧杆菌的蔗糖磷酸化酶(BaSP)、来自运动发酵单胞菌的果糖激酶(frk)、和突变果糖-6-P-氨基转移酶(EcglmS*54，如Deng等人(Biochimie 88,419-429(2006))所述)。
在这种菌株中，通过过表达来自酿酒酵母的葡糖胺-6-P-氨基转移酶(ScGNA1)，来自卵形拟杆菌的N-乙酰葡糖胺-2-差向异构酶(BoAGE)和来自空肠弯曲杆菌的唾液酸合酶(CjneuB)来实施本发明所述的产生唾液酸的方法。分别在位置nagABCDE和manXYZ上表达ScGNA1和BoAGE。由质粒pCX-CjneuB-NmneuA-Pdbst表达CjneuB。
通过利用CMP唾液酸合成酶，例如来自脑膜炎奈瑟氏球菌的NmneuA，随后利用唾液酸转移酶，例如来自美人鱼发光杆菌的Pdbst，将唾液酸转化为CMP-唾液酸，进行乳-N-新四糖部分的唾液酸化。由高拷贝质粒pCX-CjneuB-NmneuA-Pdbst表达这些基因(NmneuA和Pdbst)。
如实施例1(材料和方法)所述培养所述菌株。简而言之，接种5mLLB预培养物，并使之在37℃生长过夜。将该培养物在使用100mL培养基的摇瓶实验中用作接种体，所述培养基含有10g/L蔗糖作为碳和能量源、10g/L乳糖作为前体，并根据实施例1的描述制备。采集常规样品并分析。这种菌株产生大量唾液酸化乳-N-新四糖。
替代性糖基转移酶是可能的。例如，如果表达EcWgbO(来自大肠杆菌O55:H7)而不是NmlgtB，则获得唾液酸化的乳-N-四糖的产生。
实施例9：用枯草芽孢杆菌产生唾液酸
在另一个实施方案中，本发明可用于在枯草芽孢杆菌即另一种细菌生产宿主中产生N-乙酰神经氨酸。
根据本发明，从能够在细胞内过量产生葡糖胺-6-磷酸的菌株开始，获得产生N-乙酰神经氨酸的菌株。为此，过表达天然果糖-6-P-氨基转移酶(BsglmS)。通过敲除基因nagA、nagB和gamA破坏以下酶活性：N-乙酰葡糖胺-6-磷酸脱乙酰酶和葡糖胺-6-磷酸异构酶。
在这种菌株中，通过过表达来自酿酒酵母的葡糖胺-6-P-氨基转移酶(ScGNA1)、来自卵形拟杆菌的N-乙酰葡糖胺-2-差向异构酶(BoAGE)和来自空肠弯曲杆菌的唾液酸合酶(CjneuB)，来实施本发明所述的产生唾液酸的方法。如实施例1所述，通过质粒引入这些基因。
如实施例1(材料和方法)所述培养所述菌株。简而言之，接种5mLLB预培养物，并使之在30℃生长过夜。该培养物在使用100mL培养基的摇瓶实验中用作接种体，所述培养基含有10g/L蔗糖，并根据实施例1的描述制备。这种菌株产生大量N-乙酰神经氨酸。
实施例10：产生6-唾液酸乳糖且不分泌GlcNAc、ManNAc或唾液酸的菌株的发酵
本发明的另一个实施例提供了所述方法和菌株产生6-唾液酸乳糖的用途。
将能够使用蔗糖作为碳源累积葡糖胺-6-磷酸的大肠杆菌菌株进一步改造，以允许N-乙酰神经氨酸产生。这种基础菌株过表达来自青春双歧杆菌的蔗糖磷酸化酶(BaSP)、来自运动发酵单胞菌的果糖激酶(Zmfrk)、突变的果糖-6-P-氨基转移酶(EcglmS*54，如Deng等人(Biochimie 88,419-429(2006)所述)。为了产生6唾液酸乳糖，破坏操纵子nagABCDE、nanATEK和manXYZ。在nagABCDE位置引入BaSP和Zmfrk，在nanATEK位置引入EcglmS*54。这些修饰按实施例1所述并且基于Datsenko&Wanner(PNAS USA 97,6640-6645(2000))的原则进行。
在这种菌株中，通过过表达来自酿酒酵母的葡糖胺-6-P-氨基转移酶(ScGNA1)、来自卵形拟杆菌的N-乙酰葡糖胺-2-差向异构酶(BoAGE)和来自脑膜炎奈瑟氏球菌的唾液酸合酶(NmneuB)，来实施本发明所述的用于产生6-唾液酸乳糖的生物合成途径。分别在位置nagABCDE和manXYZ上表达ScGNA1和BoAGE。使用高拷贝质粒pBR322-NmNeuB表达NmNeuB。通过过表达来自大肠杆菌的乳糖通透酶EclacY(如WO 2016/075243的实施例1所述和证明的，通过引用将WO 2016/075243并入本文)、来自脑膜炎奈瑟氏球菌的CMP-唾液酸合酶(NmNeuA)和来自美人鱼发光杆菌的唾液酸转移酶(Pdbst)，进一步修饰所述菌株。除此之外，破坏了lacZ。使用低拷贝质粒pSC101-NmneuA-Pdbst表达NmNeuA和Pdbst。
如实施例1(材料和方法)所述，将所述菌株培养于生物反应器中。简而言之，接种5mL LB预培养物，并使之在37℃生长过夜。该培养物在使用500mL培养基的摇瓶实验中用作接种体，所述培养基含有10g/L蔗糖，并如实施例1所述制备。这种培养物在2L生物反应器实验中用作接种体。如实施例1所述，采集常规样品并进行分析。6-唾液酸乳糖的最终浓度为30.5g/L。在发酵期间和最终的培养液中未检测到细胞外GlcNAc、ManNAc和唾液酸。
相同生物也基于作为碳源的葡萄糖、麦芽糖或甘油产生6-唾液酸乳糖。
实施例11：磷酸酶对生长和唾液酸产生的影响
另一个实例提供了几个能够产生N-乙酰神经氨酸(唾液酸)的大肠杆菌菌株的生长结果和唾液酸产生，其中如下所示菌株表达额外的磷酸酶。
基础菌株过表达突变果糖-6-P-氨基转移酶(EcglmS*54,如Deng等人(Biochimie88,419-429(2006)所述)、来自酿酒酵母的葡糖胺-6-P-氨基转移酶(ScGNA1)，来自卵形拟杆菌的N-乙酰葡糖胺-2-差向异构酶(BoAGE)和来自空肠弯曲杆菌的唾液酸合酶(CjneuB)。为了允许基因唾液酸产生，破坏操纵子nagABCDE和nanATEK。lacYZA操纵子仅被单个基因操纵子即天然lacY代替，如实施例10所述，lacY是产生唾液酸乳糖所需的。这些修饰按实施例1所述并且基于Datsenko和Wanner(PNAS USA 97,6640-6645(2000))的原则进行。
然后用携带不同磷酸酶的质粒补充这种基础菌株，用于比较磷酸酶对生长和唾液酸产生的影响。在比较时使用基础菌株作为空白。除了磷酸酶和驱动磷酸酶表达的启动子外，这些质粒还由pSC101ori和壮观霉素抗性标记组成。表达了以下磷酸酶：EcAphA(SEQ IDNO:42)、EcCof(SEQ ID NO:43)、EcHisB(SEQ ID NO:44)、EcOtsB(SEQ ID NO:45)、EcSurE(SEQ ID NO:46)、EcYaed(SEQ ID NO:47)、EcYcjU(SEQ ID NO:48)、EcYedP(SEQ ID NO:49)、EcYfbT(SEQ ID NO:50)、EcYidA(SEQ ID NO:51)、EcYigB(SEQ ID NO:52)、EcYihX(SEQID NO:53)、EcYniC(SEQ ID NO:54)、EcYqaB(SEQ ID NO:55)、EcYrbL(SEQ ID NO:56)和PsMupP(SEQ ID NO:57)。表达的其他磷酸酶是EcAppA(SEQ ID NO:58)、EcGph(SEQ ID NO:59)、EcSerB(SEQ ID NO:60)、EcNagD(SEQ ID NO:61)、EcYbhA(SEQ ID NO:62)、EcYbiV(SEQID NO:63)、EcYbjL(SEQ ID NO:64)、EcYfbR(SEQ ID NO:65)、EcYieH(SEQ ID NO:66)、EcYjgL(SEQ ID NO:67)、Ec YjjG(SEQ ID NO:68)、EcYrfG(SEQ ID NO:69)、EcYbiU(SEQ IDNO:70)、ScDOG1(SEQ ID NO:71)和BsAraL(SEQ ID NO:72)。
在第一个实验中，使用上述菌株的子集。在第二个实验中，测试了上述菌株的第二子集。
如实施例1(材料和方法)所述培养每种菌株。简而言之，该工作流程由3个生长步骤组成：首先在LB上生长，然后在具有15g/L甘油的MMsf上生长，最后是使用15g/L甘油MMsf的生长。第一步在96孔板中进行，每孔使用175μL LB，并在37℃孵育过夜。第二步使用175μL培养基在96孔板中进行，在37℃孵育24小时。最终生长步骤在以下中进行：i)在96孔板中使用175μL培养基，在37℃孵育以测定第一个实验的μMax(参见图5)和ii)在24孔深孔板中使用3mL，以测定第二个实验的唾液酸产量和光密度(见图4)。
图4和5的参考表：
基于图4和5，可以选择能使菌株比空白菌株生长更好(没有受损的生长)并比空白菌株产生更多唾液酸的磷酸酶。
基于上述，发现至少包含基序1和基序2的磷酸酶提供的菌株不会受损并比空白菌株产生更多的唾液酸。
实施例12：鉴定与本发明方法所用的磷酸酶相关的其他序列
使用数据库序列搜索工具，例如Basic Local Alignment Tool(BLAST)(Altschul等人(1990)J.Mol.Biol.215:403-410；和Altschul等人(1997)Nucleic Acids Res.25:3389-3402)，在保存于国家生物技术信息中心(NCBI)的Entrez Nucleotides数据库中的那些序列中，鉴定与SEQ ID NO:43、44、45、47、48、49、50、51、52、54、55和57相关的序列(多肽)。该程序用于通过将核酸或多肽序列与序列数据库进行比较并通过计算匹配的统计显著性来找到序列之间的局部相似性区域。通过成对比较来观察分析的输出，并根据概率得分(E值)进行排序，其中得分反映了具体比对偶然发生的概率(E值越低，命中越显著)。除了E值之外，还通过同一性百分比对比较进行评分。同一性百分比是指在具体长度上两条比较的多肽序列之间的相同氨基酸的数量。在某些情况下，可以调整默认参数，以修改搜索的严格性。例如，可以增加E值以显示不太严格的匹配。这样可以鉴别短的几乎完全的匹配。
表1A至1K分别提供了与SEQ ID NO:43、44、45、47、48、50、51、52、54、55和57相关的同源多肽序列的列表。
表1A：与Ec Cof(SEQ ID NO:43)相关的多肽的实例，显示了与SEQ ID 43的序列同一性：
表1B：与Ec HisB(SEQ ID NO:44)相关的多肽的实例，显示与SEQ ID 44的序列同一性：
表1C：与Ec OtsB(SEQ ID NO:45)相关的多肽的实例，显示了与SEQ ID 45的序列同一性：
表1D：与Ec Yaed(SEQ ID NO:47)相关的多肽的实例，显示了与SEQ ID 47的序列同一性：
表1E：与Ec YcjUB(SEQ ID NO:48)相关的多肽的实例，显示了与SEQ ID NO:48的序列同一性：
表1F：与Ec YfbT(SEQ ID NO:50)相关的多肽的实例，显示了与SEQ ID NO:50的序列同一性：
表1G：与Ec YidA(SEQ ID NO:51)相关的多肽的实例，显示了与SEQ ID NO:51的序列同一性：
表1H：与Ec YigB(SEQ ID NO:52)相关的多肽的实例，显示了与SEQ ID NO:52的序列同一性：
表1I：与Ec YniC(SEQ ID NO:54)相关的多肽的实例，显示了与SEQ ID NO:54的序列同一性：
表1J：与Ec YqaB(SEQ ID NO:55)相关的多肽的实例，显示了与SEQ ID NO:55的序列同一性：
表1K：与Ps MupP(SEQ ID NO:57)相关的多肽的实例，显示了与SEQ ID NO:57的序列同一性：
研究机构，例如基因组研究所(Institute for Genomic Research，TIGR；从TA开始)已经试验性组装和公开披露了序列。真核基因直系同源物(EGO)数据库可用于通过关键词搜索或通过使用BLAST算法鉴别与目的核酸序列或多肽序列相关的这类序列。例如联合基因组研究所(Joint Genome Institute)已经为特定生物创建了专门的核酸序列数据库。
实施例13：鉴定可用于实施本发明方法的多肽序列所含的结构域和基序
蛋白质家族、结构域和位点的综合资源(InterPro)数据库是用于基于文本和序列搜索的常用签名数据库的集成界面。InterPro数据库结合了这些数据库，这些数据库使用不同的方法和不同程度的关于良好表征的蛋白质的生物学信息来获得蛋白质特征。合作数据库包括SWISS-PROT、PROSITE、TrEMBL、PRINTS、ProDom和Pfam、Smart和TIGRFAM。Pfam是多个序列比对和隐藏马尔可夫模型的大集合，涵盖许多常见的蛋白质结构域和家族。Pfam托管在英国的SangerInstitute服务器上。Interpro由英国的欧洲生物信息学研究(EuropeanBioinformatics Institute)所主办。
表2显示了SEQ ID NO:43、44、45、47、48、49、50、51、52、54和55所示的多肽序列的InterPro扫描结果。
表2：SEQ ID NO:43、44、45、47、48、49、50、51、52、54和55所示的多肽序列的InterPro扫描结果(主要登录号)。
数据库 登录号 登录名称
Interpro IPR023214 HAD超家族
进行测试的磷酸酶多肽的比对，图6显示部分比对。基序1和基序12用方框表示。使用clustalomega进行比对。
实施例14：磷酸酶对酿酒酵母的生长和唾液酸产生的影响
能够产生N-乙酰神经氨酸(唾液酸)的几种酿酒酵母菌株的唾液酸产生的另一个实例，其中所述菌株表达如下所示的额外磷酸酶。
这里使用的菌株衍生自实施例4所述的菌株。根据本发明，为了增强酿酒酵母的生长和唾液酸产生，通过2-micron质粒(Chan 2013(Plasmid 70(2013)2-17))引入磷酸酶基因，并且如实施例1中所述，使用合成的组成型启动子(Blazeck 2012(Biotechnology andBioengineering,Vol.109,No.11))表达所述基因。该实施方案所用的具体质粒是p2a_2μ_sia_glmS-phospha。这种基于质粒p2a_2μ_sia_glmS质粒的质粒描述于实施例1中。使用Gietz和Woods(2002,PMID 12073338)描述的转化技术将其引入酿酒酵母中，并获得突变菌株。如实施例11所述评估磷酸酶表达对这些突变体的生长和唾液酸产生的影响。
实施例15：磷酸酶对枯草芽孢杆菌的生长和唾液酸产生的影响
在另一个实施方案中，本发明可用于增强枯草芽孢杆菌(即另一种细菌生产宿主)的生长和唾液酸产生。
此处所用的菌株衍生自实施例9所述的菌株。除了实施例9所述的改变之外，如实施例1所述在质粒中过表达磷酸酶基因EcAphA(SEQ ID NO:42)、EcCof(SEQ ID NO:43)、EcHisB(SEQ ID NO:44)、EcOtsB(SEQ ID NO:45)、EcSurE(SEQ ID NO:46)、EcYaed(SEQ IDNO:47)、EcYcjU(SEQ ID NO:48)、EcYedP(SEQ ID NO:49)、EcYfbT(SEQ ID NO:50)、EcYidA(SEQ ID NO:51)、EcYigB(SEQ ID NO:52)、EcYihX(SEQ ID NO:53)、EcYniC(SEQ ID NO:54)、EcYqaB(SEQ ID NO:55)、EcYrbL(SEQ ID NO:56)、PsMupP(SEQ ID NO:57)、EcAppA(SEQID NO:58)、EcGph(SEQ ID NO:59)、EcSerB(SEQ ID NO:60)、EcNagD(SEQ ID NO:61)、EcYbhA(SEQ ID NO:62)、EcYbiV(SEQ ID NO:63)、EcYbjL(SEQ ID NO:64)、EcYfbR(SEQ IDNO:65)、EcYieH(SEQ ID NO:66)、EcYjgL(SEQ ID NO:67)、Ec YjjG(SEQ ID NO:68)、EcYrfG(SEQ ID NO:69)、EcYbiU(SEQ ID NO:70)、ScDOG1(SEQ ID NO:71)和BsAraL(SEQ ID NO:72)。随后，将这种质粒引入枯草芽孢杆菌中。如实施例11所述，评估磷酸酶表达对所创制的突变体的生长和唾液酸产生的影响。
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<223> 合成的DNA
<400> 22
gctgacaaag tgcgatttgt tc 22
<210> 23
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 23
gtcgccctgt aattcgtaac 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 24
ctttcggtca gaccaccaac 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 25
acgcctctga tttggcaaag 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 26
agccagtgcg cttaataacc 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 27
gctgaacagc aatccacttg 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 28
tgaacgatat ggtgagctgg 20
<210> 29
<211> 500
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 29
agatggaatg gcgattttca ctctaaattt taaaaattgc ctctttacaa tagcgaattt 60
cctaaccctt tttttttttt gttgattgcc tattgctcgt tcacttccca tttattttct 120
ctcgaatttc accaaaagtt gatgtggata atcaatcatc gggcctattc ctgcgggtaa 180
aacgcagggc ccaactcagg atagggttta atattatttt agaggactta caagaaggaa 240
gttatatggt ttaaaaattg taacaaagtt agaacacatt tatttagcag gtctaattta 300
gggctgcaac tatctttttg gttattcata taaaatataa ttttttattt atatagagaa 360
tacaagtgga atcatcttta acgccagctt gtagtgcgca ttgcagaata atggaagttc 420
aaaaattaaa agcgaaggag aagtgatagt agaaagacgg atgggaggct gggggacgaa 480
gagaaagtaa aagggttaat 500
<210> 30
<211> 500
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 30
ttttttataa aagttgctgt tgatttgctc gagaacttat tgcttatttg gccctgataa 60
ctatataaga aaagaaatac agttattcct tgtttatgct ggctttttgt ccactttttc 120
tcaactatat aactatgatg ttggaaagga caccggttct gtaactttgc agtgaaaata 180
agtgtgatgg atgactgaga atgctttctt gtaagcgaaa agaagtacgt gttccaaaaa 240
taaagcagaa aggcgaaaag ggtcgaatgt aagacactaa ataaatattt taagaagagg 300
aaaagtcgcc tcagaaacgc taaaatgcat ccgatttccc aaagaggaag tctaatgttt 360
tcgatttgtg aaaaaaagat aaaaatcgaa gaaaatgtag ggagccgcgc gttacccgga 420
ttgatatttg agtgatcgac ggcgtcacaa aagaaagaat gcttggctaa tcaagaaaag 480
tatgtggttt gtttcatcta 500
<210> 31
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 31
gcaggtctaa tttagggctg 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 32
agaaagcatt ctcagtcatc 20
<210> 33
<211> 1000
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 33
gactgcaaga tttcggcctg ggcggacggg aatcgtcagt tttgtaattt ctgtatcaat 60
gattttcatg gtctcttcct caagtccgag ccggtcgtat tgcttgccct gctcccagag 120
ttcaagattc atgacaatcg tgattcgttt attgcttctg accgcgccag cgccaaatag 180
cgtcatcaca ttgataatgc caaggcccct gatctcaaga aggtgctcaa ttaattccgg 240
agcgtttccc acaagagtat cctgatcctc ctgccgtatt tcaacgcaat catcggcaac 300
aaggcgatgc cctcttttca caagctctag cgctgtttcg ctttttccga cgccgctttt 360
tcctgtgatc agcacgccga caccatatat atcgacaaga acgccatgaa ttgctgtggt 420
aggcgccagc ctgctctcaa ggaagttggt taaacggctt gacagtcttg tcgttttcag 480
cggcgatctg aggacaggca ccccattttt ctcggaggcg tcaatcagct cctgcgggat 540
gggcatatct ctagaaagaa taatagctgg tgttacatca gtgcacagag aatccattcg 600
ctgctttttc tcctcttcag gaagctgttc aaagaaagaa agctctgttt ttccgagaag 660
ctgcacgcgc tccctcgggt aatatgtaaa atatccggca atttcaatac ctggtcttga 720
taggtcactc attgtaatcg ggcggttaat tccttcttct ccgctgatta attccaaatt 780
gaactgttcc attacgtctt ttgtgcgaac ctttgccacg atatgttcct cctgttccgg 840
gctgccccga gcttgctcac aatactttca ttttatcact ttcgggcttg aacctaaaac 900
agattttata aaagggggga aaacacctca gctggtctag atcactagtc tgaaaaagag 960
taaaataaag gtattcaaat tccagaaagg cggatcatct 1000
<210> 34
<211> 1000
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 34
aaggaacatg ctgacttatg aatatcaata aacaatcgcc tattccgatt tactatcaga 60
ttatggagca attaaaaacc caaattaaga acggagagct gcagccggat atgcctcttc 120
cttctgagcg cgaatatgcc gaacaattcg ggatcagccg gatgacagtt cgccaggcgc 180
tttctaattt agttaatgaa ggcttgctct atcgcctgaa agggcggggc acctttgtca 240
gcaagccaaa aatggaacaa gcacttcaag ggctgacaag ctttaccgag gatatgaaaa 300
gccgcgggat gacaccgggc agcaggctca ttgattatca gcttattgat tcaactgagg 360
agctcgcggc tatattaggc tgcgggcacc cctcctctat ccataaaatc actcgggtgc 420
ggctggcaaa tgatattccg atggcgattg agtcctcaca tattccgttt gagcttgcgg 480
gtgaattgaa cgaatcgcat tttcagtcgt cgatctatga tcatattgaa aggtacaaca 540
gcataccgat ttcccgtgca aaacaggagc ttgagccaag cgctgccacc acggaagaag 600
cgaatattct tggtattcaa aagggagcgc ctgtcctatt aattaaacga acaacatatc 660
tgcagaacgg aactgctttt gagcatgcaa aatccgtata cagaggcgac cgttatacat 720
ttgtccacta tatggatcgt ctttcataaa aaaagcctcc aacccttttt aaggattgga 780
gacatggcga aaatcaaact ggtctggtgc cggacgatat gtttcttttt tcgtcttgaa 840
cttccagatc ggtgatttcg ttttgccgtt aaaactgtct tccactataa tgtaccaata 900
ataaacagac tgcggttcaa gatgatccca gcggaattca gctgtgtccc cgctcttcac 960
ttgctcccgt tttccgagct cttcattggt atatacgtta 1000
<210> 35
<211> 1000
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 35
tggcggacat ggaataaatc acaaacgaca aagatgacgc cggcaagaat agagttaatc 60
aaatagagca cgggcgcaac gaacaagaaa gaaaactcaa ccggttctgt aattccggtc 120
agcatagatg tgagcgccgc agaaatcatc acgccggaga tcattttttt cttttccgga 180
cgcgcggtat ggataatggc aagagcaacg gccggcagac agaaaatcat gtaagggaaa 240
tcccccatca taaagcgccc ggctgtcggg tctcccgcga aaaaccttgt caggtcgccg 300
gttacggtgt tgcctgttga tgggtctgtg tattctccca tcataaaata gaaaggcgta 360
taaaaaatat gatgcaggcc aaaaggaatc agcaaacgat agatcgttgc ataaaagaac 420
aggccgactg ttgaatcggc aattaaactg ctggctgcgt taattccgtt ttggatcagc 480
ggccaaacga atgagaaaat gacgccgatc accaatgaac tgacggaagt aatgatcggg 540
acaaagcgtt ttccagagaa aaatccaagg accggatgca gctcgattga tgaaaatcgc 600
ttatataaat aggcggcgag aagcccgata atgattcctc cgaaaacccc catatcaatc 660
aggtgctcgg ctccttcata cggaggctga aggccgagta attttcccat attgtcgagg 720
gtgacggtta aaattaagta tccgatgaca gcggcaagtc cggctacacc ttctccgccg 780
gctaatccga tcgcgacccc cacggcgaaa atcagcggaa ggttatcgaa tacaacgccg 840
cccgcatcct ttataatagg gatgttcagt aaatccttgt ctccgaaacg gagcaaaaga 900
cctgctgccg gcaggacggc aaccggagtc atcaacgcgc ggccaagctg ctgcagaatt 960
tgaaatgcct ttttaaacat gacagtctcc ttttattgtg 1000
<210> 36
<211> 1000
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 36
gtgacacccc ctcaaagaga tagacaagca ccatatttgt tatgaccaat ttatgatact 60
tgtcattacg aatttagcac cgcccttatc aaactgtcaa tattaatttc tgaaaatttg 120
ttataaaaga aggatacaaa tctttcatat tgggagggca aatggtatta tggtctcaat 180
gaaaaagaac ggattgcata cagaatgggg agaatgaaat gacagcttta tattctgtta 240
tcaagtttaa aatcattgag ttaattaaat cgggcaaata tcaggcgaat gatcagctgc 300
cgacggagag tgagttttgc gaacaatatg atgtcagcag aacaactgtg agactggctc 360
tgcagcagct agagcttgag ggatatatta aaagaattca aggaaaaggg acatttgtat 420
cggcggccaa aatacaaacg ccgattccgc ataagattac gagctttgca gaacaaatga 480
gaggacttcg ttctgaatca aaagtgcttg agcttgtggt gattcctgcc gatcattcca 540
tcgccgagct tttgaaaatg aaagagaatg aacctgtcaa caagcttgtc agagtcagat 600
acgccgaggg ggaacctttg cagtatcata cctcatatat tccctggaag gcggcaccgg 660
ggctggcgca ggaggaatgc accggctcgc tgtttgaatt gttaaggaca aaatacaata 720
ttgaaatcag caggggcacg gaatcgatcg aaccgatttt aacggatgaa acgatcagcg 780
gacacttatt aaccaatgtc ggagcgcctg cgtttttatc agaatccctt acctatgata 840
aaaatgaaga agtggtggaa tatgcgcaaa ttattacacg gggagaccga acgaaattca 900
ccgtagaaca gtcatatcat tcataaagca atgtgtttta agaagggaat ggtggttcta 960
tgtttttatt tacgaatgga aaagtgctgt ggggagcagt 1000
<210> 37
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 37
tgtaatcggg cggttaattc 20
<210> 38
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 38
gccctttcag gcgatagag 19
<210> 39
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 39
acggcgaaaa tcagcggaag 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 合成的DNA
<400> 40
tcactctccg tcggcagctg 20
<210> 41
<211> 72
<212> DNA
<213> artificial
<220>
<223> UTR1
<400> 41
aattcgccgg agggatatta aaatgaatgg aaaattgaaa catcttaatc atgctaagga 60
ggttttctaa tg 72
<210> 42
<211> 237
<212> PRT
<213> 大肠杆菌（Escherichia coli）
<400> 42
Met Arg Lys Ile Thr Gln Ala Ile Ser Ala Val Cys Leu Leu Phe Ala
1 5 10 15
Leu Asn Ser Ser Ala Val Ala Leu Ala Ser Ser Pro Ser Pro Leu Asn
20 25 30
Pro Gly Thr Asn Val Ala Arg Leu Ala Glu Gln Ala Pro Ile His Trp
35 40 45
Val Ser Val Ala Gln Ile Glu Asn Ser Leu Ala Gly Arg Pro Pro Met
50 55 60
Ala Val Gly Phe Asp Ile Asp Asp Thr Val Leu Phe Ser Ser Pro Gly
65 70 75 80
Phe Trp Arg Gly Lys Lys Thr Phe Ser Pro Glu Ser Glu Asp Tyr Leu
85 90 95
Lys Asn Pro Val Phe Trp Glu Lys Met Asn Asn Gly Trp Asp Glu Phe
100 105 110
Ser Ile Pro Lys Glu Val Ala Arg Gln Leu Ile Asp Met His Val Arg
115 120 125
Arg Gly Asp Ala Ile Phe Phe Val Thr Gly Arg Ser Pro Thr Lys Thr
130 135 140
Glu Thr Val Ser Lys Thr Leu Ala Asp Asn Phe His Ile Pro Ala Thr
145 150 155 160
Asn Met Asn Pro Val Ile Phe Ala Gly Asp Lys Pro Gly Gln Asn Thr
165 170 175
Lys Ser Gln Trp Leu Gln Asp Lys Asn Ile Arg Ile Phe Tyr Gly Asp
180 185 190
Ser Asp Asn Asp Ile Thr Ala Ala Arg Asp Val Gly Ala Arg Gly Ile
195 200 205
Arg Ile Leu Arg Ala Ser Asn Ser Thr Tyr Lys Pro Leu Pro Gln Ala
210 215 220
Gly Ala Phe Gly Glu Glu Val Ile Val Asn Ser Glu Tyr
225 230 235
<210> 43
<211> 272
<212> PRT
<213> 大肠杆菌
<400> 43
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asp His His Leu Gly Glu Lys Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Ala Leu Glu
35 40 45
Met Gln His Ile Leu Gly Ala Leu Ser Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Val His Ser Leu Glu Gly Glu Leu Leu His Arg
65 70 75 80
Asp Asp Leu Pro Ala Asp Val Ala Glu Leu Val Leu Tyr Gln Gln Trp
85 90 95
Asp Thr Arg Ala Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr
100 105 110
Gly Lys Glu Ile Pro Ala Leu Leu Gln Ala Phe Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Ile Ile Asp Val Lys Lys Met Pro Leu Gly Ser Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile
145 150 155 160
Gln Leu Tyr Glu Ala Leu Gly Glu Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Thr Asp Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala
180 185 190
Leu Thr Val Leu Thr Gln His Leu Gly Leu Ser Leu Arg Asp Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Val Ser Val Gly
210 215 220
Ser Gly Phe Ile Met Gly Asn Ala Met Pro Gln Leu Arg Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His
245 250 255
Tyr Leu Thr His Trp Leu Asp Tyr Pro His Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 44
<211> 355
<212> PRT
<213> 大肠杆菌
<400> 44
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Thr
130 135 140
Gly Leu Arg Tyr Asp Arg Glu Thr Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Arg Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Ile Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 45
<211> 266
<212> PRT
<213> 大肠杆菌
<400> 45
Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Leu Ser Ala Lys Tyr Ala
1 5 10 15
Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu Ile Lys Pro His Pro
20 25 30
Asp Gln Val Val Val Pro Asp Asn Ile Leu Gln Gly Leu Gln Leu Leu
35 40 45
Ala Thr Ala Ser Asp Gly Ala Leu Ala Leu Ile Ser Gly Arg Ser Met
50 55 60
Val Glu Leu Asp Ala Leu Ala Lys Pro Tyr Arg Phe Pro Leu Ala Gly
65 70 75 80
Val His Gly Ala Glu Arg Arg Asp Ile Asn Gly Lys Thr His Ile Val
85 90 95
His Leu Pro Asp Ala Ile Ala Arg Asp Ile Ser Val Gln Leu His Thr
100 105 110
Val Ile Ala Gln Tyr Pro Gly Ala Glu Leu Glu Ala Lys Gly Met Ala
115 120 125
Phe Ala Leu His Tyr Arg Gln Ala Pro Gln His Glu Asp Ala Leu Met
130 135 140
Thr Leu Ala Gln Arg Ile Thr Gln Ile Trp Pro Gln Met Ala Leu Gln
145 150 155 160
Gln Gly Lys Cys Val Val Glu Ile Lys Pro Arg Gly Thr Ser Lys Gly
165 170 175
Glu Ala Ile Ala Ala Phe Met Gln Glu Ala Pro Phe Ile Gly Arg Thr
180 185 190
Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val
195 200 205
Val Asn Arg Leu Gly Gly Met Ser Val Lys Ile Gly Thr Gly Ala Thr
210 215 220
Gln Ala Ser Trp Arg Leu Ala Gly Val Pro Asp Val Trp Ser Trp Leu
225 230 235 240
Glu Met Ile Thr Thr Ala Leu Gln Gln Lys Arg Glu Asn Asn Arg Ser
245 250 255
Asp Asp Tyr Glu Ser Phe Ser Arg Ser Ile
260 265
<210> 46
<211> 253
<212> PRT
<213> 大肠杆菌
<400> 46
Met Arg Ile Leu Leu Ser Asn Asp Asp Gly Val His Ala Pro Gly Ile
1 5 10 15
Gln Thr Leu Ala Lys Ala Leu Arg Glu Phe Ala Asp Val Gln Val Val
20 25 30
Ala Pro Asp Arg Asn Arg Ser Gly Ala Ser Asn Ser Leu Thr Leu Glu
35 40 45
Ser Ser Leu Arg Thr Phe Thr Phe Glu Asn Gly Asp Ile Ala Val Gln
50 55 60
Met Gly Thr Pro Thr Asp Cys Val Tyr Leu Gly Val Asn Ala Leu Met
65 70 75 80
Arg Pro Arg Pro Asp Ile Val Val Ser Gly Ile Asn Ala Gly Pro Asn
85 90 95
Leu Gly Asp Asp Val Ile Tyr Ser Gly Thr Val Ala Ala Ala Met Glu
100 105 110
Gly Arg His Leu Gly Phe Pro Ala Leu Ala Val Ser Leu Asp Gly His
115 120 125
Lys His Tyr Asp Thr Ala Ala Ala Val Thr Cys Ser Ile Leu Arg Ala
130 135 140
Leu Cys Lys Glu Pro Leu Arg Thr Gly Arg Ile Leu Asn Ile Asn Val
145 150 155 160
Pro Asp Leu Pro Leu Asp Gln Ile Lys Gly Ile Arg Val Thr Arg Cys
165 170 175
Gly Thr Arg His Pro Ala Asp Gln Val Ile Pro Gln Gln Asp Pro Arg
180 185 190
Gly Asn Thr Leu Tyr Trp Ile Gly Pro Pro Gly Gly Lys Cys Asp Ala
195 200 205
Gly Pro Gly Thr Asp Phe Ala Ala Val Asp Glu Gly Tyr Val Ser Ile
210 215 220
Thr Pro Leu His Val Asp Leu Thr Ala His Ser Ala Gln Asp Val Val
225 230 235 240
Ser Asp Trp Leu Asn Ser Val Gly Val Gly Thr Gln Trp
245 250
<210> 47
<211> 191
<212> PRT
<213> 大肠杆菌
<400> 47
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Lys Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Ser Val Glu Glu Phe Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Leu Ser Ala Arg Asp Tyr Leu His Ile Asp Met
115 120 125
Ala Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Val Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Ile Thr Pro Glu Ala Glu Asn Ala Ala Asp Trp Val Leu Asn Ser Leu
165 170 175
Ala Asp Leu Pro Gln Ala Ile Lys Lys Gln Gln Lys Pro Ala Gln
180 185 190
<210> 48
<211> 219
<212> PRT
<213> 大肠杆菌
<400> 48
Met Lys Leu Gln Gly Val Ile Phe Asp Leu Asp Gly Val Ile Thr Asp
1 5 10 15
Thr Ala His Leu His Phe Gln Ala Trp Gln Gln Ile Ala Ala Glu Ile
20 25 30
Gly Ile Ser Ile Asp Ala Gln Phe Asn Glu Ser Leu Lys Gly Ile Ser
35 40 45
Arg Asp Glu Ser Leu Arg Arg Ile Leu Gln His Gly Gly Lys Glu Gly
50 55 60
Asp Phe Asn Ser Gln Glu Arg Ala Gln Leu Ala Tyr Arg Lys Asn Leu
65 70 75 80
Leu Tyr Val His Ser Leu Arg Glu Leu Thr Val Asn Ala Val Leu Pro
85 90 95
Gly Ile Arg Ser Leu Leu Ala Asp Leu Arg Ala Gln Gln Ile Ser Val
100 105 110
Gly Leu Ala Ser Val Ser Leu Asn Ala Pro Thr Ile Leu Ala Ala Leu
115 120 125
Glu Leu Arg Glu Phe Phe Thr Phe Cys Ala Asp Ala Ser Gln Leu Lys
130 135 140
Asn Ser Lys Pro Asp Pro Glu Ile Phe Leu Ala Ala Cys Ala Gly Leu
145 150 155 160
Gly Val Pro Pro Gln Ala Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly
165 170 175
Ile Asp Ala Ile Asn Ala Ser Gly Met Arg Ser Val Gly Ile Gly Ala
180 185 190
Gly Leu Thr Gly Ala Gln Leu Leu Leu Pro Ser Thr Glu Ser Leu Thr
195 200 205
Trp Pro Arg Leu Ser Ala Phe Trp Gln Asn Val
210 215
<210> 49
<211> 271
<212> PRT
<213> 大肠杆菌
<400> 49
Met Phe Ser Ile Gln Gln Pro Leu Leu Val Phe Ser Asp Leu Asp Gly
1 5 10 15
Thr Leu Leu Asp Ser His Ser Tyr Asp Trp Gln Pro Ala Ala Pro Trp
20 25 30
Leu Thr Arg Leu Arg Glu Ala Asn Val Pro Val Ile Leu Cys Ser Ser
35 40 45
Lys Thr Ser Ala Glu Met Leu Tyr Leu Gln Lys Thr Leu Gly Leu Gln
50 55 60
Gly Leu Pro Leu Ile Ala Glu Asn Gly Ala Val Ile Gln Leu Ala Glu
65 70 75 80
Gln Trp Gln Glu Ile Asp Gly Phe Pro Arg Ile Ile Ser Gly Ile Ser
85 90 95
His Gly Glu Ile Ser Leu Val Leu Asn Thr Leu Arg Glu Lys Glu His
100 105 110
Phe Lys Phe Thr Thr Phe Asp Asp Val Asp Asp Ala Thr Ile Ala Glu
115 120 125
Trp Thr Gly Leu Ser Arg Ser Gln Ala Ala Leu Thr Gln Leu His Glu
130 135 140
Ala Ser Val Thr Leu Ile Trp Arg Asp Ser Asp Glu Arg Met Ala Gln
145 150 155 160
Phe Thr Ala Arg Leu Asn Glu Leu Gly Leu Gln Phe Met Gln Gly Ala
165 170 175
Arg Phe Trp His Val Leu Asp Ala Ser Ala Gly Lys Asp Gln Ala Ala
180 185 190
Asn Trp Ile Ile Ala Thr Tyr Gln Gln Leu Ser Gly Lys Arg Pro Thr
195 200 205
Thr Leu Gly Leu Gly Asp Gly Pro Asn Asp Ala Pro Leu Leu Glu Val
210 215 220
Met Asp Tyr Ala Val Ile Val Lys Gly Leu Asn Arg Glu Gly Val His
225 230 235 240
Leu His Asp Glu Asp Pro Ala Arg Val Trp Arg Thr Gln Arg Glu Gly
245 250 255
Pro Glu Gly Trp Arg Glu Gly Leu Asp His Phe Phe Ser Ala Arg
260 265 270
<210> 50
<211> 216
<212> PRT
<213> 大肠杆菌
<400> 50
Met Arg Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Ser Asn Trp Ala Arg Arg His
20 25 30
Gly Leu Ala Pro Glu Glu Val Leu Ala Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Ala Gly Lys Ser Glu Ala Asp Ile
50 55 60
Ala Ala Glu Phe Thr Arg Leu Glu His Ile Glu Ala Thr Glu Thr Glu
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Ile Ala Leu Leu Ser His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Lys Ile Ala Gly Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Ala Pro Gln Glu Cys Val Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Arg Leu Asn Glu
180 185 190
Val Asp Leu Val Leu His Ser Leu Glu Gln Ile Thr Val Thr Lys Gln
195 200 205
Pro Asn Gly Asp Val Ile Ile Gln
210 215
<210> 51
<211> 270
<212> PRT
<213> 大肠杆菌
<400> 51
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Asn Ala Ile Ala Ala Ala
20 25 30
Arg Ala Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Asn Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ala Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Phe Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Thr Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Phe Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Asn Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Ala Ile Leu Asp Gln Ala Ile Ala Arg Ile Pro Gln Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Val Leu
195 200 205
Gly Ile Lys Pro Glu Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Val Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Val Ala Asn Phe Val Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Phe Ala Ile Glu Lys Tyr Val Leu Asn
260 265 270
<210> 52
<211> 238
<212> PRT
<213> 大肠杆菌
<400> 52
Met Arg Phe Tyr Arg Pro Leu Gly Arg Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Pro Val Ile Leu Arg Thr Glu
20 25 30
Arg Glu Ala Leu Thr Phe Val Gln Asn Tyr His Pro Ala Leu Arg Ser
35 40 45
Phe Gln Asn Glu Asp Leu Gln Arg Leu Arg Gln Ala Val Arg Glu Ala
50 55 60
Glu Pro Glu Ile Tyr His Asp Val Thr Arg Trp Arg Phe Arg Ser Ile
65 70 75 80
Glu Gln Ala Met Leu Asp Ala Gly Leu Ser Ala Glu Glu Ala Ser Ala
85 90 95
Gly Ala His Ala Ala Met Ile Asn Phe Ala Lys Trp Arg Ser Arg Ile
100 105 110
Asp Val Pro Gln Gln Thr His Asp Thr Leu Lys Gln Leu Ala Lys Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Glu Phe Val Leu Arg Ala Gly Pro His Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr Phe Leu Ala Ala Glu Lys Leu
165 170 175
Asn Val Pro Ile Gly Glu Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Gly Gly Ala Ile Arg Ser Gly Met Gln Ala Cys Trp Ile Arg
195 200 205
Pro Glu Asn Gly Asp Leu Met Gln Thr Trp Asp Ser Arg Leu Leu Pro
210 215 220
His Leu Glu Ile Ser Arg Leu Ala Ser Leu Thr Ser Leu Ile
225 230 235
<210> 53
<211> 199
<212> PRT
<213> 大肠杆菌
<400> 53
Met Leu Tyr Ile Phe Asp Leu Gly Asn Val Ile Val Asp Ile Asp Phe
1 5 10 15
Asn Arg Val Leu Gly Ala Trp Ser Asp Leu Thr Arg Ile Pro Leu Ala
20 25 30
Ser Leu Lys Lys Ser Phe His Met Gly Glu Ala Phe His Gln His Glu
35 40 45
Arg Gly Glu Ile Ser Asp Glu Ala Phe Ala Glu Ala Leu Cys His Glu
50 55 60
Met Ala Leu Pro Leu Ser Tyr Glu Gln Phe Ser His Gly Trp Gln Ala
65 70 75 80
Val Phe Val Ala Leu Arg Pro Glu Val Ile Ala Ile Met His Lys Leu
85 90 95
Arg Glu Gln Gly His Arg Val Val Val Leu Ser Asn Thr Asn Arg Leu
100 105 110
His Thr Thr Phe Trp Pro Glu Glu Tyr Pro Glu Ile Arg Asp Ala Ala
115 120 125
Asp His Ile Tyr Leu Ser Gln Asp Leu Gly Met Arg Lys Pro Glu Ala
130 135 140
Arg Ile Tyr Gln His Val Leu Gln Ala Glu Gly Phe Ser Pro Ser Asp
145 150 155 160
Thr Val Phe Phe Asp Asp Asn Ala Asp Asn Ile Glu Gly Ala Asn Gln
165 170 175
Leu Gly Ile Thr Ser Ile Leu Val Lys Asp Lys Thr Thr Ile Pro Asp
180 185 190
Tyr Phe Ala Lys Val Leu Cys
195
<210> 54
<211> 222
<212> PRT
<213> 大肠杆菌
<400> 54
Met Ser Thr Pro Arg Gln Ile Leu Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Asp Val
20 25 30
Met Ala Ser Leu Gly Val Asp Ile Ser Arg Arg Asn Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Asp Leu Trp Tyr Ala Arg
50 55 60
Gln Pro Trp Asn Gly Pro Ser Arg Gln Glu Val Val Glu Arg Val Ile
65 70 75 80
Ala Arg Ala Ile Ser Leu Val Glu Glu Thr Arg Pro Leu Leu Pro Gly
85 90 95
Val Arg Glu Ala Val Ala Leu Cys Lys Glu Gln Gly Leu Leu Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Thr Met
115 120 125
Phe Asp Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala Glu Lys Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Leu Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Val Asp Pro Leu Thr Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Ile Ala Ser Lys Ala Ala Arg Met Arg Ser Ile Val Val Pro
180 185 190
Ala Pro Glu Ala Gln Asn Asp Pro Arg Phe Val Leu Ala Asp Val Lys
195 200 205
Leu Ser Ser Leu Thr Glu Leu Thr Ala Lys Asp Leu Leu Gly
210 215 220
<210> 55
<211> 188
<212> PRT
<213> 大肠杆菌
<400> 55
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
20 25 30
His Tyr Gly Leu Gln Tyr Asp Ile Gln Ala Met Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Arg
65 70 75 80
Ser Met Leu Leu Asp Ser Val Glu Pro Leu Pro Leu Val Asp Val Val
85 90 95
Lys Ser Trp His Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
115 120 125
Phe Asp Ala Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Gln Arg Met Gly Val Gln Pro Thr
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Ile Gln Ala Ala Arg
165 170 175
Ala Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 56
<211> 188
<212> PRT
<213> 大肠杆菌
<400> 56
Met Ser Lys Ala Gly Ala Ser Leu Ala Thr Cys Tyr Gly Pro Val Ser
1 5 10 15
Ala Asp Val Ile Ala Lys Ala Glu Asn Ile Arg Leu Leu Ile Leu Asp
20 25 30
Val Asp Gly Val Leu Ser Asp Gly Leu Ile Tyr Met Gly Asn Asn Gly
35 40 45
Glu Glu Leu Lys Ala Phe Asn Val Arg Asp Gly Tyr Gly Ile Arg Cys
50 55 60
Ala Leu Thr Ser Asp Ile Glu Val Ala Ile Ile Thr Gly Arg Lys Ala
65 70 75 80
Lys Leu Val Glu Asp Arg Cys Ala Thr Leu Gly Ile Thr His Leu Tyr
85 90 95
Gln Gly Gln Ser Asn Lys Leu Ile Ala Phe Ser Asp Leu Leu Glu Lys
100 105 110
Leu Ala Ile Ala Pro Glu Asn Val Ala Tyr Val Gly Asp Asp Leu Ile
115 120 125
Asp Trp Pro Val Met Glu Lys Val Gly Leu Ser Val Ala Val Ala Asp
130 135 140
Ala His Pro Leu Leu Ile Pro Arg Ala Asp Tyr Val Thr Arg Ile Ala
145 150 155 160
Gly Gly Arg Gly Ala Val Arg Glu Val Cys Asp Leu Leu Leu Leu Ala
165 170 175
Gln Gly Lys Leu Asp Glu Ala Lys Gly Gln Ser Ile
180 185
<210> 57
<211> 223
<212> PRT
<213> 恶臭假单胞菌（Pseudomonas putida）
<400> 57
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Cys Gln Ala Met Leu Ala Glu Arg
20 25 30
Gly Leu Pro Ala Val Asp Asp Asn Leu Ile Arg Gly Val Ile Ser Gly
35 40 45
Gly Ala Arg Ala Met Val Ala Thr Ala Phe Ala Met Asp Pro Glu Ala
50 55 60
Asp Gly Phe Glu Ala Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Val His Ser Lys Leu Phe Glu Gly Met Ala Glu Leu Leu
85 90 95
Ala Asp Ile Glu Lys Gly Asn Leu Leu Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Glu Pro Ile Met Gln Gln Leu Gly Leu Ala Glu
115 120 125
Arg Ser Ala Leu Leu Ile Cys Pro Asp His Val Lys Asn Ser Lys Pro
130 135 140
Asp Pro Glu Pro Leu Ile Leu Ala Cys Lys Thr Leu Asn Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Arg Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Glu Asp Asn Pro Asn Asn Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Ile Asp Ser Ala Leu Cys Gly Cys
210 215 220
<210> 58
<211> 432
<212> PRT
<213> 大肠杆菌
<400> 58
Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu Leu Ile Pro Leu Thr
1 5 10 15
Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser
20 25 30
Val Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr
35 40 45
Gln Leu Met Gln Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val
50 55 60
Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu
65 70 75 80
Gly His Tyr Gln Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Lys
85 90 95
Lys Gly Cys Pro Gln Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp
100 105 110
Glu Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro
115 120 125
Asp Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp
130 135 140
Pro Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala
145 150 155 160
Asn Val Thr Asp Ala Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp
165 170 175
Phe Thr Gly His Arg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu
180 185 190
Asn Phe Pro Gln Ser Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu
195 200 205
Ser Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala
210 215 220
Asp Asn Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr
225 230 235 240
Glu Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp
245 250 255
Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His
260 265 270
Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser
275 280 285
Arg Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His
290 295 300
Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu
305 310 315 320
Phe Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu
325 330 335
Glu Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly
340 345 350
Gly Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln
355 360 365
Trp Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp
370 375 380
Lys Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr
385 390 395 400
Leu Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala
405 410 415
Gly Phe Thr Gln Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
420 425 430
<210> 59
<211> 252
<212> PRT
<213> 大肠杆菌
<400> 59
Met Asn Lys Phe Glu Asp Ile Arg Gly Val Ala Phe Asp Leu Asp Gly
1 5 10 15
Thr Leu Val Asp Ser Ala Pro Gly Leu Ala Ala Ala Val Asp Met Ala
20 25 30
Leu Tyr Ala Leu Glu Leu Pro Val Ala Gly Glu Glu Arg Val Ile Thr
35 40 45
Trp Ile Gly Asn Gly Ala Asp Val Leu Met Glu Arg Ala Leu Thr Trp
50 55 60
Ala Arg Gln Glu Arg Ala Thr Gln Arg Lys Thr Met Gly Lys Pro Pro
65 70 75 80
Val Asp Asp Asp Ile Pro Ala Glu Glu Gln Val Arg Ile Leu Arg Lys
85 90 95
Leu Phe Asp Arg Tyr Tyr Gly Glu Val Ala Glu Glu Gly Thr Phe Leu
100 105 110
Phe Pro His Val Ala Asp Thr Leu Gly Ala Leu Gln Ala Lys Gly Leu
115 120 125
Pro Leu Gly Leu Val Thr Asn Lys Pro Thr Pro Phe Val Ala Pro Leu
130 135 140
Leu Glu Ala Leu Asp Ile Ala Lys Tyr Phe Ser Val Val Ile Gly Gly
145 150 155 160
Asp Asp Val Gln Asn Lys Lys Pro His Pro Asp Pro Leu Leu Leu Val
165 170 175
Ala Glu Arg Met Gly Ile Ala Pro Gln Gln Met Leu Phe Val Gly Asp
180 185 190
Ser Arg Asn Asp Ile Gln Ala Ala Lys Ala Ala Gly Cys Pro Ser Val
195 200 205
Gly Leu Thr Tyr Gly Tyr Asn Tyr Gly Glu Ala Ile Asp Leu Ser Gln
210 215 220
Pro Asp Val Ile Tyr Gln Ser Ile Asn Asp Leu Leu Pro Ala Leu Gly
225 230 235 240
Leu Pro His Ser Glu Asn Gln Glu Ser Lys Asn Asp
245 250
<210> 60
<211> 322
<212> PRT
<213> 大肠杆菌
<400> 60
Met Pro Asn Ile Thr Trp Cys Asp Leu Pro Glu Asp Val Ser Leu Trp
1 5 10 15
Pro Gly Leu Pro Leu Ser Leu Ser Gly Asp Glu Val Met Pro Leu Asp
20 25 30
Tyr His Ala Gly Arg Ser Gly Trp Leu Leu Tyr Gly Arg Gly Leu Asp
35 40 45
Lys Gln Arg Leu Thr Gln Tyr Gln Ser Lys Leu Gly Ala Ala Met Val
50 55 60
Ile Val Ala Ala Trp Cys Val Glu Asp Tyr Gln Val Ile Arg Leu Ala
65 70 75 80
Gly Ser Leu Thr Ala Arg Ala Thr Arg Leu Ala His Glu Ala Gln Leu
85 90 95
Asp Val Ala Pro Leu Gly Lys Ile Pro His Leu Arg Thr Pro Gly Leu
100 105 110
Leu Val Met Asp Met Asp Ser Thr Ala Ile Gln Ile Glu Cys Ile Asp
115 120 125
Glu Ile Ala Lys Leu Ala Gly Thr Gly Glu Met Val Ala Glu Val Thr
130 135 140
Glu Arg Ala Met Arg Gly Glu Leu Asp Phe Thr Ala Ser Leu Arg Ser
145 150 155 160
Arg Val Ala Thr Leu Lys Gly Ala Asp Ala Asn Ile Leu Gln Gln Val
165 170 175
Arg Glu Asn Leu Pro Leu Met Pro Gly Leu Thr Gln Leu Val Leu Lys
180 185 190
Leu Glu Thr Leu Gly Trp Lys Val Ala Ile Ala Ser Gly Gly Phe Thr
195 200 205
Phe Phe Ala Glu Tyr Leu Arg Asp Lys Leu Arg Leu Thr Ala Val Val
210 215 220
Ala Asn Glu Leu Glu Ile Met Asp Gly Lys Phe Thr Gly Asn Val Ile
225 230 235 240
Gly Asp Ile Val Asp Ala Gln Tyr Lys Ala Lys Thr Leu Thr Arg Leu
245 250 255
Ala Gln Glu Tyr Glu Ile Pro Leu Ala Gln Thr Val Ala Ile Gly Asp
260 265 270
Gly Ala Asn Asp Leu Pro Met Ile Lys Ala Ala Gly Leu Gly Ile Ala
275 280 285
Tyr His Ala Lys Pro Lys Val Asn Glu Lys Ala Glu Val Thr Ile Arg
290 295 300
His Ala Asp Leu Met Gly Val Phe Cys Ile Leu Ser Gly Ser Leu Asn
305 310 315 320
Gln Lys
<210> 61
<211> 250
<212> PRT
<213> 大肠杆菌
<400> 61
Met Thr Ile Lys Asn Val Ile Cys Asp Ile Asp Gly Val Leu Met His
1 5 10 15
Asp Asn Val Ala Val Pro Gly Ala Ala Glu Phe Leu His Gly Ile Met
20 25 30
Asp Lys Gly Leu Pro Leu Val Leu Leu Thr Asn Tyr Pro Ser Gln Thr
35 40 45
Gly Gln Asp Leu Ala Asn Arg Phe Ala Thr Ala Gly Val Asp Val Pro
50 55 60
Asp Ser Val Phe Tyr Thr Ser Ala Met Ala Thr Ala Asp Phe Leu Arg
65 70 75 80
Arg Gln Glu Gly Lys Lys Ala Tyr Val Val Gly Glu Gly Ala Leu Ile
85 90 95
His Glu Leu Tyr Lys Ala Gly Phe Thr Ile Thr Asp Val Asn Pro Asp
100 105 110
Phe Val Ile Val Gly Glu Thr Arg Ser Tyr Asn Trp Asp Met Met His
115 120 125
Lys Ala Ala Tyr Phe Val Ala Asn Gly Ala Arg Phe Ile Ala Thr Asn
130 135 140
Pro Asp Thr His Gly Arg Gly Phe Tyr Pro Ala Cys Gly Ala Leu Cys
145 150 155 160
Ala Gly Ile Glu Lys Ile Ser Gly Arg Lys Pro Phe Tyr Val Gly Lys
165 170 175
Pro Ser Pro Trp Ile Ile Arg Ala Ala Leu Asn Lys Met Gln Ala His
180 185 190
Ser Glu Glu Thr Val Ile Val Gly Asp Asn Leu Arg Thr Asp Ile Leu
195 200 205
Ala Gly Phe Gln Ala Gly Leu Glu Thr Ile Leu Val Leu Ser Gly Val
210 215 220
Ser Ser Leu Asp Asp Ile Asp Ser Met Pro Phe Arg Pro Ser Trp Ile
225 230 235 240
Tyr Pro Ser Val Ala Glu Ile Asp Val Ile
245 250
<210> 62
<211> 272
<212> PRT
<213> 大肠杆菌
<400> 62
Met Thr Thr Arg Val Ile Ala Leu Asp Leu Asp Gly Thr Leu Leu Thr
1 5 10 15
Pro Lys Lys Thr Leu Leu Pro Ser Ser Ile Glu Ala Leu Ala Arg Ala
20 25 30
Arg Glu Ala Gly Tyr Gln Leu Ile Ile Val Thr Gly Arg His His Val
35 40 45
Ala Ile His Pro Phe Tyr Gln Ala Leu Ala Leu Asp Thr Pro Ala Ile
50 55 60
Cys Cys Asn Gly Thr Tyr Leu Tyr Asp Tyr His Ala Lys Thr Val Leu
65 70 75 80
Glu Ala Asp Pro Met Pro Val Ile Lys Ala Leu Gln Leu Ile Glu Met
85 90 95
Leu Asn Glu His His Ile His Gly Leu Met Tyr Val Asp Asp Ala Met
100 105 110
Val Tyr Glu His Pro Thr Gly His Val Ile Arg Thr Ser Asn Trp Ala
115 120 125
Gln Thr Leu Pro Pro Glu Gln Arg Pro Thr Phe Thr Gln Val Ala Ser
130 135 140
Leu Ala Glu Thr Ala Gln Gln Val Asn Ala Val Trp Lys Phe Ala Leu
145 150 155 160
Thr His Asp Asp Leu Pro Gln Leu Gln His Phe Gly Lys His Val Glu
165 170 175
His Glu Leu Gly Leu Glu Cys Glu Trp Ser Trp His Asp Gln Val Asp
180 185 190
Ile Ala Arg Gly Gly Asn Ser Lys Gly Lys Arg Leu Thr Lys Trp Val
195 200 205
Glu Ala Gln Gly Trp Ser Met Glu Asn Val Val Ala Phe Gly Asp Asn
210 215 220
Phe Asn Asp Ile Ser Met Leu Glu Ala Ala Gly Thr Gly Val Ala Met
225 230 235 240
Gly Asn Ala Asp Asp Ala Val Lys Ala Arg Ala Asn Ile Val Ile Gly
245 250 255
Asp Asn Thr Thr Asp Ser Ile Ala Gln Phe Ile Tyr Ser His Leu Ile
260 265 270
<210> 63
<211> 271
<212> PRT
<213> 大肠杆菌
<400> 63
Met Ser Val Lys Val Ile Val Thr Asp Met Asp Gly Thr Phe Leu Asn
1 5 10 15
Asp Ala Lys Thr Tyr Asn Gln Pro Arg Phe Met Ala Gln Tyr Gln Glu
20 25 30
Leu Lys Lys Arg Gly Ile Lys Phe Val Val Ala Ser Gly Asn Gln Tyr
35 40 45
Tyr Gln Leu Ile Ser Phe Phe Pro Glu Leu Lys Asp Glu Ile Ser Phe
50 55 60
Val Ala Glu Asn Gly Ala Leu Val Tyr Glu His Gly Lys Gln Leu Phe
65 70 75 80
His Gly Glu Leu Thr Arg His Glu Ser Arg Ile Val Ile Gly Glu Leu
85 90 95
Leu Lys Asp Lys Gln Leu Asn Phe Val Ala Cys Gly Leu Gln Ser Ala
100 105 110
Tyr Val Ser Glu Asn Ala Pro Glu Ala Phe Val Ala Leu Met Ala Lys
115 120 125
His Tyr His Arg Leu Lys Pro Val Lys Asp Tyr Gln Glu Ile Asp Asp
130 135 140
Val Leu Phe Lys Phe Ser Leu Asn Leu Pro Asp Glu Gln Ile Pro Leu
145 150 155 160
Val Ile Asp Lys Leu His Val Ala Leu Asp Gly Ile Met Lys Pro Val
165 170 175
Thr Ser Gly Phe Gly Phe Ile Asp Leu Ile Ile Pro Gly Leu His Lys
180 185 190
Ala Asn Gly Ile Ser Arg Leu Leu Lys Arg Trp Asp Leu Ser Pro Gln
195 200 205
Asn Val Val Ala Ile Gly Asp Ser Gly Asn Asp Ala Glu Met Leu Lys
210 215 220
Met Ala Arg Tyr Ser Phe Ala Met Gly Asn Ala Ala Glu Asn Ile Lys
225 230 235 240
Gln Ile Ala Arg Tyr Ala Thr Asp Asp Asn Asn His Glu Gly Ala Leu
245 250 255
Asn Val Ile Gln Ala Val Leu Asp Asn Thr Ser Pro Phe Asn Ser
260 265 270
<210> 64
<211> 561
<212> PRT
<213> 大肠杆菌
<400> 64
Met Asn Ile Asn Val Ala Glu Leu Leu Asn Gly Asn Tyr Ile Leu Leu
1 5 10 15
Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu
20 25 30
Gly Ser Ile Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu
35 40 45
Leu Leu Gly Gln Gln His Phe Ser Ile Asn Thr Asp Ala Leu Asn Leu
50 55 60
Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn
65 70 75 80
Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala
85 90 95
Leu Val Met Val Gly Ser Ala Leu Val Ile Ala Leu Gly Leu Gly Lys
100 105 110
Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser
115 120 125
Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His
130 135 140
Ser Gly Met Glu Ser Arg Gln Leu Ser Leu Ala Leu Asp Asn Leu Ser
145 150 155 160
Leu Gly Tyr Ala Leu Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Val
165 170 175
Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Gln Thr Ser
180 185 190
Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ala Asn Arg
195 200 205
Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu
210 215 220
Val Ala Trp Thr Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg
225 230 235 240
Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala
245 250 255
Asn Pro Asp Gly Asp Ala Val Leu Gln Met Gly Asp Glu Ile Ala Leu
260 265 270
Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Pro Ser Phe Arg Asn
275 280 285
Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr
290 295 300
Glu Glu Val Val Val Lys Asn His Asn Ala Val Gly Lys Arg Leu Ala
305 310 315 320
Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg
325 330 335
Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly
340 345 350
Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Thr Ile Ala
355 360 365
Asp Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu
370 375 380
Ala Phe Cys Ala Phe Phe Val Ile Gly Leu Met Ile Gly Met Ile Thr
385 390 395 400
Phe Gln Phe Ser Thr Phe Ser Phe Gly Met Gly Asn Ala Ala Gly Leu
405 410 415
Leu Phe Ala Gly Ile Met Leu Gly Phe Met Arg Ala Asn His Pro Thr
420 425 430
Phe Gly Tyr Ile Pro Gln Gly Ala Leu Ser Met Val Lys Glu Phe Gly
435 440 445
Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly Ile
450 455 460
Asn Asn Gly Leu Gly Ala Ile Gly Gly Gln Met Leu Ile Ala Gly Leu
465 470 475 480
Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr
485 490 495
Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala
500 505 510
Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser
515 520 525
Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val
530 535 540
Leu Leu Thr Leu Ala Gly Thr Ile Ile Val Met Val Trp Pro Gly Leu
545 550 555 560
Gly
<210> 65
<211> 199
<212> PRT
<213> 大肠杆菌
<400> 65
Met Lys Gln Ser His Phe Phe Ala His Leu Ser Arg Leu Lys Leu Ile
1 5 10 15
Asn Arg Trp Pro Leu Met Arg Asn Val Arg Thr Glu Asn Val Ser Glu
20 25 30
His Ser Leu Gln Val Ala Met Val Ala His Ala Leu Ala Ala Ile Lys
35 40 45
Asn Arg Lys Phe Gly Gly Asn Val Asn Ala Glu Arg Ile Ala Leu Leu
50 55 60
Ala Met Tyr His Asp Ala Ser Glu Val Leu Thr Gly Asp Leu Pro Thr
65 70 75 80
Pro Val Lys Tyr Phe Asn Ser Gln Ile Ala Gln Glu Tyr Lys Ala Ile
85 90 95
Glu Lys Ile Ala Gln Gln Lys Leu Val Asp Met Val Pro Glu Glu Leu
100 105 110
Arg Asp Ile Phe Ala Pro Leu Ile Asp Glu His Ala Tyr Ser Asp Glu
115 120 125
Glu Lys Ser Leu Val Lys Gln Ala Asp Ala Leu Cys Ala Tyr Leu Lys
130 135 140
Cys Leu Glu Glu Leu Ala Ala Gly Asn Asn Glu Phe Leu Leu Ala Lys
145 150 155 160
Thr Arg Leu Glu Ala Thr Leu Glu Ala Arg Arg Ser Gln Glu Met Asp
165 170 175
Tyr Phe Met Glu Ile Phe Val Pro Ser Phe His Leu Ser Leu Asp Glu
180 185 190
Ile Ser Gln Asp Ser Pro Leu
195
<210> 66
<211> 221
<212> PRT
<213> 大肠杆菌
<400> 66
Met Ser Arg Ile Glu Ala Val Phe Phe Asp Cys Asp Gly Thr Leu Val
1 5 10 15
Asp Ser Glu Val Ile Cys Ser Arg Ala Tyr Val Thr Met Phe Gln Glu
20 25 30
Phe Gly Ile Thr Leu Asp Pro Glu Glu Val Phe Lys Arg Phe Lys Gly
35 40 45
Val Lys Leu Tyr Glu Ile Ile Asp Ile Val Ser Leu Glu His Gly Val
50 55 60
Thr Leu Ala Lys Thr Glu Ala Glu His Val Tyr Arg Ala Glu Val Ala
65 70 75 80
Arg Leu Phe Asp Ser Glu Leu Glu Ala Ile Glu Gly Ala Gly Ala Leu
85 90 95
Leu Ser Ala Ile Thr Ala Pro Met Cys Val Val Ser Asn Gly Pro Asn
100 105 110
Asn Lys Met Gln His Ser Met Gly Lys Leu Asn Met Leu His Tyr Phe
115 120 125
Pro Asp Lys Leu Phe Ser Gly Tyr Asp Ile Gln Arg Trp Lys Pro Asp
130 135 140
Pro Ala Leu Met Phe His Ala Ala Lys Ala Met Asn Val Asn Val Glu
145 150 155 160
Asn Cys Ile Leu Val Asp Asp Ser Val Ala Gly Ala Gln Ser Gly Ile
165 170 175
Asp Ala Gly Met Glu Val Phe Tyr Phe Cys Ala Asp Pro His Asn Lys
180 185 190
Pro Ile Val His Pro Lys Val Thr Thr Phe Thr His Leu Ser Gln Leu
195 200 205
Pro Glu Leu Trp Lys Ala Arg Gly Trp Asp Ile Thr Ala
210 215 220
<210> 67
<211> 604
<212> PRT
<213> 大肠杆菌
<400> 67
Met Ser Lys Ile Ser Asp Leu Asn Tyr Ser Gln His Ile Thr Leu Ala
1 5 10 15
Asp Asn Phe Lys Gln Lys Ser Glu Val Leu Asn Thr Trp Arg Val Gly
20 25 30
Met Asn Asp Phe Ala Arg Ile Ala Gly Gly Gln Asp Asn Arg Arg Asn
35 40 45
Ile Leu Ser Pro Gly Ala Phe Leu Glu Phe Leu Ala Lys Ile Phe Thr
50 55 60
Leu Gly Tyr Val Asp Phe Ser Lys Arg Ser Asn Glu Ala Gly Arg Asn
65 70 75 80
Met Met Ala His Ile Lys Ser Ser Ser Tyr Ser Lys Asp Thr Asn Gly
85 90 95
Asn Glu Lys Met Lys Phe Tyr Met Asn Asn Pro Val Gly Glu Arg Ala
100 105 110
Asp Ser Pro Lys Val Ile Ile Glu Ile Ser Leu Ser Thr Ile Thr Thr
115 120 125
Met Gly Thr Arg Gln Gly His Thr Ala Ile Ile Phe Pro Gln Pro Asp
130 135 140
Gly Ser Thr Asn Arg Tyr Glu Gly Lys Ser Phe Glu Arg Lys Asp Glu
145 150 155 160
Ser Ser Leu His Leu Ile Thr Asn Lys Val Leu Ala Cys Tyr Gln Ser
165 170 175
Glu Ala Asn Lys Lys Ile Ala Arg Leu Leu Asn Asn Asn Gln Glu Leu
180 185 190
Asn Asn Leu Gln Lys Leu Asn Asn Leu Gln Lys Leu Asn Asn Leu Leu
195 200 205
Lys Leu Asn Asn Ile Gln Gly Leu Asn Asn Pro Gln Glu Leu Asn Asn
210 215 220
Pro Gln Asn Leu Asn Asp Ser Gln Glu Leu Asn Asn Ser Gln Glu Leu
225 230 235 240
Asn Ser Pro Gln Glu Leu Asn Asp Pro Gln Glu Leu Asn Asn Ser Gln
245 250 255
Asp Leu Asn Asn Ser Lys Val Ser Cys Thr Val Ser Val Asp Ser Thr
260 265 270
Ile Thr Gly Leu Leu Lys Glu Pro Leu Asn Asn Ala Leu Leu Ala Ile
275 280 285
Arg Asn Glu His Leu Leu Leu Met Pro His Val Cys Asp Glu Ser Ile
290 295 300
Ser Tyr Leu Leu Gly Glu Lys Gly Ile Leu Glu Glu Ile Asp Lys Leu
305 310 315 320
Tyr Ala Leu Asn Asp His Gly Ile Asp Asn Asp Lys Val Gly Asn Asn
325 330 335
Glu Ile Asn Asp Ile Lys Val Asn Leu Ser His Ile Leu Ile Asp Ser
340 345 350
Leu Asp Asp Ala Lys Val Asn Leu Thr Pro Val Ile Asp Ser Ile Leu
355 360 365
Glu Thr Phe Ser Lys Ser Pro Tyr Ile Asn Asp Val Arg Ile Leu Asp
370 375 380
Trp Cys Phe Asn Lys Ser Met Gln Tyr Phe Asp Asp Thr Lys Lys Ile
385 390 395 400
Lys His Ala Cys Ser Val Ile Asn His Ile Asn Leu Arg Ser Asp Gln
405 410 415
Ser Lys Ile Ala Glu Thr Leu Phe Phe Asn Leu Asp Lys Glu Pro Tyr
420 425 430
Lys Asn Ser Pro Glu Leu Gln Gly Leu Ile Trp Asn Lys Leu Val Val
435 440 445
Tyr Val Asn Glu Phe Asn Leu Ser Asn Arg Glu Lys Thr Asn Leu Ile
450 455 460
Gln Arg Leu Phe Asp Asn Val Glu Ser Ile Phe Asn Glu Val Pro Val
465 470 475 480
Ser Ile Leu Val Asn Asp Ile Phe Met Asn Asp Phe Phe Met Lys Asn
485 490 495
Pro Glu Met Ile Asn Trp Tyr Phe Pro Gln Leu Leu Lys Ser Tyr Glu
500 505 510
Gly Glu Lys Ile Tyr Phe Asp Asn Leu Lys Tyr Asp Leu Asn Asp Asn
515 520 525
Asp Lys Glu Ser Asn Lys Glu Ile Leu Lys Asn Gln Pro Asp Asn Val
530 535 540
Ile Lys Glu Lys Leu Asn Asn Glu Tyr Lys Leu Arg Phe Arg Met Met
545 550 555 560
Gln Thr Ile Leu Gln Ser Arg Val Asn Val Leu Pro Tyr Ile Asn Glu
565 570 575
Gln Arg Leu Asn Lys Leu Asn Pro Pro Glu Asn Leu Arg Ile Ala Ile
580 585 590
Glu His Phe Gly Trp Lys Asn Arg Pro Ile Thr Ala
595 600
<210> 68
<211> 225
<212> PRT
<213> 大肠杆菌
<400> 68
Met Lys Trp Asp Trp Ile Phe Phe Asp Ala Asp Glu Thr Leu Phe Thr
1 5 10 15
Phe Asp Ser Phe Thr Gly Leu Gln Arg Met Phe Leu Asp Tyr Ser Val
20 25 30
Thr Phe Thr Ala Glu Asp Phe Gln Asp Tyr Gln Ala Val Asn Lys Pro
35 40 45
Leu Trp Val Asp Tyr Gln Asn Gly Ala Ile Thr Ser Leu Gln Leu Gln
50 55 60
His Gly Arg Phe Glu Ser Trp Ala Glu Arg Leu Asn Val Glu Pro Gly
65 70 75 80
Lys Leu Asn Glu Ala Phe Ile Asn Ala Met Ala Glu Ile Cys Thr Pro
85 90 95
Leu Pro Gly Ala Val Ser Leu Leu Asn Ala Ile Arg Gly Asn Ala Lys
100 105 110
Ile Gly Ile Ile Thr Asn Gly Phe Ser Ala Leu Gln Gln Val Arg Leu
115 120 125
Glu Arg Thr Gly Leu Arg Asp Tyr Phe Asp Leu Leu Val Ile Ser Glu
130 135 140
Glu Val Gly Val Ala Lys Pro Asn Lys Lys Ile Phe Asp Tyr Ala Leu
145 150 155 160
Glu Gln Ala Gly Asn Pro Asp Arg Ser Arg Val Leu Met Val Gly Asp
165 170 175
Thr Ala Glu Ser Asp Ile Leu Gly Gly Ile Asn Ala Gly Leu Ala Thr
180 185 190
Cys Trp Leu Asn Ala His His Arg Glu Gln Pro Glu Gly Ile Ala Pro
195 200 205
Thr Trp Thr Val Ser Ser Leu His Glu Leu Glu Gln Leu Leu Cys Lys
210 215 220
His
225
<210> 69
<211> 222
<212> PRT
<213> 大肠杆菌
<400> 69
Met His Ile Asn Ile Ala Trp Gln Asp Val Asp Thr Val Leu Leu Asp
1 5 10 15
Met Asp Gly Thr Leu Leu Asp Leu Ala Phe Asp Asn Tyr Phe Trp Gln
20 25 30
Lys Leu Val Pro Glu Thr Trp Gly Ala Lys Asn Gly Val Thr Pro Gln
35 40 45
Glu Ala Met Glu Tyr Met Arg Gln Gln Tyr His Asp Val Gln His Thr
50 55 60
Leu Asn Trp Tyr Cys Leu Asp Tyr Trp Ser Glu Gln Leu Gly Leu Asp
65 70 75 80
Ile Cys Ala Met Thr Thr Glu Met Gly Pro Arg Ala Val Leu Arg Glu
85 90 95
Asp Thr Ile Pro Phe Leu Glu Ala Leu Lys Ala Ser Gly Lys Gln Arg
100 105 110
Ile Leu Leu Thr Asn Ala His Pro His Asn Leu Ala Val Lys Leu Glu
115 120 125
His Thr Gly Leu Asp Ala His Leu Asp Leu Leu Leu Ser Thr His Thr
130 135 140
Phe Gly Tyr Pro Lys Glu Asp Gln Arg Leu Trp His Ala Val Ala Glu
145 150 155 160
Ala Thr Gly Leu Lys Ala Glu Arg Thr Leu Phe Ile Asp Asp Ser Glu
165 170 175
Ala Ile Leu Asp Ala Ala Ala Gln Phe Gly Ile Arg Tyr Cys Leu Gly
180 185 190
Val Thr Asn Pro Asp Ser Gly Ile Ala Glu Lys Gln Tyr Gln Arg His
195 200 205
Pro Ser Leu Asn Asp Tyr Arg Arg Leu Ile Pro Ser Leu Met
210 215 220
<210> 70
<211> 421
<212> PRT
<213> 大肠杆菌
<400> 70
Met Ala Ser Thr Phe Thr Ser Asp Thr Leu Pro Ala Asp His Lys Ala
1 5 10 15
Ala Ile Arg Gln Met Lys His Ala Leu Arg Ala Gln Leu Gly Asp Val
20 25 30
Gln Gln Ile Phe Asn Gln Leu Ser Asp Asp Ile Ala Thr Arg Val Ala
35 40 45
Glu Ile Asn Ala Leu Lys Ala Gln Gly Asp Ala Val Trp Pro Val Leu
50 55 60
Ser Tyr Ala Asp Ile Lys Ala Gly His Val Thr Ala Glu Gln Arg Glu
65 70 75 80
Gln Ile Lys Arg Arg Gly Cys Ala Val Ile Lys Gly His Phe Pro Arg
85 90 95
Glu Gln Ala Leu Gly Trp Asp Gln Ser Met Leu Asp Tyr Leu Asp Arg
100 105 110
Asn Arg Phe Asp Glu Val Tyr Lys Gly Pro Gly Asp Asn Phe Phe Gly
115 120 125
Thr Leu Ser Ala Ser Arg Pro Glu Ile Tyr Pro Ile Tyr Trp Ser Gln
130 135 140
Ala Gln Met Gln Ala Arg Gln Ser Glu Glu Met Ala Asn Ala Gln Ser
145 150 155 160
Phe Leu Asn Arg Leu Trp Thr Phe Glu Ser Asp Gly Lys Gln Trp Phe
165 170 175
Asn Pro Asp Val Ser Val Ile Tyr Pro Asp Arg Ile Arg Arg Arg Pro
180 185 190
Pro Gly Thr Thr Ser Lys Gly Leu Gly Ala His Thr Asp Ser Gly Ala
195 200 205
Leu Glu Arg Trp Leu Leu Pro Ala Tyr Gln Arg Val Phe Ala Asn Val
210 215 220
Phe Asn Gly Asn Leu Ala Gln Tyr Asp Pro Trp His Ala Ala His Arg
225 230 235 240
Thr Glu Val Glu Glu Tyr Thr Val Asp Asn Thr Thr Lys Cys Ser Val
245 250 255
Phe Arg Thr Phe Gln Gly Trp Thr Ala Leu Ser Asp Met Leu Pro Gly
260 265 270
Gln Gly Leu Leu His Val Val Pro Ile Pro Glu Ala Met Ala Tyr Val
275 280 285
Leu Leu Arg Pro Leu Leu Asp Asp Val Pro Glu Asp Glu Leu Cys Gly
290 295 300
Val Ala Pro Gly Arg Val Leu Pro Val Ser Glu Gln Trp His Pro Leu
305 310 315 320
Leu Ile Glu Ala Leu Thr Ser Ile Pro Lys Leu Glu Ala Gly Asp Ser
325 330 335
Val Trp Trp His Cys Asp Val Ile His Ser Val Ala Pro Val Glu Asn
340 345 350
Gln Gln Gly Trp Gly Asn Val Met Tyr Ile Pro Ala Ala Pro Met Cys
355 360 365
Glu Lys Asn Leu Ala Tyr Ala His Lys Val Lys Ala Ala Leu Glu Lys
370 375 380
Gly Ala Ser Pro Gly Asp Phe Pro Arg Glu Asp Tyr Glu Thr Asn Trp
385 390 395 400
Glu Gly Arg Phe Thr Leu Ala Asp Leu Asn Ile His Gly Lys Arg Ala
405 410 415
Leu Gly Met Asp Val
420
<210> 71
<211> 246
<212> PRT
<213> 酿酒酵母（Saccharomyces cerevisiae）
<400> 71
Met Ala Glu Phe Ser Ala Asp Leu Cys Leu Phe Asp Leu Asp Gly Thr
1 5 10 15
Ile Val Ser Thr Thr Val Ala Ala Glu Lys Ala Trp Thr Lys Leu Cys
20 25 30
Tyr Glu Tyr Gly Val Asp Pro Ser Glu Leu Phe Lys His Ser His Gly
35 40 45
Ala Arg Thr Gln Glu Val Leu Arg Arg Phe Phe Pro Lys Leu Asp Asp
50 55 60
Thr Asp Asn Lys Gly Val Leu Ala Leu Glu Lys Asp Ile Ala His Ser
65 70 75 80
Tyr Leu Asp Thr Val Ser Leu Ile Pro Gly Ala Glu Asn Leu Leu Leu
85 90 95
Ser Leu Asp Val Asp Thr Glu Thr Gln Lys Lys Leu Pro Glu Arg Lys
100 105 110
Trp Ala Ile Val Thr Ser Gly Ser Pro Tyr Leu Ala Phe Ser Trp Phe
115 120 125
Glu Thr Ile Leu Lys Asn Val Gly Lys Pro Lys Val Phe Ile Thr Gly
130 135 140
Phe Asp Val Lys Asn Gly Lys Pro Asp Pro Glu Gly Tyr Ser Arg Ala
145 150 155 160
Arg Asp Leu Leu Arg Gln Asp Leu Gln Leu Thr Gly Lys Gln Asp Leu
165 170 175
Lys Tyr Val Val Phe Glu Asp Ala Pro Val Gly Ile Lys Ala Gly Lys
180 185 190
Ala Met Gly Ala Ile Thr Val Gly Ile Thr Ser Ser Tyr Asp Lys Ser
195 200 205
Val Leu Phe Asp Ala Gly Ala Asp Tyr Val Val Cys Asp Leu Thr Gln
210 215 220
Val Ser Val Val Lys Asn Asn Glu Asn Gly Ile Val Ile Gln Val Asn
225 230 235 240
Asn Pro Leu Thr Arg Ala
245
<210> 72
<211> 272
<212> PRT
<213> 枯草芽孢杆菌（Bacillus subtilis）
<400> 72
Met Arg Ile Met Ala Ser His Asp Thr Pro Val Ser Pro Ala Gly Ile
1 5 10 15
Leu Ile Asp Leu Asp Gly Thr Val Phe Arg Gly Asn Glu Leu Ile Glu
20 25 30
Gly Ala Arg Glu Ala Ile Lys Thr Leu Arg Arg Met Gly Lys Lys Ile
35 40 45
Val Phe Leu Ser Asn Arg Gly Asn Ile Ser Arg Ala Met Cys Arg Lys
50 55 60
Lys Leu Leu Gly Ala Gly Ile Glu Thr Asp Val Asn Asp Ile Val Leu
65 70 75 80
Ser Ser Ser Val Thr Ala Ala Phe Leu Lys Lys His Tyr Arg Phe Ser
85 90 95
Lys Val Trp Val Leu Gly Glu Gln Gly Leu Val Asp Glu Leu Arg Leu
100 105 110
Ala Gly Val Gln Asn Ala Ser Glu Pro Lys Glu Ala Asp Trp Leu Val
115 120 125
Ile Ser Leu His Glu Thr Leu Thr Tyr Asp Asp Leu Asn Gln Ala Phe
130 135 140
Gln Ala Ala Ala Gly Gly Ala Arg Ile Ile Ala Thr Asn Lys Asp Arg
145 150 155 160
Ser Phe Pro Asn Glu Asp Gly Asn Ala Ile Asp Val Ala Gly Met Ile
165 170 175
Gly Ala Ile Glu Thr Ser Ala Gln Ala Lys Thr Glu Leu Val Val Gly
180 185 190
Lys Pro Ser Trp Leu Met Ala Glu Ala Ala Cys Thr Ala Met Gly Leu
195 200 205
Ser Ala His Glu Cys Met Ile Ile Gly Asp Ser Ile Glu Ser Asp Ile
210 215 220
Ala Met Gly Lys Leu Tyr Gly Met Lys Ser Ala Leu Val Leu Thr Gly
225 230 235 240
Ser Ala Lys Gln Gly Glu Gln Arg Leu Tyr Thr Pro Asp Tyr Val Leu
245 250 255
Asp Ser Ile Lys Asp Val Thr Lys Leu Ala Glu Glu Gly Ile Leu Ile
260 265 270
<210> 73
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 基序1: hDxDx[TV]
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X是疏水氨基酸 (A, I, L, M, F, V, P或G)
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> X可以是T或V
<400> 73
Xaa Asp Xaa Asp Xaa Xaa
1 5
<210> 74
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 基序2: [GSTDE][DSEN]x[hP]x[DGTS]
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X可以是G, S, T, D或E
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X可以是D, S, E或N
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> X可以是疏水氨基酸 (A, I, L, M, F, V, P或G)或P
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> X可以是D, G, T或S
<400> 74
Xaa Xaa Xaa Xaa Xaa Xaa
1 5
<210> 75
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> [GSTDE][DSEN]xx[hP]x[DGTS]
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X可以是G, S, T, D或E
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X可以是D, S, E或N
<220>
<221> MISC_FEATURE
<222> (3)..(4)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> X可以是疏水氨基酸 (A, I, L, M, F, V, P或G)或P
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> X可以是D, G, T或S
<400> 75
Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5
<210> 76
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> [GSTDE][DSEN]x[hP]xx[DGTS]
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X可以是G, S, T, D或E
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X可以是D, S, E或N
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> X可以是疏水氨基酸 (A, I, L, M, F, V, P或G)或P
<220>
<221> MISC_FEATURE
<222> (5)..(6)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> X可以是D, G, T或S
<400> 76
Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5
<210> 77
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> [GSTDE][DSEN]xx[hP]xx[DGTS]
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X可以是G, S, T, D或E
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X可以是D, S, E或N
<220>
<221> MISC_FEATURE
<222> (3)..(4)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> X可以是疏水氨基酸 (A, I, L, M, F, V, P或G)或P
<220>
<221> MISC_FEATURE
<222> (6)..(7)
<223> X可以是任意氨基酸
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> X可以是a D, G, T或S
<400> 77
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5
<210> 78
<211> 272
<212> PRT
<213> 弗氏志贺氏菌（Shigella flexneri）
<400> 78
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asp His His Leu Gly Glu Lys Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Ala Leu Glu
35 40 45
Met Gln His Ile Leu Gly Ala Leu Ser Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Val His Ser Leu Glu Gly Glu Leu Leu His Arg
65 70 75 80
Asp Asp Leu Pro Ala Asp Val Ala Glu Leu Val Leu Tyr Gln Gln Trp
85 90 95
Asp Thr Arg Ala Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr
100 105 110
Gly Lys Glu Ile Pro Ala Leu Leu Gln Ala Phe Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Ile Ile Asp Val Lys Lys Met Pro Leu Gly Ser Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile
145 150 155 160
Gln Leu Tyr Glu Ala Leu Gly Glu Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Thr Asp Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala
180 185 190
Leu Thr Val Leu Thr Gln His Leu Asp Leu Ser Leu Arg Asp Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Val Ser Val Gly
210 215 220
Ser Gly Phe Ile Met Gly Asn Ala Met Pro Gln Leu Arg Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His
245 250 255
Tyr Leu Thr His Trp Leu Asp Tyr Pro His Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 79
<211> 272
<212> PRT
<213> 鲍氏志贺氏菌（Shigella boydii）
<400> 79
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asp His His Leu Gly Glu Lys Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Ala Leu Glu
35 40 45
Met Gln His Ile Leu Gly Ala Leu Ser Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Val His Ser Leu Glu Gly Glu Leu Leu His Arg
65 70 75 80
Asp Asp Leu Pro Ala Asp Val Ala Glu Leu Val Leu Tyr Gln Gln Trp
85 90 95
Asp Thr Arg Ala Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr
100 105 110
Gly Lys Glu Ile Pro Ala Leu Leu Gln Ala Phe Val Tyr Asn Gly Phe
115 120 125
Arg Tyr Gln Ile Ile Asp Val Lys Lys Met Pro Leu Gly Ser Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile
145 150 155 160
Gln Leu Tyr Glu Ala Leu Gly Glu Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Thr Asp Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala
180 185 190
Leu Thr Val Leu Thr Gln His Leu Gly Leu Ser Leu Arg Asp Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Gly Ser Val Gly
210 215 220
Ser Gly Phe Ile Met Gly Asn Ala Met Pro Gln Leu Arg Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His
245 250 255
Tyr Leu Thr His Trp Leu Asp Tyr Pro His Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 80
<211> 272
<212> PRT
<213> 弗格森埃希氏菌（Escherichia fergusonii）
<400> 80
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asp His His Leu Gly Glu Lys Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Ala Leu Glu
35 40 45
Met Gln His Ile Leu Gly Ala Leu Ser Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Val His Ser Leu Glu Gly Glu Leu Leu His Arg
65 70 75 80
Asp Asp Leu Pro Ala Asp Val Ala Glu Leu Val Leu Tyr Gln Gln Trp
85 90 95
Asp Thr Arg Ala Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr
100 105 110
Gly Lys Glu Ile Pro Ala Leu Leu Gln Ala Phe Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Ile Ile Asp Val Lys Lys Met Pro Leu Asp Arg Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile
145 150 155 160
Gln Leu His Glu Ala Leu Gly Glu Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Thr Asp Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala
180 185 190
Leu Thr Val Leu Thr Gln His Leu Gly Leu Ser Leu Arg Glu Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Gly Ser Val Gly
210 215 220
Ser Gly Phe Ile Met Gly Asn Ala Met Pro Gln Leu Arg Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His
245 250 255
Tyr Leu Thr His Trp Leu Asp Tyr Pro His Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 81
<211> 245
<212> PRT
<213> 金黄色葡萄球菌（Staphylococcus aureus）
<400> 81
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Val Pro
1 5 10 15
Asp His His Leu Gly Glu Lys Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Ala Leu Glu
35 40 45
Met Gln His Ile Leu Gly Ala Leu Ser Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Val His Ser Leu Glu Gly Glu Leu Leu His Arg
65 70 75 80
Asp Asp Leu Pro Ala Asp Val Ala Glu Leu Val Leu Tyr Gln Gln Trp
85 90 95
Asp Thr Arg Ala Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr
100 105 110
Gly Lys Glu Ile Pro Ala Leu Leu Gln Ala Phe Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Ile Ile Asp Val Lys Lys Met Pro Leu Gly Ser Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile
145 150 155 160
Gln Leu Tyr Glu Ala Leu Gly Glu Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Thr Asp Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala
180 185 190
Leu Thr Val Leu Thr Gln His Leu Gly Leu Ser Leu Arg Asp Cys Thr
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Val Ser Val Gly
210 215 220
Ser Gly Phe Ile Met Gly Asn Ala Met Pro Gln Leu Arg Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val
245
<210> 82
<211> 272
<212> PRT
<213> unknown
<220>
<223> 阿尔伯蒂埃希氏菌（Escherichia albertii）
<400> 82
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asp His His Leu Gly Glu Lys Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Asp Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Ala Leu Glu
35 40 45
Met Arg His Ile Leu Gly Thr Phe Ala Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Val His Ser Gln Glu Gly Asp Leu Leu Tyr Arg
65 70 75 80
Asp Asp Leu Pro Ala Asp Val Ala Glu Arg Val Leu His Gln Lys Trp
85 90 95
Asp Thr Gln Ala Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr
100 105 110
Gly Gln Glu Met Pro Ser Leu Leu Gln Ala Phe Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Ile Ile Asp Val Lys Lys Met Pro Leu Asp Arg Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile
145 150 155 160
Gln Leu Asn Glu Ala Leu Gly Asp Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Ile Asn Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala
180 185 190
Leu Ala Val Leu Ala Asp Tyr Leu Gly Phe Ser Leu Arg Asp Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Gly Ser Val Gly
210 215 220
Asn Gly Phe Ile Met Gly Asn Ala Met Pro Gln Leu Leu Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His
245 250 255
Tyr Leu Thr His Trp Leu Asp Asn Pro His Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 83
<211> 272
<212> PRT
<213> 无丙二酸柠檬酸杆菌（Citrobacter amalonaticus）
<400> 83
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asn His Leu Leu Gly Asp Glu Thr Leu Ser Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Val Leu Glu
35 40 45
Met Arg His Ile Leu Gly Thr Phe Ser Leu Asp Ala Phe Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Ile His Ser Leu Glu Gly Glu Val Leu His Arg
65 70 75 80
Gln Asp Leu Glu Pro Ala Val Ala Glu Ile Val Leu His Gln Arg Trp
85 90 95
Asp Thr Gln Ala Ser Met His Ile Phe Asn Asp Asn Gly Trp Phe Thr
100 105 110
Gly Gln Glu Ile Pro Glu Met Leu His Ala His Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Ile Val Asp Val Ala Arg Ile Pro Ala Asp Arg Val Thr
130 135 140
Lys Val Cys Phe Cys Gly Asp His Asp Asp Leu Thr Arg Leu Lys Ile
145 150 155 160
Gln Leu Glu Glu Val Leu Gly Ala Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Val Asp Cys Leu Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ser Ala
180 185 190
Leu Glu Val Leu Ser Gly His Leu Gly Leu Ser Leu Ala Glu Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Gly Ser Val Gly
210 215 220
Arg Gly Leu Ile Met Gly Asn Ala Met Pro Gln Leu Ile Ala Glu Leu
225 230 235 240
Pro His Leu Pro Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His
245 250 255
Phe Leu Thr His Trp Leu Asp Tyr Pro Asn Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 84
<211> 272
<212> PRT
<213> 肠沙门氏菌（Salmonella enterica）
<400> 84
Met Ala Arg Leu Ala Ala Phe Asp Met Asp Gly Thr Leu Leu Met Pro
1 5 10 15
Asp His His Leu Gly Lys Glu Thr Leu Ala Thr Leu Ala Arg Leu Arg
20 25 30
Glu Arg Asp Ile Thr Leu Thr Phe Ala Thr Gly Arg His Val Leu Glu
35 40 45
Met Arg His Ile Leu Gly Thr Leu Ser Leu Asp Ala Tyr Leu Ile Thr
50 55 60
Gly Asn Gly Thr Arg Ile His Ser Leu Glu Gly Asp Val Leu His Arg
65 70 75 80
Gln Asp Leu Asp Pro Gln Val Ala Asp Thr Val Met His His Ala Trp
85 90 95
Asp Thr Arg Ala Ser Met His Val Phe Asn Asp Asn Gly Trp Phe Thr
100 105 110
Gly Gln Glu Ile Pro Ala Leu Leu Gln Ala His Val Tyr Ser Gly Phe
115 120 125
Arg Tyr Gln Val Ile Asp Ile Lys Ser Ile Pro Ala His Gln Val Thr
130 135 140
Lys Ile Cys Phe Cys Gly Asp His Asp Asp Leu Ile Arg Leu Arg Ile
145 150 155 160
Gln Leu Asn Glu Ala Leu Glu Glu Arg Ala His Leu Cys Phe Ser Ala
165 170 175
Val Asp Cys Leu Glu Val Leu Pro Leu Gly Cys Asn Lys Gly Ser Ala
180 185 190
Leu Ala Val Leu Ser Asn His Leu Gly Leu Ser Leu Ala Asp Cys Met
195 200 205
Ala Phe Gly Asp Ala Met Asn Asp Arg Glu Met Leu Gly Ser Val Gly
210 215 220
Arg Gly Leu Ile Met Gly Asn Ala Met Pro Gln Leu Ile Ala Ala Leu
225 230 235 240
Pro His Leu Ser Val Ile Gly His Cys Gly Asn Gln Ala Val Ser His
245 250 255
Phe Leu Thr His Trp Leu Asp Asn Pro His Leu Pro Tyr Ser Pro Glu
260 265 270
<210> 85
<211> 220
<212> PRT
<213> 大肠杆菌
<400> 85
Leu Gly Ala Leu Ser Leu Asp Ala Tyr Leu Ile Thr Gly Asn Gly Thr
1 5 10 15
Arg Val His Ser Leu Glu Gly Glu Leu Leu His Arg Asp Asp Leu Pro
20 25 30
Ala Asp Val Ala Glu Leu Val Leu Tyr Gln Gln Trp Asp Thr Arg Ala
35 40 45
Ser Met His Ile Phe Asn Asp Asp Gly Trp Phe Thr Gly Lys Glu Ile
50 55 60
Pro Ala Leu Leu Gln Ala Phe Val Tyr Ser Gly Phe Arg Tyr Gln Ile
65 70 75 80
Ile Asp Val Lys Lys Met Pro Leu Gly Ser Val Thr Lys Ile Cys Phe
85 90 95
Cys Gly Asp His Asp Asp Leu Thr Arg Leu Gln Ile Gln Leu Tyr Glu
100 105 110
Ala Leu Gly Glu Arg Ala His Leu Cys Phe Ser Ala Thr Asp Cys Leu
115 120 125
Glu Val Leu Pro Val Gly Cys Asn Lys Gly Ala Ala Leu Thr Val Leu
130 135 140
Thr Gln His Leu Gly Leu Ser Leu Arg Asp Cys Met Ala Phe Gly Asp
145 150 155 160
Ala Met Asn Asp Arg Glu Met Leu Gly Ser Val Gly Ser Gly Phe Ile
165 170 175
Met Gly Asn Ala Met Pro Gln Leu Arg Ala Glu Leu Pro His Leu Pro
180 185 190
Val Ile Gly His Cys Arg Asn Gln Ala Val Ser His Tyr Leu Thr His
195 200 205
Trp Leu Asp Tyr Pro His Leu Pro Tyr Ser Pro Glu
210 215 220
<210> 86
<211> 355
<212> PRT
<213> 弗氏志贺氏菌
<400> 86
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Thr
130 135 140
Gly Leu Arg Tyr Asp Arg Glu Thr Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Ile Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Met Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 87
<211> 355
<212> PRT
<213> 阿尔伯蒂埃希氏菌
<400> 87
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Thr
130 135 140
Gly Leu Arg Tyr Asp Arg Glu Thr Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Asp Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Ile Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Met Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 88
<211> 355
<212> PRT
<213> 弗氏志贺氏菌
<400> 88
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Gly Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Asn
130 135 140
Gly Leu Arg Tyr Asp Arg Glu Thr Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Ile Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Leu Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 89
<211> 355
<212> PRT
<213> 索氏志贺氏菌（Shigella sonnei）
<400> 89
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro Pro Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Asn
130 135 140
Gly Leu Arg Tyr Asp Arg Glu Ile Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Met Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 90
<211> 355
<212> PRT
<213> 大肠杆菌
<400> 90
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Gln Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ser Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Asn
130 135 140
Gly Leu Arg Tyr Asp Arg Glu Thr Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Ile Lys Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Ile Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 91
<211> 355
<212> PRT
<213> 痢疾志贺氏菌（Shigella dysenteriae）
<400> 91
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Asn
130 135 140
Gly Leu Arg Tyr Asp Cys Glu Ile Leu Ser Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asp Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Arg Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Met Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 92
<211> 355
<212> PRT
<213> 旱獭埃希氏菌（Escherichia marmotae）
<400> 92
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Gly Val Ile Pro Glu Leu Leu Lys Leu Gln Lys Ala Gly Tyr
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Arg Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Ile Phe
65 70 75 80
Thr Ser Gln Gly Val Gln Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Asp Asp Glu Cys Asp Cys Arg Lys Pro Lys Val Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Arg Ala Ser Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Ile Gln Leu Ala Glu Asn Met Gly Ile Asn
130 135 140
Gly Leu Arg Tyr Asn Arg Glu Thr Leu Asn Trp Pro Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asn Arg Tyr Ala His Val Val Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Gln Val Trp Leu Asp Arg Glu Gly Gly Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Glu Ile Asn Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Ile Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 93
<211> 355
<212> PRT
<213> 猪霍乱沙门氏菌（Salmonella bongori）
<400> 93
Met Ser Gln Lys Tyr Leu Phe Ile Asp Arg Asp Gly Thr Leu Ile Ser
1 5 10 15
Glu Pro Pro Ser Asp Phe Gln Val Asp Arg Phe Asp Lys Leu Ala Phe
20 25 30
Glu Pro Glu Val Ile Pro Val Leu Leu Lys Leu Gln Lys Ala Gly Phe
35 40 45
Lys Leu Val Met Ile Thr Asn Gln Asp Gly Leu Gly Thr Gln Ser Phe
50 55 60
Pro Gln Ala Asp Phe Asp Gly Pro His Asn Leu Met Met Gln Val Phe
65 70 75 80
Thr Ser Gln Gly Val His Phe Asp Glu Val Leu Ile Cys Pro His Leu
85 90 95
Pro Ala Asp Glu Cys Asp Cys Arg Lys Pro Lys Ile Lys Leu Val Glu
100 105 110
Arg Tyr Leu Ala Glu Gln Ala Met Asp Ser Ala Asn Ser Tyr Val Ile
115 120 125
Gly Asp Arg Ala Thr Asp Val Gln Leu Ala Asp Asn Met Gly Ile Thr
130 135 140
Gly Leu Arg Tyr His Arg Glu Thr Leu Asn Trp Ser Met Ile Gly Glu
145 150 155 160
Gln Leu Thr Lys Arg Asp Arg Tyr Ala His Val Ile Arg Asn Thr Lys
165 170 175
Glu Thr Gln Ile Asp Val Arg Val Trp Leu Asp Arg Glu Gly Asn Ser
180 185 190
Lys Ile Asn Thr Gly Val Gly Phe Phe Asp His Met Leu Asp Gln Ile
195 200 205
Ala Thr His Gly Gly Phe Arg Met Asp Val Thr Val Lys Gly Asp Leu
210 215 220
Tyr Ile Asp Asp His His Thr Val Glu Asp Thr Gly Leu Ala Leu Gly
225 230 235 240
Glu Ala Leu Lys Leu Ala Leu Gly Asp Lys Arg Gly Ile Cys Arg Phe
245 250 255
Gly Phe Val Leu Pro Met Asp Glu Cys Leu Ala Arg Cys Ala Leu Asp
260 265 270
Ile Ser Gly Arg Pro His Leu Glu Tyr Lys Ala Glu Phe Thr Tyr Gln
275 280 285
Arg Val Gly Asp Leu Ser Thr Glu Met Ile Glu His Phe Phe Arg Ser
290 295 300
Leu Ser Tyr Thr Met Gly Val Thr Leu His Leu Lys Thr Lys Gly Lys
305 310 315 320
Asn Asp His His Arg Val Glu Ser Leu Phe Lys Ala Phe Gly Arg Thr
325 330 335
Leu Arg Gln Ala Ile Arg Val Glu Gly Asp Thr Leu Pro Ser Ser Lys
340 345 350
Gly Val Leu
355
<210> 94
<211> 266
<212> PRT
<213> 索氏志贺氏菌
<400> 94
Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Leu Ser Ala Lys Tyr Ala
1 5 10 15
Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu Ile Lys Pro His Pro
20 25 30
Asp Gln Val Val Val Pro Asp Asn Ile Leu Gln Gly Leu Gln Leu Leu
35 40 45
Ala Thr Ala Ser Asp Gly Ala Leu Ala Leu Ile Ser Gly Arg Ser Met
50 55 60
Val Glu Leu Asp Ala Leu Ala Lys Pro Tyr Arg Phe Pro Leu Ala Gly
65 70 75 80
Val His Gly Ala Glu Arg Arg Asp Ile Asn Gly Lys Thr His Ile Val
85 90 95
His Leu Pro Asp Ala Ile Ala Arg Asp Ile Ser Val Gln Leu His Thr
100 105 110
Val Ile Ala Gln Tyr Pro Gly Ala Glu Leu Glu Ala Lys Gly Met Ala
115 120 125
Phe Ala Leu His Tyr Arg Gln Ala Pro Gln His Glu Asp Ala Leu Met
130 135 140
Thr Leu Ala Gln Arg Ile Thr Gln Ile Trp Pro Gln Met Ala Leu Gln
145 150 155 160
Gln Gly Lys Cys Val Val Glu Ile Lys Pro Arg Gly Thr Ser Lys Gly
165 170 175
Glu Ala Ile Ala Ala Phe Met Gln Glu Ala Pro Phe Ile Gly Arg Thr
180 185 190
Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val
195 200 205
Val Asn Arg Leu Gly Gly Met Ser Val Lys Ile Gly Thr Gly Ala Thr
210 215 220
Gln Ala Ser Trp Arg Leu Ala Gly Val Pro Asp Val Trp Ser Trp Leu
225 230 235 240
Glu Met Ile Ser Thr Ala Leu Gln Gln Lys Arg Glu Asn Asn Arg Ser
245 250 255
Asp Asp Tyr Glu Ser Phe Ser Arg Ser Ile
260 265
<210> 95
<211> 266
<212> PRT
<213> 大肠杆菌
<400> 95
Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Leu Ser Ala Lys Tyr Ala
1 5 10 15
Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu Ile Lys Pro His Pro
20 25 30
Asp Gln Val Val Val Pro Asp Asn Ile Leu Gln Gly Leu Gln Leu Leu
35 40 45
Ala Thr Ala Ser Asp Gly Ala Leu Ala Leu Ile Ser Gly Arg Ser Met
50 55 60
Val Glu Leu Asp Ala Leu Ala Lys Pro Tyr Arg Phe Pro Leu Ala Gly
65 70 75 80
Val His Gly Ala Glu Arg Arg Asp Ile Asn Gly Lys Thr His Ile Val
85 90 95
His Leu Pro Asp Ala Ile Ala Arg Asp Ile Ser Val Gln Leu His Thr
100 105 110
Val Ile Ala Gln Tyr Pro Gly Ala Glu Leu Glu Ala Lys Gly Met Ala
115 120 125
Phe Ala Leu His Tyr Arg Gln Ala Pro Gln His Glu Asp Ala Leu Met
130 135 140
Thr Leu Ala Gln Arg Ile Thr Gln Ile Trp Pro Gln Met Ala Leu Gln
145 150 155 160
Gln Gly Lys Cys Val Val Glu Ile Lys Pro Arg Gly Thr Ser Lys Gly
165 170 175
Glu Ala Ile Ala Ala Phe Met Gln Glu Ala Pro Phe Ile Gly Arg Thr
180 185 190
Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val
195 200 205
Val Asn Arg Leu Gly Gly Met Ser Ile Lys Ile Gly Thr Gly Ala Thr
210 215 220
Gln Ala Ser Trp Arg Leu Ala Gly Val Pro Asp Val Trp Ser Trp Leu
225 230 235 240
Glu Met Ile Thr Thr Ala Leu Gln Gln Lys Arg Glu Asn Asn Arg Ser
245 250 255
Asp Asp Tyr Glu Ser Phe Ser Arg Ser Ile
260 265
<210> 96
<211> 266
<212> PRT
<213> 弗氏志贺氏菌
<400> 96
Met Thr Glu Pro Leu Thr Glu Thr Pro Glu Leu Ser Ala Lys Tyr Ala
1 5 10 15
Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu Ile Lys Pro His Pro
20 25 30
Asp Gln Val Val Val Pro Asp Asn Ile Leu Gln Gly Leu Gln Leu Leu
35 40 45
Ala Thr Ala Ser Asp Gly Ala Leu Ala Leu Ile Ser Gly Arg Ser Met
50 55 60
Val Glu Leu Asp Ala Leu Ala Lys Pro Tyr Arg Phe Pro Leu Ala Gly
65 70 75 80
Val His Gly Ala Glu Arg Arg Asp Ile Asn Gly Lys Thr His Ile Val
85 90 95
His Leu Pro Asp Ala Ile Ala Arg Asp Ile Ser Val Gln Leu His Thr
100 105 110
Val Ile Ala Gln Tyr Pro Gly Ala Glu Leu Glu Ala Lys Gly Met Ala
115 120 125
Phe Ala Leu His Tyr Arg Gln Ala Pro Gln His Glu Asp Ala Leu Met
130 135 140
Thr Leu Ala Gln Arg Ile Thr Gln Ile Trp Pro Gln Met Ala Leu Gln
145 150 155 160
Gln Gly Lys Cys Val Val Glu Ile Lys Pro Arg Gly Thr Ser Lys Gly
165 170 175
Glu Ala Ile Ala Ala Phe Met Gln Glu Ala Pro Phe Ile Gly Arg Thr
180 185 190
Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val
195 200 205
Val Asn Arg Leu Gly Gly Met Ser Val Lys Ile Gly Thr Gly Ala Thr
210 215 220
Gln Ala Ser Trp Arg Leu Ala Gly Val Pro Asp Val Trp Ser Trp Leu
225 230 235 240
Lys Met Ile Thr Ala Ala Leu Gln Gln Lys Arg Glu Asn Asn Arg Ser
245 250 255
Asp Asp Tyr Glu Ser Phe Ser Arg Ser Ile
260 265
<210> 97
<211> 265
<212> PRT
<213> 阿尔伯蒂埃希氏菌
<400> 97
Met Thr Glu Pro Leu Thr Ala Leu Pro Glu Leu Ser Ala Lys Tyr Ala
1 5 10 15
Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu Ile Lys Pro His Pro
20 25 30
Asp Gln Val Ala Val Pro Asp Lys Ile Leu Gln Gly Leu Gln Arg Leu
35 40 45
Ala Thr Ile Ser His Gly Ala Leu Ala Leu Ile Ser Gly Arg Ser Met
50 55 60
Val Glu Leu Asp Ala Leu Val Asn Pro Tyr Arg Phe Pro Leu Ala Gly
65 70 75 80
Val His Gly Ala Glu Arg Arg Asp Ile Asn Gly Lys Thr His Ile Val
85 90 95
His Leu Pro Lys Ala Met Ala Arg Asp Ile Cys Val Gln Leu His Met
100 105 110
Ala Ile Ala Arg Phe Pro Gly Ala Glu Leu Glu Glu Lys Gly Met Ala
115 120 125
Phe Ala Leu His Tyr Arg Gln Val Pro Gln Tyr Glu Asp Ala Leu Leu
130 135 140
Thr Leu Ala Arg His Ile Thr Gln Thr Trp Pro Gln Met Val Leu Gln
145 150 155 160
Gln Gly Lys Cys Val Val Glu Ile Lys Pro Gly Gly Thr Ser Lys Gly
165 170 175
Glu Ala Ile Val Ala Phe Met Gln Glu Thr Pro Phe Ile Gly Arg Lys
180 185 190
Pro Val Phe Leu Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val
195 200 205
Val Asn Arg Leu Gly Gly Ile Ser Val Lys Ile Gly Thr Gly Ala Thr
210 215 220
Gln Ala Ser Trp Arg Met Ala Gly Val Pro Asp Val Trp Arg Trp Leu
225 230 235 240
Glu Met Ile Thr His Ala Leu Gln Lys Arg Ala Asn Asp Arg Ser Asp
245 250 255
Asn Asp Glu Pro Phe Ser Arg Ser Ile
260 265
<210> 98
<211> 267
<212> PRT
<213> 弗格森埃希氏菌
<400> 98
Met Thr Lys Pro Leu Thr Glu Ala Pro Glu Leu Ser Ala Lys Tyr Ala
1 5 10 15
Trp Phe Phe Asp Leu Asp Gly Thr Leu Ala Glu Ile Lys Pro His Pro
20 25 30
Asp Gln Val Ala Ile Pro Asp Ala Ile Leu Gln Gly Leu Gln Gln Leu
35 40 45
Ala Val His Ser Asp Gly Ala Leu Ala Leu Ile Ser Gly Arg Ser Met
50 55 60
Val Glu Leu Asp Thr Leu Ala Lys Pro Tyr Arg Phe Pro Leu Ala Gly
65 70 75 80
Val His Gly Ala Glu Arg Arg Asp Ile Asn Gly Lys Thr His Ile Val
85 90 95
Arg Leu Pro Asp Ala Met Val Arg Asp Ile Ser Val Gln Leu His Thr
100 105 110
Thr Leu Ala Gly Leu Thr Gly Val Glu Ile Glu Glu Lys Gly Met Ala
115 120 125
Phe Ala Leu His Tyr Arg Gln Ala Pro Gln His Glu Ala Leu Leu Phe
130 135 140
Thr Leu Ala Gln Arg Ile Thr Gln Ile Trp Pro Gln Met Ala Leu Gln
145 150 155 160
Gln Gly Lys Cys Val Val Glu Ile Lys Pro Arg Gly Thr Cys Lys Gly
165 170 175
Asp Ala Ile Ala Glu Phe Met Gln Glu Thr Pro Phe Val Gly Arg Ile
180 185 190
Pro Ile Phe Trp Gly Asp Asp Leu Thr Asp Glu Ser Gly Phe Ala Val
195 200 205
Val Asn Gln Ala Gly Gly Ile Ser Val Lys Ile Gly Ala Gly Glu Thr
210 215 220
Gln Ala Lys Trp Arg Leu Ala Gly Val Pro Asp Val Trp Arg Trp Leu
225 230 235 240
Ala Val Ile Thr Asn Ser Leu Gln Glu Gln Lys Leu Glu Glu Asn Arg
245 250 255
Ser Asp Asp Tyr Glu Ser Phe Ser Arg Ser Ile
260 265
<210> 99
<211> 191
<212> PRT
<213> 弗格森埃希氏菌
<400> 99
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Lys Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Ser Val Glu Glu Phe Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Leu Ser Ala Arg Asp Tyr Leu His Ile Asp Met
115 120 125
Ala Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Thr Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Ile Thr Pro Glu Ala Glu Asn Ala Ala Asp Trp Val Leu Asn Ser Leu
165 170 175
Ala Asp Leu Pro Gln Ala Ile Lys Lys Gln Gln Lys Pro Ala Gln
180 185 190
<210> 100
<211> 191
<212> PRT
<213> 索氏志贺氏菌
<400> 100
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Lys Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Ser Val Glu Glu Phe Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Leu Ser Ala Arg Asp Tyr Leu His Ile Asp Met
115 120 125
Ala Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Val Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Ile Thr Pro Glu Ala Glu Asn Ala Ala Asp Trp Met Leu Asn Ser Leu
165 170 175
Ala Asp Leu Pro Gln Ala Ile Lys Lys Gln Gln Lys Pro Ala Gln
180 185 190
<210> 101
<211> 191
<212> PRT
<213> 大肠杆菌
<400> 101
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Lys Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Ser Val Glu Glu Phe Arg Gln Leu Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Leu Ser Ala Arg Asp Tyr Leu His Ile Asp Met
115 120 125
Ala Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Val Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Ile Thr Pro Glu Ala Glu Asn Ala Ala Asp Trp Val Leu Asn Ser Leu
165 170 175
Ala Asp Leu Pro Gln Ala Ile Lys Lys Gln Gln Lys Pro Ala Arg
180 185 190
<210> 102
<211> 188
<212> PRT
<213> 阿尔伯蒂埃希氏菌
<400> 102
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Lys Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Ser Val Glu Glu Phe Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Ile Ser Ala Arg Asp Tyr Leu His Ile Asp Met
115 120 125
Thr Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Ser Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Val Thr Pro Glu Ala Glu Asp Ala Ala Asp Trp Val Leu Asn Ser Leu
165 170 175
Ala Asp Leu Pro Gln Ala Ile Lys Lys Gln Gln Lys
180 185
<210> 103
<211> 188
<212> PRT
<213> 中间克吕沃尔氏菌（Kluyvera intermedia）
<400> 103
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Glu Gly Val Ile Asp Ala Met Arg Glu Leu Lys Ala Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Thr Val Glu Glu Phe Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Ile Ser Ala Arg Asp Tyr Leu His Ile Asp Met
115 120 125
Ser Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Ala Ala Ala Asp Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Val Thr Pro Glu Ala Glu Asn Ala Ala Asp Trp Val Ile Asn Ser Leu
165 170 175
Ala Asp Leu Pro Ala Ala Ile Lys Lys Gln Gln Lys
180 185
<210> 104
<211> 188
<212> PRT
<213> 柯氏柠檬酸杆菌(Citrobacter koseri)
<400> 104
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Glu Phe Glu Phe Ile
20 25 30
Glu Gly Val Ile Asp Ala Met Arg Glu Leu Lys Lys Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Asp Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Ser Val Glu Glu Phe Arg Gln Ala Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Ile Ser Ala Arg Asp Phe Leu His Ile Asp Met
115 120 125
Ala Ala Ser Tyr Met Val Gly Asp Lys Ile Glu Asp Met Gln Ala Ala
130 135 140
Ala Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Val Thr Pro Glu Ala Glu Asn Ala Ala Asp Trp Val Leu Asn Ser Leu
165 170 175
Ala Asp Leu Pro Ser Ala Ile Arg Lys Gln Gln Lys
180 185
<210> 105
<211> 188
<212> PRT
<213> Kosakonia arachidis
<400> 105
Met Thr Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asn Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Glu Met Gly Tyr Ala
35 40 45
Leu Val Leu Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Gly Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Thr Val Glu Glu Phe Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Ile Ser Ala Arg Asp Phe Leu His Ile Asp Met
115 120 125
Ser Ala Ser Tyr Met Val Gly Asp Lys Ile Glu Asp Met Gln Ala Ala
130 135 140
Gln Ala Ala Gln Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Ile Thr Pro Glu Ala Glu Lys Ser Ala Asp Trp Val Ile Asn Ser Leu
165 170 175
Ala Glu Leu Pro Leu Ala Ile Lys Lys His Gln Lys
180 185
<210> 106
<211> 187
<212> PRT
<213> 栖冷克吕沃尔氏菌(Kluyvera cryocrescens)
<400> 106
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Asp Phe Glu Phe Ile
20 25 30
Asp Gly Val Ile Asp Ala Met Arg Glu Leu Lys Glu Met Gly Tyr Ala
35 40 45
Leu Val Leu Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Gly Val Asp Leu Asp Gly Ile Tyr Phe Cys Pro His His Pro
85 90 95
Gln Gly Thr Val Glu Glu Tyr Arg Gln Val Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Leu Lys Ser Ala Gln Glu Tyr Leu His Ile Asp Met
115 120 125
Ser Ser Ser Tyr Met Val Gly Asp Lys Ile Glu Asp Met Gln Ala Ala
130 135 140
Ala Ala Ala Asn Val Gly Thr Lys Val Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Val Thr Glu Asp Ala Glu Lys Ala Ala Asp Trp Val Ile Asn Ser Leu
165 170 175
Ala Asp Leu Pro Ala Met Ile Lys Lys Gln Lys
180 185
<210> 107
<211> 188
<212> PRT
<213> 非脱羧勒克氏菌(Leclercia adecarboxylata)
<400> 107
Met Ala Lys Ser Val Pro Ala Ile Phe Leu Asp Arg Asp Gly Thr Ile
1 5 10 15
Asn Val Asp His Gly Tyr Val His Glu Ile Asp Glu Phe Glu Phe Ile
20 25 30
Glu Gly Val Ile Glu Ala Met Arg Glu Leu Lys Glu Met Gly Phe Ala
35 40 45
Leu Val Val Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Lys Phe Thr
50 55 60
Glu Ala Gln Phe Glu Thr Leu Thr Glu Trp Met Asp Trp Ser Leu Ala
65 70 75 80
Asp Arg Gly Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Pro
85 90 95
Gln Gly Thr Val Glu Glu Tyr Arg Gln Thr Cys Asp Cys Arg Lys Pro
100 105 110
His Pro Gly Met Phe Ile Ser Ala Gln Glu Phe Leu His Ile Asp Met
115 120 125
Ser Ala Ser Tyr Met Val Gly Asp Lys Leu Glu Asp Met Gln Ala Ala
130 135 140
Ala Ala Ala Gly Val Gly His Lys Ile Leu Val Arg Thr Gly Lys Pro
145 150 155 160
Val Thr Pro Glu Ala Glu Asn Ala Ala Asp Cys Val Ile Asn Ser Leu
165 170 175
Ala Ala Leu Pro Glu Thr Ile Lys Lys Gln Gln Lys
180 185
<210> 108
<211> 219
<212> PRT
<213> 索氏志贺氏菌
<400> 108
Met Lys Leu Gln Gly Val Ile Phe Asp Leu Asp Gly Val Ile Thr Asp
1 5 10 15
Thr Ala His Leu His Phe Gln Ala Trp Gln Gln Ile Ala Ala Glu Ile
20 25 30
Gly Ile Ser Ile Asp Ala Gln Phe Asn Glu Ser Leu Lys Gly Ile Ser
35 40 45
Arg Asp Glu Ser Leu Arg Arg Ile Leu Gln His Gly Gly Lys Glu Gly
50 55 60
Asp Phe Asn Ser Gln Glu Arg Ala Gln Leu Ala Tyr Arg Lys Asn Leu
65 70 75 80
Leu Tyr Val His Ser Leu Arg Glu Leu Thr Val Asn Ala Val Leu Pro
85 90 95
Gly Ile Arg Ser Leu Leu Ala Asp Leu Arg Ala Gln Gln Ile Ser Val
100 105 110
Gly Leu Ala Ser Val Ser Leu Asn Ala Pro Thr Ile Leu Ala Ala Leu
115 120 125
Glu Leu Arg Lys Phe Phe Thr Phe Cys Ala Asp Ala Ser Gln Leu Lys
130 135 140
Asn Ser Lys Pro Asp Pro Glu Ile Phe Leu Ala Ala Cys Ala Gly Leu
145 150 155 160
Gly Val Pro Pro Gln Ala Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly
165 170 175
Ile Asp Ala Ile Asn Ala Ser Gly Met Arg Ser Val Gly Ile Gly Ala
180 185 190
Gly Leu Thr Gly Ala Gln Leu Leu Leu Pro Ser Thr Glu Ser Leu Thr
195 200 205
Trp Pro Arg Leu Ser Ala Phe Trp Gln Asn Val
210 215
<210> 109
<211> 219
<212> PRT
<213> 大肠杆菌
<400> 109
Met Lys Leu Gln Gly Val Ile Phe Asp Leu Asp Gly Val Ile Thr Asp
1 5 10 15
Thr Ala His Leu His Phe Lys Ala Trp Gln Gln Ile Ala Ala Glu Ile
20 25 30
Gly Ile Ser Ile Asp Ala Gln Phe Asn Glu Ser Leu Lys Gly Ile Ser
35 40 45
Arg Asp Glu Ser Leu Arg Arg Ile Leu Gln His Gly Gly Lys Glu Gly
50 55 60
Asp Phe Asn Pro Gln Glu Arg Ala Gln Leu Ala Tyr Arg Lys Asn Leu
65 70 75 80
Leu Tyr Val His Ser Leu Arg Glu Leu Thr Val Asn Ala Val Leu Pro
85 90 95
Gly Ile Arg Asn Leu Leu Ala Asp Leu Arg Ala Gln Gln Ile Pro Val
100 105 110
Gly Leu Ala Ser Val Ser Leu Asn Ala Pro Thr Ile Leu Ala Ala Leu
115 120 125
Glu Leu Arg Glu Phe Phe Thr Phe Cys Ala Asp Ala Ser Gln Leu Lys
130 135 140
Asn Ser Lys Pro Asp Pro Glu Ile Phe Leu Ala Ala Cys Ala Gly Leu
145 150 155 160
Gly Val Pro Pro Gln Ala Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly
165 170 175
Ile Asp Ala Ile Asn Ala Ser Gly Met Arg Ser Val Gly Ile Gly Ala
180 185 190
Gly Leu Thr Gly Ala Gln Leu Leu Leu Pro Ser Thr Asp Ser Leu Thr
195 200 205
Trp Pro Arg Leu Ser Ala Phe Trp Gln Asn Val
210 215
<210> 110
<211> 219
<212> PRT
<213> 大肠杆菌
<400> 110
Met Lys Leu Gln Gly Val Ile Phe Asp Leu Asp Gly Val Ile Thr Asp
1 5 10 15
Thr Ala His Leu His Phe Gln Ala Trp Gln Gln Ile Ala Ala Glu Ile
20 25 30
Gly Ile Gly Ile Asp Val Gln Phe Asn Glu Thr Leu Lys Gly Ile Ser
35 40 45
Arg Asp Glu Ser Leu Arg Arg Ile Leu Gln His Gly Gly Lys Glu Gly
50 55 60
Asp Phe Asn Val Gln Glu Arg Ala Gln Leu Ala Tyr Arg Lys Asn Leu
65 70 75 80
Leu Tyr Val His Ser Leu Arg Glu Leu Thr Val Asn Ala Val Leu Pro
85 90 95
Gly Ile Arg Pro Leu Leu Ala Asp Leu Arg Ala Gln Gly Ile Pro Val
100 105 110
Gly Leu Ala Ser Val Ser Leu Asn Ala Pro Thr Ile Leu Ala Ala Leu
115 120 125
Glu Leu Arg Glu Phe Phe Thr Phe Cys Ala Asp Ala Ser Gln Leu Lys
130 135 140
His Ser Lys Pro Asp Pro Glu Ile Phe Leu Ala Ala Cys Ala Gly Leu
145 150 155 160
Gly Val Pro Pro Gln Ala Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly
165 170 175
Ile Asp Ala Ile Asn Ala Ser Gly Met Arg Ser Val Gly Ile Gly Thr
180 185 190
Ser Leu Thr Gly Ala Gln Leu Leu Leu Pro Ser Thr Glu Ser Leu Thr
195 200 205
Trp Pro Arg Leu Ser Ala Phe Trp Gln Asn Val
210 215
<210> 111
<211> 208
<212> PRT
<213> 弗氏志贺氏菌
<400> 111
Met Lys Leu Gln Gly Val Ile Phe Asp Leu Asp Gly Val Ile Thr Asp
1 5 10 15
Thr Ala His Leu His Phe Gln Ala Trp Gln Gln Ile Ala Ala Glu Ile
20 25 30
Gly Ile Ser Ile Asp Ala Gln Phe Asn Glu Ser Leu Lys Gly Ile Ser
35 40 45
Arg Asp Glu Ser Leu Arg Arg Ile Leu Gln His Gly Gly Lys Glu Gly
50 55 60
Asp Phe Asn Pro Gln Glu Arg Ala Gln Leu Ala Tyr Arg Lys Asn Leu
65 70 75 80
Leu Tyr Val His Ser Leu Arg Glu Leu Thr Val Asn Ala Val Leu Pro
85 90 95
Gly Ile Arg Asn Leu Leu Ala Glu Leu Arg Ala Gln Gln Ile Pro Val
100 105 110
Gly Leu Ala Ser Val Ser Leu Asn Ala Pro Thr Ile Leu Ala Ala Leu
115 120 125
Glu Leu Arg Glu Phe Phe Thr Phe Cys Ala Asp Ala Ser Gln Leu Lys
130 135 140
Asn Ser Lys Pro Asp Pro Glu Ile Phe Leu Ala Ala Cys Ala Gly Leu
145 150 155 160
Gly Val Pro Pro Gln Ala Cys Ile Gly Ile Glu Asp Ala Gln Ala Gly
165 170 175
Ile Asp Ala Ile Asn Ala Ser Gly Met Arg Ser Val Gly Ile Gly Ala
180 185 190
Gly Leu Thr Gly Ala Gln Leu Leu Leu Pro Ser Thr Asp Ser Leu Thr
195 200 205
<210> 112
<211> 216
<212> PRT
<213> 索氏志贺氏菌
<400> 112
Met Arg Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Ser Asn Trp Ser Arg Arg His
20 25 30
Gly Leu Ala Pro Glu Glu Val Leu Ala Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Ala Gly Lys Ser Glu Ala Asp Ile
50 55 60
Ala Ala Glu Phe Thr Arg Leu Glu His Ile Glu Ala Thr Glu Thr Glu
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Ile Ala Leu Leu Ser His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Lys Ile Ala Gly Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Ala Pro Gln Glu Cys Val Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Gly Leu Asn Glu
180 185 190
Val Asp Leu Val Leu His Ser Leu Glu Gln Ile Thr Val Thr Lys Gln
195 200 205
Pro Asn Gly Asp Val Ile Ile Gln
210 215
<210> 113
<211> 215
<212> PRT
<213> 魏氏柠檬酸杆菌(Citrobacter werkmanii) NBRC 105721
<400> 113
Met Gln Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Cys Asn Trp Ala Asp Arg Phe
20 25 30
Gly Leu Asp His Ala Glu Val Leu Gly Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Val Gly Lys Ser Glu Ala Glu Ile
50 55 60
Ala Ala Glu Phe Thr Arg Leu Glu Gln Ile Glu Ala Thr Glu Thr Ala
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Val Asp Leu Leu Asn His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Arg Val Ala Gly Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Ala Pro Gln Glu Cys Ala Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Arg Leu Asp Glu
180 185 190
Val Asp Phe Ser Leu Thr Ser Leu Glu Gln Ile Ser Val Thr Lys Gln
195 200 205
Pro Asn Gly Asn Val Val Val
210 215
<210> 114
<211> 215
<212> PRT
<213> 弗氏柠檬酸杆菌(Citrobacter freundii)
<400> 114
Met Gln Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Cys Asn Trp Ala Asp Arg Phe
20 25 30
Gly Leu Asp His Ala Glu Val Leu Gly Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Val Gly Lys Ser Glu Ala Glu Ile
50 55 60
Ala Ala Glu Phe Thr Arg Leu Glu Gln Ile Glu Ala Thr Glu Thr Ala
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Val Asp Leu Leu Asn His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Arg Val Ala Gly Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Val Ala Pro Gln Glu Cys Ala Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Arg Leu Asp Glu
180 185 190
Val Asp Phe Ser Leu Thr Ser Leu Glu His Ile Ser Val Thr Lys Gln
195 200 205
Pro Asn Gly Asn Val Val Val
210 215
<210> 115
<211> 215
<212> PRT
<213> 无丙二酸柠檬酸杆菌
<400> 115
Met Gln Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Cys Asn Trp Ala Asp Arg Phe
20 25 30
Gly Leu Ala His Asp Asp Val Leu Ser Phe Val His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Ala Gly Lys Pro Glu Ala Glu Ile
50 55 60
Val Ala Glu Phe Thr Arg Leu Glu Lys Ile Glu Ala Thr Glu Thr Ala
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Val Ala Leu Leu Asn His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Gln Val Ala Asn Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Ser Pro His Glu Cys Val Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Arg Leu Asp Glu
180 185 190
Val Asp Phe Val Leu Thr Ser Leu Glu Gln Leu Ser Val Thr Lys Gln
195 200 205
Pro Asn Gly Asp Val Val Val
210 215
<210> 116
<211> 215
<212> PRT
<213> 肠沙门氏菌
<400> 116
Val Gln Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Cys Ser Trp Ala Asp Arg Phe
20 25 30
Asn Leu Ala His Asp Glu Val Leu Gly Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Ala Gly Lys Ser Glu Ala Glu Ile
50 55 60
Ala Ala Glu Phe Thr Arg Leu Glu Gln Ile Glu Ala Thr Glu Thr Ala
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Val Asp Leu Leu Asn His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Gln Val Ala Gly Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Val Pro Gln Glu Cys Val Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Arg Leu Ala Asp
180 185 190
Val Asp Phe Ala Leu Asn Ser Leu Thr Gln Leu Ser Val Ala Lys Gln
195 200 205
Pro Asn Gly Asp Val Val Val
210 215
<210> 117
<211> 216
<212> PRT
<213> 弗格森埃希氏菌
<400> 117
Met Arg Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Cys Asn Trp Ala Asp Arg Phe
20 25 30
Gly Ile Asp His His Glu Leu Leu Ser Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Pro Gly Arg Pro Glu Glu Glu Ile
50 55 60
Leu Ala Glu Phe Thr Arg Leu Glu Gln Ile Glu Ala Thr Gln Thr Glu
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Ile Glu Leu Leu Thr His Leu Asn
85 90 95
Lys Ser Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Gln Val Ala Gly Leu Pro Phe Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Glu Pro Lys Glu Cys Val Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Thr Pro Ala Asp Thr Pro Arg Leu Ser Glu
180 185 190
Val Asp Phe Val Leu Thr Ser Leu Gln Gln Ile Thr Val Thr Lys Gln
195 200 205
Pro Asn Gly Glu Val Ile Ile Gln
210 215
<210> 118
<211> 215
<212> PRT
<213> 肠沙门氏菌肠道亚种Hadar血清型
<400> 118
Val Gln Cys Lys Gly Phe Leu Phe Asp Leu Asp Gly Thr Leu Val Asp
1 5 10 15
Ser Leu Pro Ala Val Glu Arg Ala Trp Cys Ser Trp Ala Asp Arg Phe
20 25 30
Asn Leu Ala His Asp Glu Val Leu Gly Phe Ile His Gly Lys Gln Ala
35 40 45
Ile Thr Ser Leu Arg His Phe Met Ala Gly Lys Ser Glu Ala Glu Ile
50 55 60
Ala Ala Glu Phe Thr Arg Leu Glu Gln Ile Glu Ala Thr Glu Thr Ala
65 70 75 80
Gly Ile Thr Ala Leu Pro Gly Ala Val Asp Leu Leu Asn His Leu Asn
85 90 95
Lys Ala Gly Ile Pro Trp Ala Ile Val Thr Ser Gly Ser Met Pro Val
100 105 110
Ala Arg Ala Arg His Gln Val Ala Gly Leu Pro Ala Pro Glu Val Phe
115 120 125
Val Thr Ala Glu Arg Val Lys Arg Gly Lys Pro Glu Pro Asp Ala Tyr
130 135 140
Leu Leu Gly Ala Gln Leu Leu Gly Leu Ser Pro Gln Glu Cys Val Val
145 150 155 160
Val Glu Asp Ala Pro Ala Gly Val Leu Ser Gly Leu Ala Ala Gly Cys
165 170 175
His Val Ile Ala Val Asn Ala Pro Ala Asp Thr Pro Arg Leu Ala Asp
180 185 190
Val Asp Phe Ala Leu Asp Ser Leu Thr Gln Leu Ser Val Ala Lys Gln
195 200 205
Pro Asn Gly Asp Val Val Val
210 215
<210> 119
<211> 270
<212> PRT
<213> 大肠杆菌
<400> 119
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Asn Ala Ile Ala Ala Ala
20 25 30
Arg Ala Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Asn Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ala Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Phe Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Thr Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Phe Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Asn Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Ala Ile Leu Asp Gln Ala Ile Ala Arg Ile Pro Gln Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Val Leu
195 200 205
Gly Ile Lys Pro Glu Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Ile Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Val Ala Asn Phe Val Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Phe Ala Ile Glu Lys Tyr Val Leu Asn
260 265 270
<210> 120
<211> 270
<212> PRT
<213> 弗格森埃希氏菌
<400> 120
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Asn Ala Ile Ala Ala Ala
20 25 30
Arg Ala Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Asn Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ala Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Glu Asp Tyr Arg Phe Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Thr Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Phe Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Asn Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Thr Ile Leu Asp Gln Ala Ile Ala Arg Ile Pro Gln Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Val Leu
195 200 205
Gly Ile Lys Pro Glu Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Val Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Val Ala Asn Phe Val Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Phe Ala Ile Glu Lys Tyr Val Leu Asn
260 265 270
<210> 121
<211> 270
<212> PRT
<213> 索氏志贺氏菌
<400> 121
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Asn Ala Ile Ala Ala Ala
20 25 30
Arg Ala Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Asn Tyr Leu Lys Glu Leu His Met Glu Leu Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ala Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Phe Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Thr Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Phe Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Asn Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Ala Ile Leu Asp Gln Ala Ile Ala Arg Ile Pro Gln Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Ser Thr Gly Val Lys Ser Leu Ala Asp Val Leu
195 200 205
Gly Ile Lys Pro Glu Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Val Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Val Ala Asn Phe Val Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Phe Ala Ile Glu Lys Tyr Val Leu Asn
260 265 270
<210> 122
<211> 269
<212> PRT
<213> 关岛特拉布斯氏菌(Trabulsiella guamensis)
<400> 122
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Asn Ala Ile Ala Ala Ala
20 25 30
Arg Met Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His His Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ser Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Phe Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Asn Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Tyr Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Ala Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Val Val Leu Asp Lys Ala Ile Ala Arg Ile Pro Ala Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Val Leu
195 200 205
Gly Ile Gln Pro Asp Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Val Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Val Ala Asn Phe Ile Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Tyr Ala Ile Glu Lys Tyr Val Leu
260 265
<210> 123
<211> 270
<212> PRT
<213> 无丙二酸柠檬酸杆菌
<400> 123
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Arg Ala Ile Ala Ala Ala
20 25 30
Arg Glu Lys Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Ser Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Gly Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Tyr Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Thr Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Tyr Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Lys Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Ala Ile Leu Asp Gln Ala Ile Ala Arg Ile Pro Ala Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Ala Leu
195 200 205
Gly Ile Lys Pro Glu Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Leu Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Ile Ala Asn Phe Val Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Tyr Ala Ile Glu Gln His Val Leu Lys
260 265 270
<210> 124
<211> 269
<212> PRT
<213> 肺炎克雷伯氏菌(Klebsiella pneumoniae)
<400> 124
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Thr Ala Ile Ala Ala Ala
20 25 30
Arg Glu Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Ser Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Gly Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Phe Leu
85 90 95
Glu Gln Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Asn Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Tyr Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Glu Ile Gln Leu Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Ala Ile Leu Asp Gln Ala Ile Ala Arg Ile Pro Ala Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Ala Leu
195 200 205
Gly Ile Lys Pro Glu Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Phe Ala Gly Val Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ala Val Lys Glu Ala Ala Asn Phe Ile Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Phe Ala Ile Glu Lys Tyr Val Leu
260 265
<210> 125
<211> 269
<212> PRT
<213> 土白蚁特拉布尔希氏菌(Trabulsiella odontotermitis)
<400> 125
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Asn Ala Ile Ala Ala Ala
20 25 30
Arg Met Arg Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His His Tyr Leu Lys Glu Leu His Met Glu Gln Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ser Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Ala Leu Ser Tyr Asp Asp Tyr Arg Tyr Leu
85 90 95
Glu Lys Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Asn Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Tyr Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Ala Thr Gln Phe Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Val Val Leu Asp Lys Ala Ile Ala Arg Ile Pro Ala Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Glu Ala Leu
195 200 205
Gly Ile Gln Pro Asp Glu Ile Met Ala Ile Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Val Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Pro Ser Val Lys Glu Val Ala Asn Phe Ile Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Tyr Ala Ile Glu Lys Tyr Val Leu
260 265
<210> 126
<211> 270
<212> PRT
<213> 神户肠杆菌(Enterobacter kobei)
<400> 126
Met Ala Ile Lys Leu Ile Ala Ile Asp Met Asp Gly Thr Leu Leu Leu
1 5 10 15
Pro Asp His Thr Ile Ser Pro Ala Val Lys Lys Ala Ile Ala Ala Ala
20 25 30
Arg Glu Lys Gly Val Asn Val Val Leu Thr Thr Gly Arg Pro Tyr Ala
35 40 45
Gly Val His Asn Tyr Leu Arg Glu Leu His Met Asp Lys Pro Gly Asp
50 55 60
Tyr Cys Ile Thr Tyr Asn Gly Ala Leu Val Gln Lys Ala Ser Asp Gly
65 70 75 80
Ser Thr Val Ala Gln Thr Thr Leu Ser Tyr Asp Asp Tyr Arg Tyr Leu
85 90 95
Glu Gln Leu Ser Arg Glu Val Gly Ser His Phe His Ala Leu Asp Arg
100 105 110
Asn Thr Leu Tyr Thr Ala Asn Arg Asp Ile Ser Tyr Tyr Thr Val His
115 120 125
Glu Ser Tyr Val Ala Thr Ile Pro Leu Val Phe Cys Glu Ala Glu Lys
130 135 140
Met Asp Pro Ala Ile Gln Leu Leu Lys Val Met Met Ile Asp Glu Pro
145 150 155 160
Ala Ile Leu Asp Lys Ala Ile Ala Arg Ile Pro Ala Glu Val Lys Glu
165 170 175
Lys Tyr Thr Val Leu Lys Ser Ala Pro Tyr Phe Leu Glu Ile Leu Asp
180 185 190
Lys Arg Val Asn Lys Gly Thr Gly Val Lys Ser Leu Ala Asp Thr Leu
195 200 205
Gly Ile Thr Pro Asp Glu Ile Met Thr Leu Gly Asp Gln Glu Asn Asp
210 215 220
Ile Ala Met Ile Glu Tyr Ala Gly Leu Gly Val Ala Met Asp Asn Ala
225 230 235 240
Ile Asp Ser Val Lys Glu Val Ala Asp Phe Val Thr Lys Ser Asn Leu
245 250 255
Glu Asp Gly Val Ala Tyr Ala Ile Glu Lys Phe Val Leu Asn
260 265 270
<210> 127
<211> 238
<212> PRT
<213> 索氏志贺氏菌
<400> 127
Met Arg Phe Tyr Arg Pro Leu Gly Arg Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Pro Val Ile Leu Arg Thr Glu
20 25 30
Arg Glu Ala Leu Thr Phe Val Gln Asn Tyr His Pro Ala Leu Arg Ser
35 40 45
Phe Gln Asn Glu Asp Leu Gln Arg Leu Arg Gln Ala Val Arg Glu Ala
50 55 60
Glu Pro Glu Ile Tyr His Asp Val Thr Arg Trp Arg Phe Arg Ser Ile
65 70 75 80
Glu Gln Ala Met Leu Asp Ala Gly Leu Ser Ala Glu Glu Ala Ser Ala
85 90 95
Gly Ala His Ala Ala Met Ile Asn Phe Ala Lys Trp Arg Ser Arg Ile
100 105 110
Asp Val Pro Gln Gln Thr His Asp Thr Leu Lys Gln Leu Ala Lys Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Glu Phe Val Leu Arg Ala Gly Pro His Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr Phe Leu Ala Ala Glu Lys Leu
165 170 175
Asn Val Pro Ile Gly Glu Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Gly Gly Ala Ile Arg Ser Gly Met Gln Ala Cys Trp Ile Lys
195 200 205
Pro Glu Asn Gly Asp Leu Met Gln Thr Trp Asp Ser Arg Leu Leu Pro
210 215 220
His Leu Glu Ile Ser Arg Leu Ala Ser Leu Thr Ser Leu Ile
225 230 235
<210> 128
<211> 223
<212> PRT
<213> 索氏志贺氏菌
<400> 128
Met Arg Phe Tyr Arg Pro Leu Gly Arg Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Pro Val Ile Leu Arg Thr Glu
20 25 30
Arg Glu Ala Leu Thr Phe Val Gln Asn Tyr His Pro Ala Leu Arg Ser
35 40 45
Phe Gln Asn Glu Asp Leu Gln Arg Leu Arg Gln Ala Val Arg Glu Ala
50 55 60
Glu Pro Glu Ile Tyr His Asp Val Thr Arg Trp Arg Phe Arg Ser Ile
65 70 75 80
Glu Gln Ala Met Leu Asp Ala Gly Leu Ser Ala Glu Glu Ala Ser Ala
85 90 95
Gly Ala His Ala Ala Met Ile Asn Phe Ala Lys Trp Arg Ser Arg Ile
100 105 110
Asp Val Pro Gln Gln Thr His Asp Thr Leu Lys Gln Leu Ala Lys Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Glu Phe Val Leu Arg Ala Gly Pro His Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr Phe Leu Ala Ala Glu Lys Leu
165 170 175
Asn Val Pro Ile Gly Glu Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Gly Gly Ala Ile Arg Ser Gly Met Gln Ala Cys Trp Ile Arg
195 200 205
Pro Glu Asn Gly Asp Leu Met Gln Thr Trp Asp Ser Arg Leu Leu
210 215 220
<210> 129
<211> 238
<212> PRT
<213> 肠沙门氏菌
<400> 129
Met Arg Phe Tyr Arg Pro Leu Gly Arg Ile Ala Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Pro Val Ile Leu Arg Thr Glu
20 25 30
Gln Glu Ala Leu Ala Phe Met Gln Asn Tyr His Pro Ser Leu Arg Ser
35 40 45
Phe Gln Asn Val Asp Leu Gln Arg Ile Arg Gln Ala Val Arg Glu Ala
50 55 60
Glu Pro Glu Ile Tyr His Asp Val Thr Arg Trp Arg His Arg Ala Ile
65 70 75 80
Glu Gln Ala Met Arg Asp Ala Gly Leu Ser Ala Gln Glu Ala Ile Ala
85 90 95
Gly Ala Asn Ala Ala Met Met His Phe Ala Lys Trp Arg Ser Gln Ile
100 105 110
Glu Val Pro Gln Ala Thr His Glu Thr Leu Gln Gln Leu Ala Lys Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Lys Phe Val Leu Arg Ala Gly Pro Asp Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr Phe Leu Val Ala Glu Lys Leu
165 170 175
His Val Pro Ile Gly Glu Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Ala Gly Ala Ile Arg Cys Gly Met Gln Ala Cys Trp Ile Lys
195 200 205
Pro Glu Asn Ala Asp Leu Met Arg Thr Gln Asp Ser Arg Leu Leu Pro
210 215 220
His Ile Glu Ile Ser Arg Leu Ala Ser Leu Thr Ser Leu Ile
225 230 235
<210> 130
<211> 238
<212> PRT
<213> 布氏柠檬酸杆菌(Citrobacter braakii)
<400> 130
Met Arg Phe Tyr Arg Pro Leu Gly Arg Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Pro Val Ile Thr Arg Thr Glu
20 25 30
Gln Glu Ala Leu Ala Phe Val Gln Asn Tyr His Pro Ala Leu Asn Ser
35 40 45
Leu Gln Asn Ser Asp Leu Gln Arg Leu Arg Gln Ala Val Arg Asp Ala
50 55 60
Glu Pro Glu Ile Tyr His Asp Val Thr Gln Trp Arg His Arg Ala Val
65 70 75 80
Glu Arg Ala Met Leu Glu Ala Gly Leu Ser Glu Ala Glu Ala Lys Met
85 90 95
Gly Ala Asn Ala Ala Met Met Asn Phe Ala Lys Trp Arg Ser Arg Ile
100 105 110
Asp Val Pro Gln Ser Thr His Asp Thr Leu Lys Thr Leu Ala Arg Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Glu Phe Val Leu Arg Ala Gly Pro Asp Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr Ala Leu Ala Ala Glu Lys Leu
165 170 175
Lys Met Pro Val Gly Glu Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Ala Gly Ala Ile Arg Cys Gly Met Gln Ala Cys Trp Ile Lys
195 200 205
Pro Glu Asn Ala Asp Leu Met Gln Thr Ala Asp Ser Arg Leu Leu Pro
210 215 220
His Ile Glu Ile Ser Gln Leu Ala Ser Leu Thr Ser Leu Ile
225 230 235
<210> 131
<211> 238
<212> PRT
<213> 霍氏肠杆菌(Enterobacter hormaechei)
<400> 131
Met Arg Phe Tyr Arg Pro Leu Gly Gln Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Pro Val Ile Leu Arg Thr Glu
20 25 30
Gln Glu Ser Leu Ala Phe Val Gln Asn Tyr His Pro Ala Leu Lys Thr
35 40 45
Met Gln Asn Lys Asp Phe Gln Lys Leu Arg Gln Ser Leu Arg Glu Thr
50 55 60
Glu Pro Asp Ile Tyr His Asp Val Thr Glu Trp Arg Arg Arg Ala Val
65 70 75 80
Glu Gln Ala Met Leu Asn Val Gly Leu Ser Ser Gln Asp Ala Ala Ile
85 90 95
Gly Ala Glu Ala Ala Met Glu Asn Phe Ala Lys Trp Arg Ser Arg Val
100 105 110
Asp Val Pro Gln Glu Thr His Asp Thr Leu Ala Lys Leu Ala Glu Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Glu Phe Val Leu Arg Ala Gly Pro His Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr His Leu Ala Ala Glu Lys Leu
165 170 175
Asn Leu Pro Leu Gly Glu Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Ala Gly Ala Ile Arg Cys Gly Met Gln Ala Cys Trp Ile Lys
195 200 205
Pro Glu Asn Ala Ser Leu Met Thr Thr Pro Asp Ser Arg Leu Leu Pro
210 215 220
His Leu Glu Ile Ser Arg Leu Ala Ser Leu Thr Thr Leu Ile
225 230 235
<210> 132
<211> 238
<212> PRT
<213> Lelliottia amnigena
<400> 132
Met Arg Phe Tyr Arg Pro Leu Gly Gln Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Gln Val Ile Leu Arg Thr Glu
20 25 30
Gln Glu Ala Leu Ala Phe Val Gln Asn Tyr His Pro Ser Leu Lys Thr
35 40 45
Leu Gln Asn Thr Asp Phe Gln Arg Leu Arg Gln Ala Leu Arg Glu Thr
50 55 60
Glu Pro Asp Ile Tyr His Asp Val Thr Glu Trp Arg Arg Arg Ala Val
65 70 75 80
Glu Gln Ala Met Ile Asn Ala Gly Leu Thr Ala Ala Glu Ala Ala Leu
85 90 95
Gly Ala Glu Ala Ser Met Ala Asn Phe Ala Lys Trp Arg Ser Arg Ile
100 105 110
Asp Val Pro Gln Glu Thr His Asp Thr Leu Ala Lys Leu Ala Glu Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Thr Phe Val Phe Arg Ala Gly Pro His Gly
145 150 155 160
Arg Ser Lys Pro Phe Ser Asp Met Tyr His Leu Ala Ala Glu Lys Leu
165 170 175
Asp Leu Pro Leu Gly Glu Ile Leu His Val Gly Gly Asp Leu Thr Thr
180 185 190
Asp Val Ala Gly Ala Ile Arg Cys Gly Met Gln Ala Cys Trp Ile Lys
195 200 205
Pro Glu Asn Ala Asp Leu Met His Thr Ile Asp Ser Arg Leu Leu Pro
210 215 220
His Val Glu Ile Ser Arg Leu Ala Ser Leu Thr Thr Leu Ile
225 230 235
<210> 133
<211> 238
<212> PRT
<213> 非脱羧勒克氏菌 （Leclercia adecarboxylata）
<400> 133
Met Arg Phe Tyr Arg Pro Leu Gly Gln Ile Ser Ala Leu Thr Phe Asp
1 5 10 15
Leu Asp Asp Thr Leu Tyr Asp Asn Arg Gln Val Ile Leu Arg Thr Glu
20 25 30
Gln Glu Ala Leu Thr Phe Val Gln Asn Tyr His Pro Ala Leu Lys Thr
35 40 45
Leu Glu Asn Lys Glu Phe His Arg Leu Arg Gln Ala Leu Arg Gln Thr
50 55 60
Glu Pro Glu Ile Tyr His Asp Val Thr Glu Trp Arg Arg Arg Ala Val
65 70 75 80
Glu Leu Ala Met Leu Asn Ala Gly Leu Thr Ala Ala Glu Ala Ala Leu
85 90 95
Gly Ala Glu Ala Ser Met Ala His Phe Ala Gln Trp Arg Ser Arg Ile
100 105 110
Asp Val Pro Gln Glu Thr His Asp Thr Leu Ala Ala Leu Ala Glu Lys
115 120 125
Trp Pro Leu Val Ala Ile Thr Asn Gly Asn Ala Gln Pro Glu Leu Phe
130 135 140
Gly Leu Gly Asp Tyr Phe Gln Phe Val Leu Arg Ala Gly Pro His Gly
145 150 155 160
Arg Ser Lys Pro Phe Asn Asp Met Tyr His Leu Ala Ala Glu Lys Leu
165 170 175
Ser Leu Pro Leu Gly Gln Ile Leu His Val Gly Asp Asp Leu Thr Thr
180 185 190
Asp Val Ala Gly Ala Ile Arg Cys Gly Met Gln Ala Cys Trp Ile Lys
195 200 205
Pro Glu Asn Ala Asp Leu Met Gln Thr Ala Asp Ser Arg Leu Leu Pro
210 215 220
His Ile Glu Ile Ser Arg Leu Ala Ser Leu Thr Thr Leu Ile
225 230 235
<210> 134
<211> 190
<212> PRT
<213> 弗氏志贺氏菌
<400> 134
Met Ser Thr Pro Arg Gln Ile Leu Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Asp Val
20 25 30
Met Ala Ser Leu Gly Val Asp Ile Ser Arg Arg Asn Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Asp Leu Trp Tyr Ala Arg
50 55 60
Gln Pro Trp Asn Gly Pro Ser Arg Gln Glu Val Val Glu Arg Val Ile
65 70 75 80
Ala Arg Ala Ile Ser Leu Val Glu Glu Thr Arg Pro Leu Leu Pro Gly
85 90 95
Val Arg Glu Ala Val Ala Leu Cys Lys Glu Gln Gly Leu Leu Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Thr Met
115 120 125
Phe Asp Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala Glu Lys Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Leu Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Val Asp Pro Leu Thr Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Ile Ala Ser Lys Ala Ala Arg Met Arg Ser Ile Val
180 185 190
<210> 135
<211> 222
<212> PRT
<213> 蔗糖小迫氏菌 (Kosakonia sacchari)
<400> 135
Met Thr Thr Pro Arg Gln Ile Leu Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Glu Val
20 25 30
Val Ala Ser Leu Gly Val Asp Ile Thr Arg Arg His Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Glu Leu Trp Tyr Ala Gln
50 55 60
Gln Pro Trp Asn Gly Pro Asp Arg Gln Glu Val Thr Glu Cys Ile Ile
65 70 75 80
Gln Arg Ala Ile Ser Leu Val Glu Glu Lys Arg Pro Leu Leu Pro Gly
85 90 95
Val Arg Glu Ala Ile Ala Leu Cys Lys Ala Asn Gly Leu Leu Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Ala Met
115 120 125
Phe Glu Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala Glu Lys Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Leu Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Val Asp Pro Leu Thr Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Ile Ala Ser Lys Ala Ala Arg Met Arg Ser Ile Val Val Pro
180 185 190
Asp Glu Glu His Arg Ala Asp Pro Arg Tyr Val Leu Ala Asn Val Lys
195 200 205
Leu Thr Ser Leu Glu Gln Leu Thr Leu Ala His Pro Ile Gly
210 215 220
<210> 136
<211> 222
<212> PRT
<213> Enterobacter mori
<400> 136
Met Ser Thr Pro Arg Gln Ile Leu Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Asp Val
20 25 30
Met Ala Ser Leu Gly Val Asp Ile Ser Arg Arg Asn Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Asp Leu Trp Tyr Ala His
50 55 60
Gln Pro Trp Val Gly Pro Gly Arg Asp Glu Val Ile Ala Arg Ile Ile
65 70 75 80
Asn Arg Ala Ile Thr Leu Val Glu Glu Gln Lys Pro Leu Leu Pro Gly
85 90 95
Val Arg Asp Ala Ile Ala Leu Cys Lys Ala Gln Gly Leu Lys Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Ser Leu
115 120 125
Phe Glu Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala Glu Lys Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Met Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Leu Asp Pro Leu Thr Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Val Ala Ser Lys Ala Ala Arg Met Arg Ser Ile Val Val Pro
180 185 190
Ala Glu Glu Gly Arg His Asp Pro Arg Phe Ala Leu Ala Asp Val Lys
195 200 205
Leu Ala Ser Leu Glu Asp Leu Thr Val Ala His Leu Arg Gly
210 215 220
<210> 137
<211> 222
<212> PRT
<213> Lelliottia amnigena
<400> 137
Met Ser Thr Ser Arg Gln Ile Val Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Asp Val
20 25 30
Met Glu Ser Leu Gly Val Asp Ile Arg Arg Arg Asn Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Glu Leu Trp Tyr Ala His
50 55 60
Gln Pro Trp Asn Gly Pro Ser Arg Gln Glu Val Thr Asp Arg Ile Ile
65 70 75 80
Ser Arg Ala Leu Thr Leu Val Glu Ala Ser Arg Pro Leu Leu Pro Gly
85 90 95
Val Arg Glu Ala Val Ala Leu Cys Lys Ala Gln Gly Leu Lys Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Ala Met
115 120 125
Phe Asp Leu Arg Glu Ser Phe Asp Ala Leu Ala Ser Ala Glu Lys Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Met Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Val Asp Thr Leu Ala Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Ile Ala Ser Lys Ala Ala Arg Met Arg Ser Val Val Val Pro
180 185 190
Ala Glu Glu Gly Gln His Asp Pro Arg Phe Ala Leu Ala Asp Val Lys
195 200 205
Leu Ala Thr Leu Ala Asp Leu Thr Pro Ala His Leu Arg Gly
210 215 220
<210> 138
<211> 222
<212> PRT
<213> 肠杆菌属638
<400> 138
Met Ser Thr Pro His Gln Ile Val Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Asp Val
20 25 30
Met Ala Ser Leu Gly Val Asp Ile Ser Arg Arg Gly Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Glu Leu Trp Phe Ala His
50 55 60
Gln Pro Trp Ser Gly Pro Ser Arg Glu Glu Val Thr Ala Arg Val Ile
65 70 75 80
Ser Arg Ala Ile Ala Leu Val Glu Glu Lys Arg Pro Leu Leu Pro Gly
85 90 95
Ala Arg Glu Ala Ile Ala Leu Cys Lys Ala Gln Gly Leu Lys Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Glu Met
115 120 125
Phe Asp Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala Glu Lys Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Met Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Val Asp Pro Leu Ala Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Val Ala Ser Lys Ala Ala Arg Met Arg Ser Ile Val Val Pro
180 185 190
Ala Glu Glu Gly Gln His Asp Pro Arg Phe Ala Leu Ala Asn Ala Lys
195 200 205
Leu Thr Ser Leu Val Asp Leu Thr Pro Ala His Leu Phe Gly
210 215 220
<210> 139
<211> 222
<212> PRT
<213> Kosakonia radicincitans
<400> 139
Met Thr Ala Pro Arg Gln Ile Leu Ala Ala Ile Phe Asp Met Asp Gly
1 5 10 15
Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu Leu Glu Val
20 25 30
Val Ala Ser Leu Gly Val Asp Ile Asn Arg Arg His Glu Leu Pro Asp
35 40 45
Thr Leu Gly Leu Arg Ile Asp Met Val Val Glu Leu Trp Tyr Ala Gln
50 55 60
Gln Pro Trp Asn Gly Pro Asp Arg Lys Glu Val Thr Glu Arg Ile Ile
65 70 75 80
Gln Arg Ala Ile Ser Leu Val Glu Glu Lys Arg Pro Leu Leu Pro Gly
85 90 95
Val Arg Glu Ala Ile Ala Leu Cys Lys Ala Asn Gly Leu Leu Val Gly
100 105 110
Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val Leu Ala Met
115 120 125
Phe Glu Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala Glu Asn Leu
130 135 140
Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Leu Asp Cys Ala Ala Lys
145 150 155 160
Leu Gly Ile Asp Pro Leu Thr Cys Val Ala Leu Glu Asp Ser Val Asn
165 170 175
Gly Met Ile Ala Ser Lys Ala Ala Arg Met Arg Ser Ile Val Val Pro
180 185 190
Asp Glu Glu His Arg Thr Asp Pro Arg Tyr Val Leu Ala Asn Val Lys
195 200 205
Leu Thr Ser Leu Glu Gln Leu Thr Leu Ala His Leu Ile Gly
210 215 220
<210> 140
<211> 209
<212> PRT
<213> 肠沙门氏菌肠道亚种新港型
<400> 140
Met Asp Gly Leu Leu Ile Asp Ser Glu Pro Leu Trp Asp Arg Ala Glu
1 5 10 15
Leu Asp Val Met Ala Ser Leu Gly Val Asp Ile Thr Arg Arg His Glu
20 25 30
Leu Pro Asp Thr Leu Gly Leu Arg Ile Asp Met Val Val Asp Leu Trp
35 40 45
Phe Ala Gln Gln Pro Trp Asn Gly Pro Asp Arg Gln Glu Val Thr Asn
50 55 60
Arg Val Ile Ala Arg Ala Ile Thr Leu Ile Glu Glu Thr Arg Pro Leu
65 70 75 80
Leu Pro Gly Val Arg Glu Ala Val Ala Leu Cys Lys Ala Gln Gly Leu
85 90 95
Leu Val Gly Leu Ala Ser Ala Ser Pro Leu His Met Leu Glu Lys Val
100 105 110
Leu Thr Met Phe Glu Leu Arg Asp Ser Phe Asp Ala Leu Ala Ser Ala
115 120 125
Glu Lys Leu Pro Tyr Ser Lys Pro His Pro Gln Val Tyr Leu Asp Cys
130 135 140
Ala Ala Lys Leu Gly Val Asp Pro Leu Thr Cys Val Ala Leu Glu Asp
145 150 155 160
Ser Val Asn Gly Leu Ile Ala Ala Lys Ala Ala Arg Met Arg Ala Ile
165 170 175
Val Val Pro Ala Glu Glu Asn Gln His Asp Pro Arg Phe Ala Leu Ala
180 185 190
Asn Val Lys Leu Asn Ser Leu Thr Glu Leu Thr Ala Ala His Leu Leu
195 200 205
Gly
<210> 141
<211> 188
<212> PRT
<213> 弗氏志贺氏菌
<400> 141
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
20 25 30
His Tyr Gly Leu Gln Tyr Asp Val Gln Ala Met Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Arg
65 70 75 80
Ser Met Leu Leu Asp Ser Val Glu Pro Leu Pro Leu Val Glu Val Val
85 90 95
Lys Ser Trp His Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg Arg Tyr
115 120 125
Phe Asp Ala Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Gln Arg Met Gly Val Gln Pro Thr
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Ile Gln Ala Ala Arg
165 170 175
Ala Ala Gly Met Asp Ala Val Asp Ile Arg Leu Leu
180 185
<210> 142
<211> 188
<212> PRT
<213> 阿尔伯蒂埃希氏菌
<400> 142
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
20 25 30
Arg Tyr Gly Leu Arg Tyr Asn Val Gln Ala Met Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro Tyr Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Arg
65 70 75 80
Ser Met Leu Leu Asp Ser Val Glu Pro Leu Pro Leu Val Glu Val Val
85 90 95
Lys Ser Trp Tyr Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Thr His Leu Gly Leu Arg Arg Tyr
115 120 125
Phe Asp Thr Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Gln His Met Gly Val Gln Pro Ala
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Ile Gln Ala Ala Arg
165 170 175
Ala Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 143
<211> 188
<212> PRT
<213> 肠沙门氏菌
<400> 143
Met Tyr Ala Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Leu
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
20 25 30
His Tyr Gly Leu Arg Phe Asp Glu Gln Ala Met Val Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Leu Ile Ala Gln Ser Ile Ile Glu Leu Asn His Ala
50 55 60
Asp Leu Asp Pro Leu Ser Leu Ala Arg Glu Lys Thr Asp Ala Val Lys
65 70 75 80
Ser Ile Leu Leu Asp Cys Val Glu Pro Leu Pro Leu Val Glu Val Val
85 90 95
Lys Ala Trp His Gly Arg Arg Pro Met Ser Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg Arg Tyr
115 120 125
Phe Asp Ala Val Val Ala Ala Asp His Val Gln His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Gln Arg Met Gly Val Met Pro Thr
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Leu Gln Ala Ala Arg
165 170 175
Ala Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 144
<211> 188
<212> PRT
<213> 中间克吕沃尔氏菌
<400> 144
Met Tyr Glu Arg Tyr Asp Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp His Asp Val Leu Gly
20 25 30
His Tyr Gly Leu Arg Tyr Asp Ile Gln Ala Met Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ser Val Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro Tyr Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Lys
65 70 75 80
Ala Met Leu Leu Asp Thr Val Arg Pro Leu Pro Leu Ile Asp Val Val
85 90 95
Lys Ala Trp Tyr Gly Arg Arg Pro Leu Ser Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ser His Leu Gly Leu Arg His Tyr
115 120 125
Phe Ala Ala Val Val Ala Ala Glu His Val Lys Asn His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Glu Lys Met Gly Val Ala Pro Gln
145 150 155 160
Lys Cys Val Val Phe Glu Asp Ala Asp Phe Gly Leu Gln Ala Ala Arg
165 170 175
Ser Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 145
<211> 188
<212> PRT
<213> 土白蚁特拉布尔希氏菌
<400> 145
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp His Glu Val Leu Gly
20 25 30
Arg Tyr Gly Ile Arg Phe Asp Glu Gln Ser Ile Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Glu Ile Ile Thr Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Ala Leu Ala Arg Glu Lys Thr Asp Ala Val Lys
65 70 75 80
Ile Met Leu Leu Asp Ser Val Gln Pro Leu Pro Leu Ile Asp Val Val
85 90 95
Lys Ala Trp His Gly Arg Arg Pro Met Ser Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
115 120 125
Phe Ser Ala Val Val Ala Ala Asp His Val Arg His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Glu Arg Met Gly Val Glu Pro Ala
145 150 155 160
Lys Cys Ile Val Phe Glu Asp Ala Asp Phe Gly Leu Gln Ala Ala Ala
165 170 175
Ser Ala Gly Met Asp Val Val Asp Val Arg Leu Leu
180 185
<210> 146
<211> 188
<212> PRT
<213> 雷金斯堡约克氏菌(Yokenella regensburgei)
<400> 146
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Cys Lys Ala Trp His Asp Val Leu Gly
20 25 30
Arg Tyr Gly Met His Phe Asp Glu Gln Ala Met Thr Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ser Ile Ile Glu Leu Asn Gln Ala
50 55 60
Thr Leu Asp Pro His Gln Leu Ala Arg Glu Lys Thr Asp Ala Val Lys
65 70 75 80
Ala Met Leu Leu Asp Thr Val Arg Pro Leu Pro Leu Ile Asp Val Val
85 90 95
Lys Ala Trp His Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg Glu Tyr
115 120 125
Phe Thr Ala Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Glu Leu Met Gly Val Ala Ala Asn
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Leu Gln Ala Ala Arg
165 170 175
Ser Ala Gly Met Asp Val Val Asp Val Arg Leu Leu
180 185
<210> 147
<211> 188
<212> PRT
<213> 土生拉乌尔菌(Raoultella terrigena)
<400> 147
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp His Glu Val Leu Gly
20 25 30
Arg Tyr Gly Met Arg Phe Asp Glu Gln Ala Met Val Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Val Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Arg Leu Ala Leu Glu Lys Thr Asn Ala Val Lys
65 70 75 80
Ala Met Leu Leu Asp Ser Val Arg Pro Leu Pro Leu Ile Glu Val Val
85 90 95
Lys Glu Trp His Gly Arg Arg Pro Met Ser Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Val Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
115 120 125
Phe Ser Ala Val Ile Ala Ala Asp His Val Thr Asn His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Glu Arg Met Gly Val Ala Pro Glu
145 150 155 160
Lys Cys Val Val Phe Glu Asp Ala Asp Phe Gly Leu Gln Ala Ala Arg
165 170 175
Arg Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 148
<211> 188
<212> PRT
<213> 肺炎克雷伯氏菌
<400> 148
Met Tyr Glu Gly Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Gln Ala Trp Asn Glu Val Leu Gly
20 25 30
Arg Tyr Gly Met Arg Phe Asp Glu Gln Ala Met Val Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Arg Leu Ala Gln Glu Lys Thr Gln Ala Val Lys
65 70 75 80
Ala Met Leu Leu Asp Ser Val Arg Pro Leu Pro Leu Ile Glu Val Val
85 90 95
Lys Ala Trp His Gly Cys Arg Pro Met Ser Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Val Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
115 120 125
Phe Ser Ala Val Val Ala Ala Asp His Val Val Asn His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Glu Arg Met Gly Val Ala Pro Glu
145 150 155 160
Lys Cys Val Val Phe Glu Asp Ala Asp Phe Gly Leu Gln Ala Ala Lys
165 170 175
Arg Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185
<210> 149
<211> 223
<212> PRT
<213> 恶臭假单胞菌
<400> 149
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Cys Gln Ala Met Leu Ala Asp Arg
20 25 30
Gly Leu Pro Ala Ile Asp Asp Ala Arg Ile Arg Glu Val Ile Ser Gly
35 40 45
Gly Ala Arg Ala Met Val Ala Ala Thr Phe Ala Met Asn Pro Glu Ala
50 55 60
Asp Gly Phe Glu Ala Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Val His Ser Lys Leu Phe Asp Gly Met Pro Glu Leu Leu
85 90 95
Ala Asp Ile Glu Lys Gly Asn Leu Leu Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Glu Pro Ile Met Gln Arg Leu Gly Leu Ala Glu
115 120 125
Arg Ser Ala Leu Leu Ile Cys Pro Asp His Val Lys Asn Ser Lys Pro
130 135 140
Asp Pro Glu Pro Leu Ile Leu Ala Cys Lys Thr Leu Gly Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Arg Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Glu Asp Asn Pro Asn Asn Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Ile Asp Ser Ala Leu Cys Gly Cys
210 215 220
<210> 150
<211> 223
<212> PRT
<213> 假单胞菌属GM84
<400> 150
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Cys Gln Ala Met Leu Ala Glu Arg
20 25 30
Gly Leu Pro Ala Ile Asp Asp Lys Leu Ile Arg Asp Val Ile Ser Gly
35 40 45
Gly Ala Arg Ala Met Val Ala Ala Thr Phe Ala Met Asn Pro Asp Asp
50 55 60
Asp Gly Phe Glu Pro Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Val His Ser Lys Leu Phe Asp Gly Met Gly Glu Leu Leu
85 90 95
Ala Asp Ile Glu Lys Gly Asn Leu Leu Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Glu Pro Ile Met Gln Arg Leu Gly Leu Ala Glu
115 120 125
Arg Ser Ala Leu Leu Ile Cys Pro Asp His Val Lys Asn Ser Lys Pro
130 135 140
Asp Pro Glu Pro Leu Ile Leu Ala Cys Lys Thr Leu Asn Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Arg Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Glu Asp Asn Pro Asn Asn Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Ile Asp Ser Ala Leu Cys Gly Cys
210 215 220
<210> 151
<211> 223
<212> PRT
<213> 喜昆虫假单胞菌（Pseudomonas entomophila）
<400> 151
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Cys Gln Ala Met Leu Thr Asp Arg
20 25 30
Gly Leu Pro Thr Ile Asp Asp Ala Arg Ile Arg Asp Val Ile Ser Gly
35 40 45
Gly Ala Arg Ala Met Val Ala Ala Thr Phe Ala Met Asp Pro Asp Ala
50 55 60
Glu Gly Phe Glu Ala Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Val His Ser Lys Leu Phe Asp Gly Met Pro Glu Leu Leu
85 90 95
Ala Asp Ile Glu Lys Gly Asn Leu Leu Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Glu Pro Ile Met Gln Arg Leu Gly Leu Ala Glu
115 120 125
Arg Ser Ala Leu Leu Ile Cys Pro Asp His Val Lys Asn Ser Lys Pro
130 135 140
Asp Pro Glu Pro Leu Ile Leu Ala Cys Lys Thr Leu Asp Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Arg Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Glu Asp Asn Pro Asn Asn Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Ile Asp Ser Ala Leu Cys Gly Cys
210 215 220
<210> 152
<211> 223
<212> PRT
<213> 弗村假单胞菌(Pseudomonas vranovensis)
<400> 152
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Cys Gln Ala Met Leu Ala Asp Arg
20 25 30
Gly Leu Pro Ala Ile Asp Asp Gln Arg Ile Arg Asp Val Ile Ser Gly
35 40 45
Gly Ala Arg Ala Met Val Glu Val Thr Phe Gly Ile Thr Pro Glu Val
50 55 60
Ala Glu Phe Glu Ala Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Val His Ser Lys Leu Phe Asp Gly Met Ala Glu Leu Leu
85 90 95
Ala Asp Ile Glu Lys Gly Asn Leu Leu Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Glu Pro Ile Met Gln Arg Leu Gly Leu Ala Glu
115 120 125
Arg Ser Ala Leu Leu Ile Cys Pro Asp His Val Lys Asn Ser Lys Pro
130 135 140
Asp Pro Glu Pro Leu Ile Leu Ala Cys Lys Thr Leu Asn Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Arg Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Glu Asp Asn Pro Asn Asn Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Leu Asp Ser Ala Leu Cys Gly Cys
210 215 220
<210> 153
<211> 223
<212> PRT
<213> 大麻假单胞菌(Pseudomonas cannabina)
<400> 153
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Ala Gln Ala Met Leu Ala Asp Arg
20 25 30
Gly Leu Pro Ala Val Ala Asp Lys Leu Ile Arg Asp Glu Ile Ser Gly
35 40 45
Gly Ala Lys Ala Met Val Ala Ala Ala Phe Ala Leu Ser Pro Glu Ala
50 55 60
Pro Glu Phe Glu Ala Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Thr His Ser Arg Leu Phe Asn Gly Met Pro Glu Leu Leu
85 90 95
Ala Asp Ile Glu Lys Ala Gly Leu Ile Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Gln Pro Ile Met Glu Gln Leu Lys Leu Ala Glu
115 120 125
Arg Ser Ala Val Leu Ile Cys Pro Asp His Val Thr His Ser Lys Pro
130 135 140
His Pro Glu Pro Met Ile Leu Ala Cys Lys Leu Leu Asp Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Lys Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Asp Asp Asn Pro Asp His Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Leu Asp Asn Ala Leu Cys Ser Cys
210 215 220
<210> 154
<211> 223
<212> PRT
<213> 蒙氏假单胞菌(Pseudomonas monteilii)
<400> 154
Met Arg Leu Arg Ala Val Leu Phe Asp Met Asp Gly Thr Leu Leu Asp
1 5 10 15
Thr Ala Pro Asp Phe Ile Ala Ile Cys Gln Ala Met Leu Ala Glu Arg
20 25 30
Gly Leu Pro Ala Ile Asp Asp Gln Ala Ile Arg Asp Val Ile Ser Gly
35 40 45
Gly Ala Arg Ala Met Val Ala Ala Thr Phe Ala Met Asn Pro Glu Asp
50 55 60
Glu Gly Phe Glu Ala Leu Arg Leu Glu Phe Leu Glu Arg Tyr Gln Arg
65 70 75 80
Asp Cys Ala Val His Ser Lys Leu Phe Asp Gly Met Gly Glu Leu Leu
85 90 95
Ala Asp Leu Glu Lys Gly Lys Leu Leu Trp Gly Val Val Thr Asn Lys
100 105 110
Pro Val Arg Phe Ala Glu Pro Ile Met Gln Gln Leu Gly Leu Ala Glu
115 120 125
Arg Ser Ala Leu Leu Ile Cys Pro Asp His Val Lys Asn Ser Lys Pro
130 135 140
Asp Pro Glu Pro Leu Ile Leu Ala Cys Lys Thr Leu Asn Leu Asp Pro
145 150 155 160
Ala Ser Val Leu Phe Val Gly Asp Asp Leu Arg Asp Ile Glu Ser Gly
165 170 175
Arg Asp Ala Gly Thr Arg Thr Ala Ala Val Arg Tyr Gly Tyr Ile His
180 185 190
Pro Asp Asp Asn Pro Asn His Trp Gly Ala Asp Val Val Val Asp His
195 200 205
Pro Leu Glu Leu Arg Lys Val Ile Asp Ser Ala Leu Cys Gly Cys
210 215 220

Claims (23)

1.在微生物中产生唾液酸化化合物的方法，所述方法包括：
-在培养基中培养微生物，所述培养基任选地包含外源性前体，
其中所述微生物包含至少一种编码磷酸酶的核酸、至少一种编码N-乙酰甘露糖胺差向异构酶的核酸和至少一种编码唾液酸合酶的核酸，且
其中所述微生物不能i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺；和
-调节所述微生物中编码HAD类磷酸酶多肽的核酸的表达，其中所述HAD类磷酸酶多肽包含：
-至少下列基序之一：
基序1：hDxDx[TV](SEQ ID NO:73)，或
基序2：[GSTDE][DSEN]x(1-2)[hP]x(1-2)[DGTS](SEQ ID NO:74、75、76、77)，
其中h表示疏水性氨基酸(A、I、L、M、F、V、P、G)，且x可以是任何不同的氨基酸；
-或SEQ ID NO:43、44、45、47、48、50、51、52、54、55或57中任一个的与所述多肽具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的总体序列同一性的同源物或衍生物。
2.根据权利要求1所述的方法，其中所述HAD类多肽包含SEQ ID NO:43、44、45、47、48、50、51、52、54、55或57中的任何一个。
3.根据权利要求1所述的方法，其中通过在微生物中引入并表达编码HAD类多肽的核酸来实现受调节的表达。
4.根据权利要求1所述的方法，其中通过组成型启动子的作用实现受调节的表达。
5.根据前述权利要求中任一项所述的方法，其中所述唾液酸化化合物选自N-乙酰神经氨酸、唾液酸化寡糖、唾液酸化脂质、唾液酸化蛋白质、唾液酸化苷元。
6.根据前述权利要求所述的方法，其中所述唾液酸化化合物是唾液酸化寡糖。
7.根据权利要求6的方法，其中所述唾液酸化寡糖是唾液酸乳糖。
8.根据权利要求6所述的方法，其中所述唾液酸化寡糖是二唾液酸乳-N-四糖。
9.根据权利要求5所述的方法，其中所述唾液酸化化合物是N-乙酰神经氨酸。
10.根据权利要求1-9中任一项所述的方法，其中所述唾液酸化化合物是唾液酸化乳-N-三糖、乳-N-四糖或乳-N-新四糖，并且其中所述微生物还包含半乳糖基转移酶(EC2.4.1.38)活性。
11.根据权利要求10所述的方法，其中所述微生物不能表达编码UDP糖水解酶和半乳糖-1-磷酸尿苷酰基转移酶的基因。
12.根据权利要求1-11中任一项所述的方法，其中所述微生物产生的细胞外N-乙酰葡糖胺比唾液酸化化合物少50％、40％、30％、20％、10％、5％、2％。
13.产生唾液酸化寡糖的方法，所述方法包括：
a)根据权利要求1-12中任一项所述的方法培养微生物，并且其中所述微生物内部产生活化的N-乙酰神经氨酸作为唾液酸转移酶的供体底物；和
b)在包含外源性前体的培养基中培养所述微生物，所述外源性前体选自乳糖、N-乙酰乳糖胺、乳-N-二糖、半乳糖、β-半乳糖苷和α-半乳糖苷，例如但不限于红细胞三糖(Gal-α-1,4Gal-β-1,4Glc)半乳糖，其中发生所述微生物对所述外源性前体的主动摄取，并且其中所述外源性前体是用于产生所述唾液酸化寡糖的所述唾液酸转移酶的接纳体底物。
14.根据权利要求1所述的方法，其中在所述微生物中过表达所述N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任一种或两种。
15.根据权利要求1所述的方法，其中在所述微生物中引入并表达所述N-乙酰甘露糖胺差向异构酶和唾液酸合酶中的任一种或两种。
16.根据权利要求1-15中任一项所述的方法，其中所述微生物是细菌，优选大肠杆菌菌株，更优选大肠杆菌菌株是K12菌株，甚至更优选大肠杆菌K12菌株是大肠杆菌MG1655。
17.根据权利要求1-15中任一项所述的方法，其中所述微生物是酵母。
18.能够通过根据权利要求1-17中任一项所述的方法获得的微生物，其中所述微生物包含编码HAD类多肽的重组核酸。
19.用于产生唾液酸化化合物的微生物，其中所述微生物包含至少一种编码磷酸酶的核酸、至少一种编码N-乙酰甘露糖胺差向异构酶的核酸和至少一种编码唾液酸合酶的核酸，和
其中所述微生物不能i)将N-乙酰葡糖胺-6-P转化为葡糖胺-6-P，ii)将N-乙酰葡糖胺转化为N-乙酰葡糖胺-6-P，和iii)将N-乙酰神经氨酸转化为N-乙酰甘露糖胺；其特征在于
所述微生物包含编码权利要求1或2所定义的HAD类磷酸酶多肽的核酸的受调节的表达。
20.构建体，其包括：
(i)编码权利要求1或2所定义的HAD类多肽的核酸；
(ii)一种或多种能够驱动(a)的核酸序列表达的控制序列；和可选地
(iii)转录终止序列。
21.根据权利要求20所述的构建体，其中所述控制序列之一是组成型启动子。
22.权利要求20或21所述的构建体在产生唾液酸化化合物的方法中的用途。
23.权利要求1-17中任一项所述的方法产生的唾液酸化化合物，其中将所述唾液酸化化合物加入食品制剂、饲料制剂、药物制剂、化妆品制剂或农业化学制剂中。
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WO2018122225A1 (en) 2018-07-05
JP2020503066A (ja) 2020-01-30
EP3562951A1 (en) 2019-11-06
RU2019117393A3 (zh) 2021-05-20
CA3048521A1 (en) 2018-07-05
KR20230104755A (ko) 2023-07-10
BR112019013211A2 (pt) 2019-12-10
AU2017385601B2 (en) 2022-08-25
US20190338328A1 (en) 2019-11-07

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