CN113179645A - 醛脱氢酶变体及其使用方法 - Google Patents

醛脱氢酶变体及其使用方法 Download PDF

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CN113179645A
CN113179645A CN201980077426.8A CN201980077426A CN113179645A CN 113179645 A CN113179645 A CN 113179645A CN 201980077426 A CN201980077426 A CN 201980077426A CN 113179645 A CN113179645 A CN 113179645A
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阿米特·沙阿
约瑟夫·沃纳
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Abstract

本发明提供了醛脱氢酶变体的多肽和编码核酸。本发明还提供了表达醛脱氢酶变体的细胞。本发明还提供了用于生产3‑羟基丁醛(3‑HBal)和/或1,3‑丁二醇(1,3‑BDO)或其酯或酰胺的方法，其包括培养表达醛脱氢酶变体的细胞或者使用这些细胞的裂解液。本发明另外提供了用于生产4‑羟基丁醛(4‑HBal)和/或1,4‑丁二醇(1,4‑BDO)或其酯或酰胺的方法，其包括培养表达醛脱氢酶变体的细胞或者使用这些细胞的裂解液。

Description

醛脱氢酶变体及其使用方法
背景技术
本发明申请主张2018年9月26日提交的美国临时专利申请No.62/737,053和2018年10月3日提交的美国临时专利申请No.62/740,830的权益，以上专利的整个内容作为参考并入本文。
参考以下临时和国际专利申请，这些专利以其全部内容作为参考并入本文：(1)2017年3月31日提交的标题为“醛脱氢酶变体和使用方法(ALDEHYDE DEHYDROGENASEVARIANTS AND METHODS OF USE)”的美国临时专利申请No.62/480,194(代理人卷号No.12956-408-888)；(2)2017年3月31日提交的标题为“3-羟基丁酰基-COA脱氢酶变体和使用方法(3-HYDROXYBUTYRYL-COA DEHYDROGENASE VARIANTS AND METHODS OF USE)”的美国临时专利申请No.62/480,208(代理人卷号No.12956-409-888)；(3)2017年3月31日提交的标题为“从发酵液获得1,3-丁二醇的方法和系统(PROCESS AND SYSTEMS FOR OBTAINING1,3-BUTANEDIOL FROM FERMENTATION BROTHS)”的美国临时专利申请No.62/480,270(代理人卷号No.12956-407-888)；(4)2018年3月29日提交的标题为“醛脱氢酶变体和使用方法(ALDEHYDE DEHYDROGENASE VARIANTS AND METHODS OF USE)”的国际专利申请No.PCT/US2018/025122(代理人卷号No.12956-408-228)；(5)2018年3月29日提交的标题为“3-羟基丁酰基-CoA脱氢酶变体和使用方法(3-HYDROXYBUTYRYL-COA DEHYDROGENASE VARIANTSAND METHODS OF USE)”的国际专利申请No.PCT/US2018/025086(代理人卷号No.12956-409-228)；和(6)2018年3月29日提交的标题为“从发酵液获得1,3-丁二醇的方法和系统(PROCESS AND SYSTEMS FOR OBTAINING 1,3-BUTANEDIOL FROM FERMENTATION BROTHS)”的国际专利申请No.PCT/US2018/025068(代理人卷号No.12956-407-228)。
本发明申请在本文中作为参考引入了ASCII文本文件形式的序列表，其标题为“12956-462-228_SL.TXT”，创建于2019年9月17日，大小为498,106字节。
本发明一般地涉及工程化以产生所期望的产品的生物，有利于所期望的产品的生产的工程化的酶，并且更具体地涉及产生所期望的产品，如3-羟基丁醛、1,3-丁二醇、4-羟基丁醛、1,4-丁二醇和相关产品以及由此衍生的产品的酶和细胞。
多种商品化学品用于制备用于商业用途的所期望的产品。多种商品化学品来源于石油。这些商品化学品具有多种用途，包括作为溶剂、树脂、聚合物前体和专用化学品的用途。所期望的商品化学品包括4-碳分子，如1,4-丁二醇和1,3-丁二醇、上游前体和下游产品。期望开发用于商品化学品生产的方法以提供石油基产品的可再生来源和提供能量和资金不太密集的方法。
因此，仍需要有利于所期望的产品的生产的方法。本发明满足了这种需要并且还提供了相关优势。
发明概述
本发明提供了醛脱氢酶变体的多肽和编码核酸。本发明还提供了表达醛脱氢酶变体的细胞。本发明还提供了用于生产3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或其酯或酰胺的方法，其包括培养表达醛脱氢酶变体的细胞或者使用这些细胞的裂解液。本发明另外提供了用于生产4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)或其酯或酰胺的方法，其包括培养表达醛脱氢酶变体的细胞或者使用这些细胞的裂解液。
附图说明
图1显示了包含醛脱氢酶的示例性1,3-丁二醇(1,3-BDO)途径。图1显示了从乙酰乙酰-CoA至1,3-丁二醇的途径。酶是：(A)乙酰乙酰-CoA还原酶(CoA-依赖性的，醛形成)；(B)3-氧丁醛还原酶(酮还原)；(C)3-羟基丁醛还原酶，在本文中也称为1,3-丁二醇脱氢酶；(D)乙酰乙酰-CoA还原酶(CoA-依赖性的，醛形成)；(E)3-氧丁醛还原酶(醛还原)；(F)4-羟基,2-丁酮还原酶；(G)乙酰乙酰-CoA还原酶(酮还原)；(H)3-羟基丁酰基-CoA还原酶(醛形成)，在本文中也称为3-羟基丁醛脱氢酶；和(I)3-羟基丁酰基-CoA还原酶(醇形成)。
图2显示了包含醛脱氢酶的示例性1,4-丁二醇(1,4-BDO)途径。催化生物合成反应的酶是：(1)琥珀酰基-CoA合成酶；(2)非CoA依赖性琥珀酸半醛脱氢酶；(3)α-酮戊二醛脱氢酶；(4)谷氨酸：琥珀酸半醛转氨酶；(5)谷氨酸脱羧酶；(6)CoA依赖性琥珀酸半醛脱氢酶；(7)4-羟基丁酸脱氢酶(也称为4-羟基丁酸脱氢酶)；(8)α-酮戊二酸脱羧酶；(9)4-羟基丁酰基CoA：乙酰基-CoA转移酶；(10)丁酸激酶(也称为4-羟基丁酸激酶)；(11)磷酸转丁酰基酶(也称为磷酸-反式-4-羟基丁酰酶)；(12)醛脱氢酶(也称为4-羟基丁酰基-CoA还原酶)；(13)醇脱氢酶(也称为4-羟基丁醛还原酶或者4-羟基丁醛还原酶)。
图3显示了ALD-1、ALD-2和ALD-3的序列对比。所述序列分别对应于SEQ ID NO：1、2和3。图中的下划线为两个环区，第一个表示为A，第二个表示为B，两者均参与底物特异性和对映异构体特异性，如本文所确定的。ALD-1中的环A是序列LQKNNETQEYSINKKWVGKD(SEQ IDNO：124)，ALD-2中的是序列IGPKGAPDRKFVGKD(SEQ ID NO：125)并且ALD-3中的是序列ITPKGLNRNCVGKD(SEQ ID NO：126)。ALD-1中的环B是序列SFAGVGYEAEGFTTFTIA(SEQ ID NO：127)，ALD-2中的是序列TYCGTGVATNGAHSGASALTIA(SEQ ID NO：128)并且ALD-3中的是序列SYAAIGFGGEGFCTFTIA(SEQ ID NO：129)。来自ALD-2的底物特异性环A和B的序列和长度不同于ALD-1和ALD-3的那些；尽管如此，比对显示了足够的保守以帮助鉴别如本文所述的替换的相应位置，并且如果与图6所示的3D建模结合，则尤其是这样。将ALD-3用作晶体结构建模的模板；参见图6，其显示两个环区相互作用以影响底物特异性和对映异构体特异性，特别是当用如本文所述的示例性替换修饰时。ALD-1和ALD-3是51.9％相同的。ALD-1和ALD-2是35.9％相同的。ALD-3和ALD-2是40％相同的。基于ALD-1、ALD-2和ALD-3的比对的环A的一致性是IXPKG-----XXNRKXVGKD(SEQ ID NO：5)。基于ALD-1、ALD-2和ALD-3的比对的环B的一致性是SYAGXGXXXE----GFXTFTIA(SEQ ID NO：6)。应理解在共有序列中具体鉴别的氨基酸是保守残基，但是根据需要并且如本文所公开的，标记为“X”的位置是可变的并且可以对应于任何氨基酸。还将理解“-----”可以对应于不同数目的氨基酸残基的存在或不存在。在图3和4A-4C中显示了这些可变数目的氨基酸残基的实例。此外，应理解可以(例如)用保守氨基酸替换共有序列中的保守残基，如本文所述(参见，例如，图4A-4C)。
图4A-4C显示了示例性醛脱氢酶(ALD)的比对，其代表性比对证实鉴别了对应于其中可以进行本发明的替换的代表性模板ALD序列中的位置的ALD中的位置。如图3，下划线是2个环区，第一个表示为A，第二个表示为B，两者均参与底物特异性和对映异构体特异性，如本文所确定的。图4A显示了与ALD-1相比，具有40-55％的截止值的示例性ALD序列比对。所述序列对应于如图4A中所示的SEQ ID NO：1(ALD-1)、13、20和24。图4B显示了与ALD-1相比，具有75-90％的截止值的示例性ALD序列比对。所述序列对应于如图4B中所示的SEQ ID NO：1(ALD-1)、30、33和37。对环A和B添加了下划线。图4C显示了与ALD-1相比，具有90％的截止值的示例性ALD序列比对。所述序列对应于如图4C中所示的SEQ ID NO：1(ALD-1)、38、40和44。ALD-1与SEQ ID NO：38、40和44分别具有99％、97％和95％的同一性。图4A-4C表明可以在与ALD-1具有至少40％的同一性的ALD中鉴别本文所教导的替换的相应位置，特别是环A和B区，并且特别是非常保守的环B区。
图5A和5B显示了多种示例性醛脱氢酶的酶活力。图5A显示了ALD-2、ALD-1和ALD-1变体对3羟基-(R)-丁醛(柱组中的左侧柱)和3羟基-(S)-丁醛(柱组中的右侧柱)的特定活性。图5B显示了3-羟基丁醛的R形式对S形式的活力比。
图6A-6C显示了醛脱氢酶959的结构的飘带图。该图显示了3-羟基-(R)-丁醛(图6A)或3-羟基-(S)-丁醛(图6B)与959结构的对接。图6C显示了与3-羟基-(R)-丁醛(R3HB)相同的取向。
发明详述
本发明涉及具有所期望的性质并且对生产所期望的产品有用的酶变体。在具体的实施方式中，本发明涉及醛脱氢酶变体，它是与自然界中存在的野生型酶相比，具有显著不同的结构和/或功能特征的酶变体。因此，本发明的醛脱氢酶不是天然存在的酶。本发明的这些醛脱氢酶变体在已经过工程设计来生产所期望的产品的工程细胞，如微生物中有用。例如，如本文所公开的，具有代谢途径的细胞，如微生物可以生产所期望的产品。可以将具有所期望的特征的本发明的醛脱氢酶引入具有使用醛脱氢酶酶活力来生产所期望的产品的代谢途径的细胞，如微生物。这些醛脱氢酶变体另外作为体外实施我们所期望的反应的生物催化剂有用。因此，本发明的醛脱氢酶变体可以在工程细胞，如微生物中使用以生产所期望的产品或者作为体外生物催化剂来生产所期望的产品。
如本文所使用的，当用于表示本发明的细胞或微生物时，术语“非天然存在的”旨在表示所述细胞具有至少一个通常在参考种的天然存在的菌株，包括参考种的野生型菌株中不存在的遗传改变。遗传改变包括(例如)引入编码代谢多肽的可表达核酸的修饰、其它核酸的添加、核酸缺失和/或细胞基因材料的其它功能性破环。这些修饰包括(例如)其用于参考种的异源、同源或者异源和同源多肽两者的编码区和功能性片段。其它修饰包括(例如)非编码调控区，其中所述修饰改变了基因或操纵子的表达。示例性的代谢多肽包括用于生产所期望的产品的生物合成途径内的酶或蛋白。
代谢修饰是指从其天然存在的状态改变的生化反应。因此，非天然存在的细胞可以具有的编码代谢多肽或其功能性片段的核酸具有基因修饰。本文公开了示例性的代谢修饰。
当用于表示细胞或微生物时，如本文所使用的，术语“分离的”旨在表示如果该细胞在自然界存在时，基本不含参考细胞在自然界中存在时的至少一种成分的细胞。所述术语包括除去如其在自然环境中所存在的一些或所有成分的细胞。所述术语还包括除去如所述细胞在非天然存在的环境中所存在的一些或所有成分的细胞。因此，分离的细胞与如其在自然界中所存在的或者如其在非天然存在的环境中生长、储存或生存的其它物质部分或完全分离。分离的细胞的具体实例包括部分纯的细胞、基本纯的细胞和在非天然存在的培养基中培养的细胞。
如本文所使用的，术语“微生物的”或“微生物”旨在表示作为包括在古细菌、细菌或真核域(domain)内的微小细胞存在的任何生物。因此，该术语旨在涵盖具有微小尺寸的原核或真核细胞或生物，并且包括所有种的细菌、古细菌和真细菌以及真核微生物，如酵母和真菌。该术语还包括可以培养用于生物化学物质的生产的任何种的细胞培养物。
如本文所使用的，术语“CoA”或“辅酶A”旨在表示有机辅因子或辅基(酶的非蛋白质部分)，它的存在是多种酶(主酶)的活力所需要的以形成活性酶系统。辅酶A在某些柠檬酸合酶中具有功能，在乙酰基或其它酰基转移中以及在脂肪酸合成和氧化、丙酮酸盐氧化中和在其它乙酰化中起作用。
当用于表示培养或生长条件时，如本文所使用的，术语“基本厌氧的”旨在表示对于液体培养基中的溶氧，氧的量小于饱和的约10％。该术语还旨在包括维持小于约1％的氧气氛的液体或固体培养基的密封室。
如本文所使用的“外源的”旨在表示将参考分子或参考活力引入宿主细胞。可以(例如)通过将编码核酸引入到宿主遗传物质中，如通过整合到宿主染色体上或者作为非染色体遗传物质，如质粒来引入所述分子。因此，如对于编码核酸的表达所使用的，该术语是指以可表达形式向细胞中引入所述编码核酸。当用于表示生物合成活力时，所述术语是指引入宿主参考生物中的活力。所述源可以是(例如)在引入宿主细胞后，表达参考活力的同源或异源编码核酸。因此，术语“内源的”是指存在于宿主中的参考分子或活力。类似地，当用于表示编码核酸的表达时，该术语是指细胞内所含的编码核酸的表达。术语“异源的”是指来源于参考种以外的来源的分子或活力，然而“同源的”是指来源于宿主细胞的分子或活力。因此，本发明的编码核酸的外源表达可以使用异源或同源编码核酸之一或两者。
应理解当细胞中包含不止一种外源核酸时，所述不止一种外源核酸是指参考编码核酸或生物合成活性，如以上所讨论的。如本文所公开的，还应理解可以将该不止一种外源核酸引入宿主细胞的单独的核酸分子上，多顺反子核酸分子上或它们的组合，并且仍认为是不止一种外源核酸。例如，如本文所公开的，可以工程设计细胞以表达编码所期望的酶或蛋白，如途径酶或蛋白的两种或更多种外源核酸。在其中将编码所期望的活力的两种外源核酸引入宿主细胞的情况下，应理解可以将所述两种外源核酸作为单一核酸引入到(例如)单个质粒上，不同的质粒上，可以在单一位点或多个位点整合到宿主染色体上，并且仍认为是两种外源核酸。类似地，应理解可以将大于两种外源核酸以任何所期望的组合，例如，在单一质粒上，在不同的质粒上引入到宿主生物中，可以在单一位点或多个位点整合到宿主染色体上，并且仍认为是两种或更多种外源核酸，例如，三种外源核酸。因此，参考外源核酸或生物合成活力的数目是指编码核酸的数目或者生物合成活力的数目，而不是引入到宿主生物中的单独的核酸的数目。
如本文所使用的，术语“基因破坏”或其语法等价形式旨在表示使编码基因产品失活或活力减弱的基因改变。所述基因改变可以是(例如)整个基因的缺失、转录或翻译所需的调控序列的缺失、导致产生截短基因产品的基因部分的缺失或者使所编码的基因产品失活或活力减弱的任何多种突变策略。基因破环的一种特别有用的方法是全基因缺失，因为它降低或消除了本发明的非天然存在的细胞中的遗传回复的发生。基因破环还包括无效突变，它是指基因或含有基因的区域内导致基因不被转录成RNA和/或翻译成功能基因产品的突变。这种无效突变可以由多种类型的突变产生，其包括(例如)点突变失活、基因部分缺失、整个基因缺失或染色体节段缺失。
当用于表示生物化学产品的生产时，如本文所使用的，术语“生长-偶联的(growth-coupled)”旨在表示在微生物的生长期期间产生了参考生物化学产品的生物合成。在具体的实施方式中，生长-偶联的产生可以是必须的，其表示参考生物化学产品的生物合成是微生物生长期期间所产生的必须产品。
如本文所使用的，术语“减弱”或其语法等价形式旨在表示削弱、降低或减弱酶或蛋白的活力或量。如果所述减弱导致活力或量低于给定功能所需的临界水平，则酶或蛋白的活力或量的减弱可以模拟完全破环。然而，模拟完全破坏，例如，一条途径完全破环的酶或蛋白的活力或量的减弱仍可以满足其它途径继续起作用。例如，内源酶或蛋白的减弱可以足以模拟用于产生本发明所期望的产品的相同的酶或蛋白的完全破环，但是酶或蛋白剩余的活力或量仍可以足以维持其它途径，如对于宿主细胞存活、增殖或生长来说关键的途径。酶或蛋白的减弱还可以是以足以提高本发明所期望的产品的得率的量削弱、降低或减弱所述酶或蛋白的活力或量，但不必需模拟所述酶或蛋白的完全破环。
本发明的非天然存在的细胞可以含有稳定的遗传变化，它是指可以培养超过5代而无所述变化损失的细胞。通常，稳定的遗传变化包括持续超过10代的修饰，具体地，稳定修饰将持续超过约25代，并且更具体地，稳定遗传修饰将超过50代，包括无穷代。
就基因破环来说，特别有用的稳定遗传变化为基因缺失。对于降低回复至所述遗传变化之前的表型的可能性来说，使用基因缺失来引入稳定遗传变化是特别有用的。例如，可以(例如)通过在一组代谢修饰中使编码催化一个或多个反应的酶的基因缺失来实现生物化学品的稳定生长-偶联的生产。还可以通过多个缺失来提高生物化学品的生长-偶联的生产的稳定性，从而显著降低对于每种破坏的活力所发生的多种补偿回复的可能性。
本领域技术人员将理解参考适合的宿主细胞或生物，如大肠杆菌(E.coli)和它们相应的代谢反应或适合于所期望的遗传材料，如所期望的代谢途径的基因的来源细胞或生物，描述了遗传变化，包括本文中举例说明的代谢修饰。然而，考虑到多种生物的全基因组测序和基因组领域中的高技术水平，本领域技术人员将容易地能够将本文所提供的教导内容和指导应用于基本上所有其它生物。例如，可以通过引入来自参考种以外的种的相同或类似的编码核酸将本文中举例说明的大肠杆菌(E.coli)代谢变化容易地应用于其它种。这些遗传变化包括(例如)种同源物的遗传变化，一般地并且具体地，直系同源物、旁系同源物或非直系同源基因置换。
直系同源物是通过垂直传递相关并且在不同生物中负责基本相同或等同的功能的基因。例如，对于环氧化物水解的生物学功能，小鼠环氧化物酶和人环氧化物酶可以考虑是直系同源物。当(例如)它们共有足够量的序列相似性以表示它们是同源的时，基因是通过垂直传递相关的，或者是通过来自共同祖先的进化相关的。如果它们共有立体结构但不必需共有足够量的序列相似性以表示它们是从共同祖先进化而来，从而一级序列相似性不是可鉴别的，则还可以认为基因是直系同源的。直系同源的基因可以编码具有约25％至100％的氨基酸序列同一性的序列相似性的蛋白。如果它们的立体结构也显示出相似性，则编码共有小于25％的氨基酸相似性的蛋白的基因也可以认为是通过垂直传递产生的。认为酶的丝氨酸蛋白酶家族的成员，包括组织纤维蛋白溶酶原活化因子和弹性酶是通过来自共同祖先的垂直传递所产生的。
直系同源物包括通过(例如)进化在结构或整体活力方面出现变化的基因或者它们的编码基因产品。例如，当一个物种编码显示出两种功能的基因产品并且其中这些功能在第二个物种中已分成不同的基因时，三种基因和它们相应的产品被认为是直系同源物。对于生物化学产品的生产，本领域技术人员将理解选择具有要引入或要破坏的代谢活性的直系同源基因来用于非天然存在的细胞的构建。显示出可分离的活力的直系同源物的实例为其中在两个或更多个物种之间或者在单一物种内已分成不同基因产品的不同活力。具体实例是两种类型的丝氨酸蛋白酶活力：弹性酶蛋白水解作用和纤溶酶原蛋白水解作用作为纤维蛋白溶酶原活化因子和弹性酶分离成不同的分子。第二实例是支原体5'-3'核酸外切酶和果蝇(Drosophila)DNA聚合酶III活力的分离。可以将来自第一物种的DNA聚合酶认为是来自第二物种的核酸外切酶或聚合酶之一或两者的直系同源物，反之亦然。
相反，旁系同源物是通过(例如)复制，然后进化趋异相关的同源物，并且其具有类似或常规的功能，但是不具有相同的功能。旁系同源物可以起源或衍生自(例如)相同物种或者不同物种。例如，微粒体环氧化物酶(环氧化物酶I)和可溶性环氧化物酶(环氧化物酶II)可以认为是旁系同源物，因为它们代表了从共同祖先共同进化而来的两种不同的酶，其催化不同的反应并且在相同物种中具有不同的功能。旁系同源物是来自相同物种，彼此之间具有表明它们是同源的或者通过来自共同祖先的共同进化相关的显著的序列相似性的蛋白。旁系同源的蛋白家族组包括HipA同源物、荧光素酶基因、肽酶等。
非直系同源基因置换是来自一个物种的可以替代不同物种中参考基因功能的非直系同源基因。替代包括(例如)与不同物种中的参考功能相比，能够在来源物种中实施基本相同或类似的功能。尽管通常非直系同源基因置换将可鉴别为与编码参考功能的已知基因在结构上相关，但是尽管如此在结构上不太相关但是在功能上类似的基因以及它们相应的基因产品仍将属于如本文所使用的术语的含义中。与设法取代的编码所述功能的基因相比，功能相似性需要(例如)在非直系同源基因产品的活性位点或结合区中的至少一些结构相似性。因此，非直系同源基因包括(例如)旁系同源物或非相关基因。
因此，在鉴别和构建具有所期望的产品的生物合成能力的本发明的非天然存在的细胞中，通过将本文所提供的教导或指导应用于特定物种，本领域技术人员将理解代谢修饰的鉴别可以包括直系同源物的鉴别和包含或失活。在旁系同源物和/或非直系同源基因置换存在于编码催化类似或基本类似的代谢反应的酶的参考细胞中的程度上，本领域技术人员还可以使用这些进化相关的基因。类似地对于基因破环，也可以在宿主细胞中使进化相关的基因被破坏或缺失以降低或消除靶向破坏的酶促活力的功能冗余。
可以通过本领域技术人员熟知的方法确定直系同源物、旁系同源物和非直系同源基因置换。例如，对于两种多肽的核酸或氨基酸序列的检查将显示出所比较的序列之间的序列同一性和相似性。基于这些相似性，本领域技术人员可以确定所述相似性是否足够高以表明所述蛋白通过来自共同祖先的进化相关。本领域技术人员熟知的算法，如Align、BLAST、Clustal W等比较和确定了原始序列的相似性或同一性，并且还确定了可以分配权重和得分的序列中的空位的存在或意义。这些算法在本领域中也是已知的并且类似地适合于确定核苷酸序列的相似性或同一性。基于计算统计学相似性，或者在随机多肽中找到类似匹配的机会和所确定的匹配的显著性的熟知方法来计算确定关联性的足够的相似性的参数。如果需要，本领域技术人员还可以视觉地优化两条或更多条序列的计算机比较。可以预期相关基因产品或蛋白具有高相似性，例如，25％至100％的序列同一性。如果扫描大小足够的数据库，则不相关的蛋白可以具有与预期偶然发生的基本相同的同一性(约5％)。5％至24％之间的序列可以或可以不代表足够的同源性以得出所比较的序列相关的结论。考虑数据组的大小，可以实施确定这些匹配的显著性的其它统计分析以确定这些序列的相关性。
例如，使用BLAST算法，用于确定两条或更多条序列的相关性的示例性参数可以如下所示。简要地，可以使用BLASTP 2.0.8版(1999-01-05)和下列参数：Matrix：0BLOSUM62；空位开放：11；空位扩展：1；x_dropoff：50；期待值：10.0；字长：3；过滤器：开进行氨基酸序列比对。可以使用BLASTN 2.0.6版(1998-09-16)和下列参数：匹配：1；错配：-2；空位开放：5；空位扩展：2；x_dropoff：50；期待值：10.0；字长：11；过滤器：关进行核酸序列比对。本领域技术人员将已知，例如，可以对以上参数进行哪些改变以提高或降低比较的严格性和确定两条或更多条序列的相关性。
在一个实施方式中，本发明提供了醛脱氢酶，它是野生型或亲代醛脱氢酶的变体。本发明的醛脱氢酶将酰基-CoA转化为其相应的醛。这种酶也可以被称为氧化还原酶，其将酰基-CoA转化为其相应的醛。可以将本发明的这种醛脱氢酶分类为反应1.2.1.b，氧化还原酶(酰基-CoA至醛)，其中前3个数字对应于前3个酶学委员会数字，其表示与底物特异性无关的一般转化类型。本发明的醛脱氢酶的示例性酶转化包括(但不限于)3-羟基丁酰基-CoA向3-羟基丁醛(也称为3-HBal)的转化(参见图1)和4-羟基丁酰基-CoA向4-羟基丁醛的转化(参见图2)。本发明的醛脱氢酶可以用于在含有适合的代谢途径的细胞，如微生物中或体外生产所期望的产品，如3-羟基丁醛(3-HBal)、1,3-丁二醇(1,3-BDO)、4-羟基丁醛(4-HBal)、1,4-丁二醇(1,4-BDO)或者其它所期望的产品，如下游产品，包括其酯或酰胺。例如，使用(例如)脂肪酶，1,3-BDO可以在体内或体外与酸反应以转化为酯。这些酯可以具有营养、医学和食品用途，并且当使用1,3-丁二醇的R-形式时，具有优势，因为(与通过乙醛化学合成路线，从石油或乙醇制备的S-形式或外消旋混合物相比)这是动物和人作为能源的最佳利用形式(例如，酮酯，如(R)-3-羟丁基-R-1,3-丁二醇单酯(其具有美国公认安全(GRAS)批准)和(R)-3-羟基丁酸酯甘油单酯或二酯)。所述酮酯可以口服递送并且所述酯释放通过身体使用的R-1,3-丁二醇(参见，例如，WO2013150153)。因此，本发明对于提供改善的酶促路线和微生物以提供高度富集或基本对映体纯的并且相对于副产品还具有改善的纯度质量的改善的1,3-丁二醇(即R-1,3-丁二醇)的组合物是特别有用的。
1,3-丁二醇，也称为丁二醇具有其它食品相关用途，包括直接作为食品源使用、食品成分、调味剂、调味剂的溶剂或增溶剂、稳定剂、乳化剂和抗-微生物剂和防腐剂。在制药工业中，1,3-丁二醇作为肠胃外药物溶剂使用。1,3-丁二醇在化妆品中作为以下成分使用：润肤剂、防止不溶性成分结晶的稀释剂、作为水溶性低的成分(如香料)的增溶剂和作为抗-微生物剂和防腐剂。例如，它可以用作稀释剂，特别是在头发喷雾和卷发剂中；它降低了来自精油的香味的损失，防止通过微生物的腐败，并用作苯甲酸酯的溶剂。1,3-丁二醇可以在0.1％或以下至50％或以上的浓度使用。它在头发和洗浴产品、眼部和面部化妆品、香料、个人清洁产品以及剃须和皮肤护理制剂中使用(参见，例如，化妆品成分审查委员会的报告："Final Report on the Safety Assessment of Butylene Glycol,Hexylene Glycol,Ethoxydiglycol,and Dipropylene Glycol",Journal of the American College ofToxicology,4卷,5期,1985，该文献作为参考并入本文)。该报告提供了1,3-丁二醇(丁二醇)在化妆品中的具体使用和浓度；参见，例如，其中标题为“产品制剂数据(ProductFormulation Data)”的报告表2。
在一个实施方式中，本发明提供了分离的核酸分子，其选自：(a)编码被称为SEQID NO：1、2或3的或表4中所提及的氨基酸序列的核酸分子，其中所述氨基酸序列包含对应于位置I66的氨基酸替换；(b)在高严格杂交条件下杂交至(a)的核酸并且包含编码对应于位置I66的氨基酸替换的核酸序列的核酸分子；和(c)与(a)或(b)互补的核酸分子。
在本发明的核酸的一些实施方式中，位置I66的氨基酸替换是表1、2和/或3中所示的氨基酸替换。在一些实施方式中，除位置I66的替换之外，所述氨基酸序列在表1、2和/或3中所示的其它氨基酸变体位置包含一个或多个氨基酸替换。在一些实施方式中，除位置I66的替换之外，所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。
在本发明的核酸分子的一些实施方式中，除一个或多个氨基酸替换以外，所述氨基酸序列与SEQ ID NO：1、2或3或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性或者是相同的。在一些实施方式中，所述氨基酸序列包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个表1、2和/或3中所示的氨基酸替换。在一些实施方式中，所述氨基酸序列包含如表1、2和/或3中所示的变体的氨基酸替换。
在一个实施方式中，分离的核酸分子可以选自：(a)编码被称为SEQ ID NO：1、2或3的或者表4中所提及的氨基酸序列的核酸分子，其中所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换；(b)在高严格杂交条件下杂交至(a)的核酸并且包含编码表1、2和/或3中所示的一个或多个氨基酸替换的核酸序列的核酸分子；(c)编码包含环A(SEQ IDNO：5)和/或环B(SEQ ID NO：6)的共有序列的氨基酸序列的核酸分子，其中所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换；和(d)与(a)或(b)互补的核酸分子。在一个实施方式中，除所述一个或多个氨基酸替换以外，通过所述核酸分子编码的氨基酸序列与SEQ ID NO：1、2或3或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性或者是相同的。所述氨基酸序列可以包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个或者更多个表1、2和/或3中所示的氨基酸替换，例如，17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42或43个，即多至全部氨基酸位置具有替换。
本发明还提供了含有本发明的核酸分子的载体。在一个实施方式中，所述载体是表达载体。在一个实施方式中，所述载体包含双链DNA。
本发明还提供了编码本发明的醛脱氢酶多肽的核酸。编码本发明的醛脱氢酶的核酸分子还可以包括杂交至在本文中通过SEQ ID NO、GenBank和/或GI编号所公开的核酸的核酸分子或者杂交至编码在本文中通过SEQ ID NO、GenBank和/或GI编号所公开的氨基酸序列的核酸分子的核酸分子。杂交条件可以包括如本文所述的那些的本领域技术人员熟知的高度严格性、中等严格性或低严格性杂交条件。类似地，可以将可以在本发明中使用的核酸分子描述为与在本文中通过SEQ ID NO、GenBank和/或GI编号所公开的核酸或者杂交至编码在本文中通过SEQ ID NO、GenBank和/或GI编号所公开的氨基酸序列的核酸分子的核酸分子具有特定序列同一性百分比。例如，所述核酸分子可以与本文所述的核酸具有至少65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性或者是相同的。
严格杂交是指在该条件下杂交的多核苷酸稳定的条件。如本领域技术人员已知的，杂交的多核苷酸的稳定性反映在杂交物的解链温度(Tm)。一般地，杂交的多核苷酸的稳定性是盐浓度(例如，钠离子浓度)和温度的函数。杂交反应可以在严格性较低的条件下进行，然后通过不同，但更高的严格性清洗。对杂交严格性的提及涉及这些清洗条件。高度严格杂交包括仅允许在0.018M NaCl，65℃形成稳定的杂化多核苷酸的那些核酸序列杂交的条件，例如，如果杂交物在0.018M NaCl，65℃不稳定，则它在高严格性条件下将不会稳定，如本文所考虑的。可以(例如)通过在50％甲酰胺、5×Denhart溶液、5×SSPE、0.2％SDS中，在42℃杂交，然后在0.1×SSPE和0.1％SDS中，在65℃清洗来提供高严格性条件。除高严格性杂交条件以外的杂交条件也可以用于描述本文所公开的核酸序列。例如，短语中等严格性杂交是指相当于在50％甲酰胺、5×Denhart溶液、5×SSPE、0.2％SDS中，在42℃杂交，然后在0.2×SSPE和0.2％SDS中，在42℃清洗的条件。短语低严格性杂交是指相当于在10％甲酰胺、5×Denhart溶液、6×SSPE、0.2％SDS中，在22℃杂交，然后在1×SSPE、0.2％SDS中，在37℃清洗的条件。Denhart溶液含有1％聚蔗糖、1％聚乙烯吡咯烷酮和1％牛血清白蛋白(BSA)。20×SSPE(氯化钠、磷酸钠、乙二胺四乙酸(EDTA))含有3M氯化钠、0.2M磷酸钠和0.025M(EDTA)。其它适合的低、中等和高严格性杂交缓冲液和条件对于本领域技术人员是熟知的并且在(例如)Sambrook等人,Molecular Cloning:A Laboratory Manual,第3版.,Cold Spring Harbor Laboratory,New York(2001)；和Ausubel等人,Current Protocolsin Molecular Biology,John Wiley and Sons,Baltimore,MD(1999)中描述。
编码本发明的醛脱氢酶的核酸分子可以与本文所公开的核苷酸序列具有至少特定的序列同一性。因此，在本发明的一些方面，编码本发明的醛脱氢酶的核酸分子具有与在本文中通过SEQ ID NO、GenBank和/或GI编号所公开的核酸或者杂交至编码在本文中通过SEQ ID NO、GenBank和/或GI编号所公开的氨基酸序列的核酸分子的核酸分子具有至少65％的同一性，至少70％的同一性，至少75％的同一性，至少80％的同一性，至少85％的同一性，至少90％的同一性，至少91％的同一性，至少92％的同一性，至少93％的同一性，至少94％的同一性，至少95％的同一性，至少96％的同一性，至少97％的同一性，至少98％的同一性或者至少99％的同一性或者是相同的核苷酸序列。
序列同一性(也称为同源性或相似性)是指两个核酸分子之间或者两个多肽之间的序列相似性。可以通过比较每条序列中的位置来确定同一性，其可以出于比较的目的进行比对。当所比较的序列中的位置被相同碱基或氨基酸占据时，则所述分子在该位置是同一的。序列之间的同一性程度是所述序列所共有的匹配或同源位置数目的函数。可以使用本领域中已知的软件程序进行两条序列的比对以确定它们的序列同一性百分比，所述软件程序如(例如)Ausubel等人,Current Protocols in Molecular Biology,John Wiley andSons,Baltimore,MD(1999)中所描述的。优选地，使用缺省参数进行比对。可以使用的在本领域中熟知的一种比对程序是设置为缺省参数的BLAST。具体地，程序为BLASTN和BLASTP，使用以下缺省参数：遗传密码＝标准；过滤器＝无；链＝两条；截止值＝60；预期值＝10；Matrix＝BLOSUM62；描述＝50条序列；排序依据＝高分；数据库＝非冗余，GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR。这些程序的详细信息可见于国家生物技术信息中心(National Center for Biotechnology Information)(还参见Altschul等人,"J.Mol.Biol.215:403-410(1990))。
在一些实施方式中，所述核酸分子是分离的核酸分子。在一些实施方式中，所述分离的核酸分子是编码所提及的多肽的变体的核酸分子，其中(i)所提及的多肽具有SEQ IDNO：1、2或3所示的氨基酸序列或者表4中的那些(SEQ ID NO：7-123)，(ii)所述变体相对于SEQ ID NO：1、2或3或者表4中的那些包含一个或多个氨基酸替换和(iii)所述一个或多个氨基酸替换选自如表1-3中所示的氨基酸替换。表1-3提供了SEQ ID NO：1、2或3或者表4中的那些的示例性变体的非限制性列表。在一个实施方式中，对于表1-3中的每种变体，除所指明的位置外，所有位置与SEQ ID NO：1、2或3或者表4中的那些相同。通过指明原始氨基酸的身份的字母，之后指明替换的氨基酸在SEQ ID NO：1、2或3或者表4中的那些中的位置的数字，之后指明取代的氨基酸的身份的字母来表示氨基酸替换。例如：“D12A”表示SEQ IDNO：1或2中位置12处的门冬氨酸被丙氨酸替换。用于识别氨基酸的单字母代码是本领域技术人员已知的标准代码。表1-3中的一些变体包括两个或更多个替换，其通过替换列表表示。所述一个或多个氨基酸替换可以选自表1-3中所列的任一个变体或者表1-3中所列的两个或更多个变体的任意组合。当从表1-3中的单一变体选择时，所产生的变体可以包含所选变体的一个或多个替换的任意组合，其包括所有所指明的替换或者不到所有所指明的替换。当从表1-3中的两个或更多个变体中选择替换时，所产生的变体可以包含所选变体的一个或多个替换，包含来自两个或更多个所选变体中的每一个的所有所指明的替换或者不到所有所指明的替换的任意组合。例如，所产生的变体可以包含来自表1-3中的单一变体的1、2、3或4个替换。作为另一个实例，所产生的变体可以包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、20、25或更多个选自表1-3中的1、2、3、4、5个或更多个所选变体的替换。在一些实施方式中，所产生的变体包含表1-3中所选变体的全部所指明的替换。在一些实施方式中，所产生的变体与SEQ ID NO：1、2或3或者表4中的那些相差至少一个氨基酸替换，但是小于25、20、10、5、4或3个氨基酸替换。在一些实施方式中，所产生的变体包含如选自表1-3的变体所指明的序列，基本由其组成或由其组成，从而仅与SEQ ID NO：1、2或3或者表4中的那些在所指明的氨基酸替换处不同。
在一些实施方式中，所述核酸分子是分离的核酸分子，其编码参考多肽(参考多肽具有SEQ ID NO：1、2或3的氨基酸序列或者表4中的那些)的变体，其中所述变体(i)包含选自表1-3的相应变体的一个或多个氨基酸替换，和(ii)与相应变体具有至少65％、70％、75％、80％、85％、90％、95％、98％、99％或100％的序列同一性。在其中第二变体与相应变体具有100％的序列同一性的情况下，所述第二变体包含如选自表1-3的变体所指明的序列，并且可以或可以不在氨基-和羧基-末端具有一个或多个其它氨基酸。在一些实施方式中，所产生的变体与选自表1-3的相应变体具有至少80％、85％、90％或95％的序列同一性；在一些情况下，同一性为至少90％或以上。在其中所产生的变体与选自表1-3的相应变体的同一性小于100％的情况下，可以改变对相应变体所指明的一个或多个氨基酸替换的位置(例如，在一个或多个氨基酸的插入或缺失的情况下)，但所述位置仍包含在所产生的变体内。例如，可以存在对应于“D12A”的门冬氨酸向丙氨酸的替换(相对于SEQ ID NO：1或2，在位置12)，但是在所产生的变体的不同位置。如本领域中熟知的，可以通过序列对比确定氨基酸是否对应于所指明的替换，虽然其处于不同的位置。通常，比对显示出侧接所替换的氨基酸的氨基酸的同一性或相似性，从而所述侧接序列被认为与另一多肽的同源序列对齐，这将允许替换的氨基酸相对于表1-3的相应变体局部定位以确定相应位置来做出替换，即便在给定多肽链中的数字位置是被移动的。在一个实施方式中，包含至少3至15个氨基酸(包括替换的位置)的区域将以相对高百分比同一性与相应变体序列局部对其，包括在沿相应变体序列的替换的氨基酸的位置(例如，90％、95％或100％的同一性)。在一些实施方式中，如果当以缺省参数使用BLASTP比对算法时，要比较的多肽序列在沿相应变体序列所指明的位置与具有相同匹配或类似氨基酸的相应变体比对，其中类似的氨基酸是使用比对算法的缺省参数，具有足以用于与所关心的变体位置比对的化学性质的氨基酸，则通过选自表1-3的相应变体所指明的一个或多个氨基酸替换(例如，所有或不到所有氨基酸替换)被认为存在于给定变体中，即使存在于沿多肽链的不同物理位置。
在一些实施方式中，本发明的核酸分子与结合本文多个实施方式中任何一项所述的核酸互补。
应理解本发明的核酸或者本发明的多肽可以不包括野生型亲代序列，例如，亲代序列，如SEQ ID NO：1、2或3或者表4中所公开的序列。本领域技术人员将容易地理解亲代野生型序列的含义基于本领域中熟知的含义。还应理解本发明的这种核酸可以不包括编码如自然界中所存在的天然存在的氨基酸序列的核酸序列。类似地，本发明的多肽可以不包括如自然界中所存在的氨基酸序列。因此，在具体的实施方式中，本发明的核酸或多肽如本文所述，但条件是编码的氨基酸序列不是野生型亲代序列或天然存在的氨基酸序列和/或所述核酸序列不是野生型或天然存在的核酸序列。本领域技术人员将理解天然存在的氨基酸或核酸序列与如自然界中所存在的天然存在的生物中存在的序列有关。因此，在本发明的核酸和/或氨基酸序列的含义中包括了未发现与天然存在的生物中的处于相同状态或具有相同核苷酸或编码氨基酸序列的核酸或氨基酸序列。例如，在非天然存在的本发明的核酸或氨基酸序列的含义内包括在来自亲代序列的一个或多个核苷酸或氨基酸位置改变的核酸或氨基酸序列，包含如本文所述的变体。本发明的分离的核酸分子不包括含有所述核酸序列的天然存在的染色体，并且可以进一步排除如天然存在的细胞中存在的其它分子，如DNA结合蛋白，例如，蛋白，如结合至真核细胞内的染色体的组蛋白。
因此，与天然存在的核酸序列相比，本发明的分离的核酸序列具有物理和化学差异。本发明的分离或非天然存在的核酸不包含或不一定具有如自然界中所存在的天然存在的核酸序列的一些或全部化学键，所述化学键是共价的或非共价键的。因此，本发明的分离的核酸不同于天然存在的核酸，例如，具有不同于如染色体中所存在的天然存在的核酸序列的化学结构。例如，可以通过从天然存在的染色体中释放分离的核酸序列的磷酸二酯键的切割来产生不同的化学结构。本发明的分离的核酸还可以通过在原核或真核细胞中从结合至染色体DNA的蛋白分离或脱离出核酸而不同于天然存在的核酸，借此通过不同的非共价键而不同于天然存在的核酸。对于原核来源的核酸，本发明的非天然存在的核酸不一定具有(例如)结合至DNA结合蛋白，如聚合酶或染色体结构蛋白的染色体的一些或全部天然存在的化学键，或者不处于高级结构，如是超螺旋的。对于真核来源的核酸，本发明的非天然存在的核酸还不含有与染色质中所存在的相同的内部核酸化学键或与结构蛋白的化学键。例如，本发明的非天然存在的核酸未化学键合至组蛋白或骨架蛋白并且不包含在着丝点或端粒中。因此，本发明的非天然存在的核酸在化学方面不同于天然存在的核酸，因为它们缺少或包含与自然界中所存在的核酸不同的范德华相互作用、氢键、离子或静电键和/或共价键。键中的这些差异可以内部存在于核酸的单独区域内(即顺式)或者键中的这些差异可以反式存在，例如，与染色体蛋白相互作用。就真核来源的核酸来说，cDNA被认为是分离或非天然存在的核酸，因为cDNA内的化学键不同于染色体DNA上的基因的共价键，即序列。因此，本领域技术人员应理解分离或非天然存在的核酸不同于天然存在的核酸。
在一个实施方式中，本发明提供了包含如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列的分离的多肽，其中所述氨基酸序列包含对应于位置I66的氨基酸替换。在一些实施方式中，位置I66处的氨基酸替换是如表1、2和/或3中所示的氨基酸替换。在一些实施方式中，除对应于氨基酸位置I66的替换之外，所述氨基酸序列包含表1、2和/或3中所示的其它氨基酸变体位置处的一个或多个氨基酸替换。在一些实施方式中，除位置I66的替换之外，所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。
在另一个实施方式中，本发明提供了包含如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列的分离的多肽，其中所述氨基酸序列包含对应于位置I66的氨基酸替换，其中除对应于位置I66的氨基酸替换外，所述氨基酸序列与如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性或者是相同的。
在本发明的分离的多肽的一些实施方式中，位置I66处的氨基酸替换是如表1、2和/或3中所示的氨基酸替换。在一些实施方式中，除对应于氨基酸位置I66的替换之外，所述氨基酸序列包含表1、2和/或3中所示的其它氨基酸变体位置处的一个或多个氨基酸替换。在一些实施方式中，除位置I66的替换之外，所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。在一些实施方式中，所述氨基酸序列还包含1至100个氨基酸位置处的保守氨基酸替换，其中所述位置是除表1、2和/或3中所示的一个或多个氨基酸替换以外的位置。
在本发明的分离的多肽的一些实施方式中，与亲代序列相比，除表1、2和/或3中所示的一个或多个氨基酸替换以外，所述氨基酸序列在2至300个氨基酸位置处不包含修饰，其中所述位置选自在如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列的2、3、4或5条序列之间相同的那些。在一个实施方式中，所述氨基酸序列包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个表1、2和/或3中所示的氨基酸替换。在具体的实施方式中，所述氨基酸序列包含如表1、2和/或3中所示的变体的氨基酸替换。
在一个实施方式中，分离的多肽包含如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列，其中所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。在一个实施方式中，分离的多肽包含环A(SEQ ID NO：5)和/或环B(SEQ ID NO：6)的共有氨基酸序列。
在另一个实施方式中，分离的多肽包含如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列，其中所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换，其中除所述一个或多个氨基酸替换外，所述氨基酸序列与如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性或者是相同的。在一个实施方式中，所述氨基酸序列还包含1至100个氨基酸位置处的保守氨基酸替换，其中所述位置是除表1、2和/或3中所示的一个或多个氨基酸替换以外的位置。在另一个实施方式中，与亲代序列相比，除表1、2和/或3中所示的一个或多个氨基酸替换以外，所述氨基酸序列在2至300个氨基酸位置处不包含修饰，其中所述位置选自在如SEQ IDNO：1、2或3或表4中所提及的氨基酸序列的2、3、4或5条序列之间相同的那些。在一个实施方式中，所述氨基酸序列包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个或者更多个表1、2和/或3中所示的氨基酸替换，例如，17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42或43个，即多至全部氨基酸位置具有替换。
在一个实施方式中，本发明的多肽编码醛脱氢酶。在一个实施方式中，所述多肽可以将3-羟基丁酰基-CoA转化为3-羟基丁醛。在一个实施方式中，所述多肽可以将4-羟基丁酰基-CoA转化为4-羟基丁醛。在一个实施方式中，所述多肽相对于亲代多肽具有更高的活力。在一个实施方式中，所述多肽对3-羟基-(R)-丁酰基-CoA的活力高于3-羟基-(S)-丁酰基-CoA。在一个实施方式中，所述多肽对3-羟基丁酰基-CoA的特异性高于乙酰-CoA。在一个实施方式中，所述多肽对4-羟基丁酰基-CoA的特异性高于乙酰-CoA。在一个实施方式中，所述多肽在细胞或细胞提取物中产生减少的副产品。在具体的实施方式中，所述副产品是乙醇或4-羟基-2-丁酮。在一个实施方式中，所述多肽相对于亲代多肽具有更高的kcat。
在一些实施方式中，本发明提供了具有本文所公开的氨基酸序列，如SEQ ID NO：1、2或3或表4中所提及的那些的分离的多肽，其中所述氨基酸序列包含如表1、2和/或3中所示的一个或多个变体氨基酸位置。具体地，这种多肽编码醛脱氢酶，其可以将酰基-CoA转化为相应的醛，例如，将3-羟基丁酰基-CoA转化为3-羟基丁醛或者4-羟基丁酰基-CoA转化为4-羟基丁醛。在一些方面，除如表1、2和/或3中所示的一个或多个变体氨基酸位置外，本发明的分离的多肽包含与如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性或者是相同的氨基酸序列。应理解变体氨基酸位置可以包括20种天然存在的氨基酸的任一种、在变体氨基酸位置的相应位置的野生型或亲代序列的保守替换或者在表1、2和/或3中如本文所公开的那些的变体氨基酸位置处的特异性氨基酸。还将理解任何变体氨基酸位置可以组合以产生其它变体。具有两个或更多个变体氨基酸位置的组合的变体显示出大于野生型的活力。因此，如本文举例说明的，通过组合活性变体氨基酸位置来产生酶变体导致产生了具有改善的性质的酶变体。使用本领域技术人员熟知的产生具有所期望的性质的多肽的方法，本领域技术人员可以容易地产生具有单一变体位置或者变体位置组合的多肽，所述所期望的性质包括提高的活力、对于3-羟基丁酰基-CoA或3-羟基丁醛的R形式比S形式高的特异性，对于3-羟基丁酰基-CoA和/或4-羟基丁酰基-CoA比乙酰-CoA高的特异性，减少的副产品形成，如乙醇或4-羟基-2-丁酮，提高的kcat，提高的体内和/或体外稳定性等，如本文所述。
“同源性”或“同一性”或“相似性”是指两条多肽之间或者两个核酸分子之间的序列相似性。可以通过比较每条序列中的位置来确定同源性，其可以出于比较的目的进行比对。当所比较的序列中的位置被相同碱基或氨基酸占据时，则所述分子在该位置是同一的。序列之间的同源性程度是所述序列所共有的匹配或同源位置数目的函数。多肽或多肽区域(或者多核苷酸或多核苷酸区域)与另一条序列具有特定百分比(例如，65％、70％、75％、80％、85％、90％、95％、98％或99％)的“序列同一性”表示当比对时，氨基酸(或核苷酸碱基)的百分比在比较两条序列中是相同的。
在某些实施方式中，本发明提供了具有包括处于本文所公开的任意组合的至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个变体的氨基酸序列的分离的多肽。所述变体可以包括表1、2和/或3中所述的变体的任意组合。在一些实施方式中，所述分离的多肽是参考多肽的变体，其中所述参考多肽具有SEQ ID NO：1、2或3的氨基酸序列或者表4中的那些氨基酸序列，并且所述多肽变体选自表1-3并且相对于SEQ ID NO：1、2或3或者表4中的那些具有一个或多个氨基酸替换。
在一些实施方式中，所述分离的多肽是参考多肽的变体，其中所述参考多肽具有SEQ ID NO：1、2或3的氨基酸序列或者表4中的那些氨基酸序列，所述多肽变体包含相对于SEQ ID NO：1、2或3或者表4中的那些的一个或多个氨基酸替换，其中所述一个或多个氨基酸替换选自表1-3，并且所述多肽变体与选自表1-3的相应变体具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性。所述一个或多个氨基酸替换可以选自表1-3中所列变体中的任一个或者选自表1-3中所列的两个或更多个变体的任意组合。当从表1-3中的单一变体选择时，所产生的变体可以包含处于任意组合的所选变体的一个或多个替换，包括所有所指明的替换或者不到所有所指明的替换。当从表1-3中的两个或更多个变体中选择替换时，所产生的变体可以包含所选变体的一个或多个替换，包含来自两个或更多个所选变体中的每一个的处于任意组合的所有所指明的替换或者不到所有所指明的替换。例如，所产生的变体可以包含来自表1-3中的单一变体的1、2、3或4个替换。作为另一个实例，所产生的变体可以包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、20、25或更多个替换，其选自表1-3的1、2、3、4、5或更多个所选变体，包括多至全部替换的位置，如本文所公开的。在一些实施方式中，所产生的变体包括表1-3中的所选变体的全部所指明的替换。在一些实施方式中，所产生的变体与SEQ ID NO：1、2或3或者表4中的那些相差至少一个氨基酸替换，但是小于25、20、10、5、4或3个氨基酸替换。在一些实施方式中，所产生的变体包含如选自表1-3的变体所指明的序列，基本由其组成或由其组成，从而仅与SEQ ID NO：1、2或3或者表4中的那些在所指明的氨基酸替换处不同。
在一些实施方式中，所产生的变体与选自表1-3的相应变体具有至少80％、85％、90％或95％的序列同一性；在一些情况下，同一性为至少90％或以上。在其中所产生的变体与选自表1-3的相应变体的同一性小于100％的情况下，可以改变对相应变体所指明的一个或多个氨基酸替换的位置(例如，在一个或多个氨基酸的插入或缺失的情况下)，但所述位置仍包含在所产生的变体内。例如，可以存在对应于“D12A”的甘氨酸向谷氨酸的替换(相对于SEQ ID NO：1或2，在位置12)，但是在所产生的变体的不同位置。如上所述并且还如本领域中熟知的，可以通过序列对比确定氨基酸是否对应于所指明的替换，虽然处于不同的位置。在一些实施方式中，如果当以缺省参数使用BLASTP比对算法时，要比较的多肽序列在沿相应变体序列所指明的位置与具有相同匹配或类似氨基酸的相应变体比对，其中类似的氨基酸是使用比对算法的缺省参数具有足以用于与所关心的变体位置比对的化学性质的氨基酸，则通过选自表1-3的相应变体所指明的一个或多个氨基酸替换(例如，所有或不到所有氨基酸替换)被认为存在于给定变体中，即使存在于沿多肽链的不同物理位置。
所述变体单独或组合可以产生相对于参考多肽，例如，野生型(天然)酶保留或改善了活力的酶。在一些方面，本发明的多肽可以具有表1、2和/或3中所述的变体的任意组合。在一些方面，具有表1、2和/或3中所述的变体的任意组合的本发明的多肽可以将酰基-CoA转化为相应的醛，例如，将3-羟基丁酰基-CoA转化为3-羟基丁醛或者将4-羟基丁酰基-CoA转化为4-羟基丁醛。产生和测定这些多肽的方法对于本领域技术人员是熟知的。
在一些实施方式中，本发明的分离的多肽还可以包括1至100个氨基酸位置，或者作为另外一种选择2至100个氨基酸位置，或者作为另外一种选择3至100个氨基酸位置，或者作为另外一种选择4至100个氨基酸位置，或者作为另外一种选择5至100个氨基酸位置，或者作为另外一种选择6至100个氨基酸位置，或者作为另外一种选择7至100个氨基酸位置，或者作为另外一种选择8至100个氨基酸位置，或者作为另外一种选择9至100个氨基酸位置，或者作为另外一种选择10至100个氨基酸位置，或者作为另外一种选择15至100个氨基酸位置，或者作为另外一种选择20至100个氨基酸位置，或者作为另外一种选择30至100个氨基酸位置，或者作为另外一种选择40至100个氨基酸位置，或者作为另外一种选择50至100个氨基酸位置或者其中任何整数的保守氨基酸替换，其中所述位置是除表1、2和/或3中所述的变体氨基酸位置以外的。在一些方面，所述保守氨基酸序列是化学保守的或者进化保守的氨基酸替换。鉴别保守氨基酸的方法对于本领域技术人员是熟知的，其中任一种方法可以用于产生本发明的分离的多肽。
在一些实施方式中，相较于亲代(野生型)序列，本发明的分离的多肽可以在2至300个氨基酸位置，或作为另外一种选择3至300个氨基酸位置，或者作为另外一种选择4至300个氨基酸位置，或者作为另外一种选择5至300个氨基酸位置，或者作为另外一种选择10至300个氨基酸位置，或者作为另外一种选择20至300个氨基酸位置，或者作为另外一种选择30至300个氨基酸位置，或者作为另外一种选择40至300个氨基酸位置，或者作为另外一种选择50至300个氨基酸位置，或者作为另外一种选择60至300个氨基酸位置，或者作为另外一种选择80至300个氨基酸位置，或者作为另外一种选择100至300个氨基酸位置，或者作为另外一种选择150至300个氨基酸位置，或者作为另外一种选择200至300个氨基酸位置，或者作为另外一种选择250到300个氨基酸位置，或者其中任何整数处不包括修饰，其中所述位置选自在如SEQ ID NO：1、2或3或表4中所提及的氨基酸序列的2、3、4或5条之间相同的那些。
应理解如本文所公开的，变体多肽，如醛脱氢酶的多肽变体可以实施与亲代多肽类似的酶促反应，例如，将酰基-CoA转化为相应的醛，如将3-羟基丁酰基-CoA转化为3-羟基丁醛或者将4-羟基丁酰基-CoA转化为4-羟基丁醛。还将理解醛脱氢酶的多肽变体可以包括为多肽提供有益特征的变体，其包括(但不限于)提高的活力、对于3-羟基丁酰基-CoA或3-羟基丁醛的R形式比S形式高的特异性，对于3-羟基丁酰基-CoA和/或4-羟基丁酰基-CoA比乙酰-CoA高的特异性，减少的副产品形成，如乙醇或4-羟基-2-丁酮，提高的kcat，提高的体内和/或体外稳定性等(参见实施例)。在具体的实施方式中，所述醛脱氢酶变体可以显示出与野生型或亲代多肽至少相同或更高的活力，即高于无变体氨基酸位置的亲代多肽。例如，相对于野生型或亲代多肽，本发明的醛脱氢酶变体可以具有1.2、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10或甚至更高倍的变体多肽活力(参见实施例)。应理解活力是指在相同测定条件下，相对于野生型或亲代多肽，本发明的醛脱氢酶将底物转化为产品的能力。
在另一个具体的实施方式中，所述醛脱氢酶变体可以显示出对3-羟基丁酰基-CoA或3-羟基丁醛的R形式相较于其S形式的提高的特异性，例如，约2至40倍更高，例如，2至35，2至30，2至25，2至20，2至15，2至10或2至5，例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25、30、35、40或甚至更高倍的活力。可以(例如)通过3-羟基丁酰基-CoA或3-羟基丁醛的R相对于S形式的活力比来测量这种提高的特异性。
在另一个具体的实施方式中，所述醛脱氢酶变体可以显示出对于3-羟基丁酰基-CoA和/或4-羟基丁酰基-CoA高于乙酰-CoA的提高的特异性，例如，1.5至100，1.5至95，1.5至90，1.5至85，1.5至80，1.5至75，1.5至70，1.5至65，1.5至60，1.5至55，1.5至50，1.5至45，1.5至40，1.5至35，1.5至30，1.5至25，1.5至20，1.5至15，1.5至10或者1.5至5，例如，2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100-倍。可以(例如)通过3-羟基丁酰基-CoA或4-羟基丁酰基-CoA相对于乙酰-CoA的活力比来测量这种提高的特异性。通过对3HB-CoA或4HB-CoA的活力除以对乙酰-CoA的活力来表示特异性。
在另一个具体的实施方式中，所述醛脱氢酶变体可以显示出减少的副产品形成，如乙醇和/或4-羟基-2-丁酮，例如，10％、15％、20％、25％、30％、35％、40％、45％、50％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％的副产品形成减少。如上所述，这种醛脱氢酶变体可以显示出相对于野生型或亲代多肽，即无变体氨基酸位置的亲代多肽，具有减少的副产品形成的活性。
在另一个具体的实施方式中，相对于野生型或亲代多肽，即无变体氨基酸位置的亲代多肽，所述醛脱氢酶变体可以显示出提高的kcat，例如，1.25、1.5、1.75、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10-倍或以上。将理解kcat表示其在酶学中熟知的转换数的含义，其中kcat＝Vmax/[ET]，其中Vmax是具有饱和底物的酶反应速率，并且[ET]是总酶浓度(参见Segel,Enzyme Kinetics:Behavior and Analysis of RapidEquilibrium and Steady-State Enzyme Kinetics,Wiley-Interscience,New York(1975))。这种醛脱氢酶变体可以显示出相对于野生型或亲代多肽，即无变体氨基酸位置的亲代多肽具有提高的kcat的活力。
在另一个具体的实施方式中，所述醛脱氢酶变体可以相对于野生型或亲代多肽，即无变体氨基酸位置的亲代多肽，显示出提高的体外或体内稳定性或者两者。例如，所述醛脱氢酶变体可以显示出在细胞裂解液中提高的体外稳定性。
应理解，在某些实施方式中，醛脱氢酶变体可以显示出处于任意组合的如上所述的两种或更多种特征，例如，两种或更多种以下特征：(1)提高的活力，(2)对3-羟基丁酰基-CoA或3-羟基丁醛的R形式高于S形式的提高的特异性，(3)对于3-羟基丁酰基-CoA和/或4-羟基丁酰基-CoA高于乙酰-CoA的提高的特异性，(4)减少的副产品形成，如乙醇和/或4-羟基-2-丁酮，(5)提高的kcat，(6)提高的体内和/或体外稳定性等。这些组合包括(例如)特征1和2；1和3；1和4；1和5；1和6；2和3；2和4；2和5；2和6；3和4；3和5；3和6；4和5；4和6；5和6；1、2和3；1、2和4；1、2和5；1、2和6；1、3和4；1、3和5；1、3和6；1、4和5；1、4和6；1、5和6；2、3和4；2、3和5；2、3和6；2、4和5；2、4和6；2、5和6；3、4和5；3、4和6；3、5和6；4、5和6；1、2、3和4；1、2、3和5；1、2、3和6；1、2、4和5；1、2、4和6；1、2、5和6；1、3、4和5；1、3、4和6；1、3、5和6；1、4、5和6；2、3、4和5；2、3、4和6；2、3、5和6；3、4、5和6；1、2、3、4和5；1、3、4、5和6；1、2、4、5和6；1、2、3、5和6；1、2、3、4和6；2、3、4、5和6；1、2、3、4、5和6。
可以通过本领域熟知的多种方法分离本发明的多肽，例如，重组表达系统、沉淀、凝胶过滤、离子交换、反相和亲和色谱法等。其它熟知的方法描述于Deutscher等人，Guideto Protein Purification:Methods in Enzymology,182卷,(Academic Press,(1990))。作为另外一种选择，可以使用熟知的重组方法获得本发明的分离的多肽(参见，例如，Sambrook等人，如上,1989；Ausubel等人,如上,1999)。本领域技术人员可以选择用于本发明的多肽的生化纯化的方法和条件，并且(例如)通过功能测定来监测纯化。
用于制备本发明的多肽的方法的一种非限制性实例是使用本领域中熟知的方法，在适合的宿主细胞，如细菌细胞、酵母细胞或者其它适合的细胞中表达编码所述多肽的核酸，并且如本文所述，再次使用熟知的纯化方法回收所表达的多肽。可以直接从已用如本文所述的表达载体转化的细胞分离本发明的多肽。重组表达的本发明的多肽还可以作为具有适当亲合标签，如谷胱甘肽S转移酶(GST)、多聚组氨酸、抗生蛋白链菌素等的融合蛋白表达，并且如果需要，可以亲合纯化。如果需要，本发明的多肽可以保留亲合标签，或者任选地，可以使用除去亲合标签所熟知的方法从多肽上除去亲合标签，例如，使用适当的酶促或化学切割。因此，本发明提供了不具有或任选地具有亲合标签的本发明的多肽。在一些实施方式中，本发明提供了表达本文所公开的本发明的多肽的宿主细胞。还可以使用本领域技术人员熟知的多肽合成方法，通过化学合成产生本发明的多肽(Merrifield,J.Am.Chem.Soc.85:2149(1964)；Bodansky,M.,Principles of Peptide Synthesis(Springer-Verlag,1984)；Houghten,Proc.Natl.Acad.Sci.,USA 82:5131(1985)；GrantSynthetic Peptides:A User Guide.W.H.Freeman and Co.,N.Y.(1992)；Bodansky M andTrost B主编,Principles of Peptide Synthesis.Springer-Verlag Inc.,NY(1993))。
在一些实施方式中，本发明提供了使用本文所公开的多肽作为生物催化剂。如本文所使用的“生物催化剂”是指引起或改变化学反应速率的生物物质。生物催化剂可以是酶。如本文所公开的，本发明的多肽可以用于提高底物向产品转化的速率。在工业反应的背景中，在不存在表达所述多肽的宿主细胞的情况下，例如，使用体外方法，本发明的多肽可以用于改善产生3-HBal、1,3-BDO、4-HBal或1,4-BDO，或者与之相关的下游产品，如其酯或酰胺的反应。在一个实施方式中，本发明提供了本发明的多肽作为生物催化剂的使用。
在本发明的一些实施方式中，作为表达醛脱氢酶的细胞的细胞裂解液提供了编码本发明的醛脱氢酶的多肽。在这种情况下，细胞裂解液用作醛脱氢酶的来源以用于在体外反应中实施3-羟基丁酰基-CoA向3-羟基丁醛或者4-羟基丁酰基-CoA向4-羟基丁醛的转化或者逆反应。在另一个实施方式中，可以以部分纯化的形式，例如，从细胞裂解液部分纯化的形式提供醛脱氢酶。在另一个实施方式中，可以以基本纯化的形式提供醛脱氢酶，其中所述醛脱氢酶与其它组分，如细胞提取物的组分基本纯化。用于部分纯化或基本纯化编码醛脱氢酶的多肽的方法在本领域中是熟知的，如本文所述。在一些实施方式中，将醛脱氢酶固定至固体载体，例如，珠、板或膜。在具体的实施方式中，所述醛脱氢酶包含亲合标签，并且所述亲合标签用于将醛脱氢酶固定至固体载体。如本文所述，这种亲合标签可以包括(但不限于)谷胱甘肽S转移酶(GST)、多聚组氨酸、抗生蛋白链菌素等。
在一些实施方式中，本发明提供了具有本文所公开的多肽和所述多肽的至少一种底物的组合物。本文描述了并且在附图中举例说明了本文所公开的每一种多肽的底物。本发明所述的组合物内的多肽可以与底物在体外或体内条件下反应。在该背景中，体外条件是指在不存在细胞的情况下或在细胞(包括本发明的细胞)外的反应。
在一个实施方式中，本发明提供了包含本发明的多肽和所述多肽的至少一种底物的组合物。在一个实施方式中，所述多肽可以与底物在体外条件下反应。在一个实施方式中，所述底物是3-羟基丁酰基-CoA。在一个实施方式中，所述底物是3-羟基-(R)-丁酰基-CoA。在一个实施方式中，所述底物是4-羟基丁酰基-CoA。
在一些实施方式中，本发明提供了构建宿主株的方法，其与其它步骤一起可以包括将本文所公开的载体引入(例如)能够表达所述载体所编码的氨基酸序列和/或能够发酵的宿主细胞中。可以使用在本领域中熟知的技术将本发明所述的载体稳定或瞬时引入到宿主细胞中，所述技术包括(但不限于)缀合、电穿孔、化学转化、转导、转染和超声转化。本文公开了其它方法，所述方法中的任一种可以在本发明所述的方法中使用。
在其它实施方式中，本发明提供了包含本发明的多肽，即本发明的醛脱氢酶的细胞。因此，本发明提供了包含编码本发明的醛脱氢酶的多肽的非天然存在的细胞。任选地，所述细胞可以包含3-HBal或1,3-BDO途径，或者4-HBal或1,4-BDO途径，并且另外任选地包含产生与之相关的下游产品，如其酯或酰胺的途径。在一些实施方式中，所述非天然存在的细胞包含至少一种编码将酰基-CoA转化为其相应的醛的醛脱氢酶的外源核酸。本领域技术人员将理解这些仅是示例性的，并且本领域技术人员基于本文的教导内容可以容易地确定本文所公开的任何底物-产品对中适合于产生所期望的产品的、并且对其来说底物向产品的转化有可用的适当的活力。因此，在具体的实施方式中，本发明提供了含有至少一种编码醛脱氢酶的外源核酸的细胞，具体而言非天然存在的细胞，其中所述醛脱氢酶在3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中起作用，如图1和2中所示。
在一个实施方式中，本发明提供了包含含有本发明的核酸的本发明的载体的细胞。本发明还提供了包含本发明的核酸的细胞。在一个实施方式中，将所述核酸分子整合到细胞染色体中。在具体的实施方式中，所述整合是位点-特异的。在本发明的一个实施方式中，表达所述核酸分子。在一个实施方式中，本发明提供了包含本发明的多肽的细胞。
在一个实施方式中，包含载体、核酸或多肽的细胞是微生物。在具体的实施方式中，所述微生物是细菌、酵母或真菌。在具体的实施方式中，所述细胞是分离的真核细胞。
在一个实施方式中，所述细胞包含产生3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或其酯或酰胺的途径。在另一个实施方式中，所述细胞包含产生4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)或其酯或酰胺的途径。在一个实施方式中，所述细胞能够发酵。在一个实施方式中，所述细胞还包含在所述细胞中表达的本发明的多肽的至少一种底物。在具体的实施方式中，所述底物是3-羟基丁酰基-CoA。在具体的实施方式中，所述底物是3-羟基-(R)-丁酰基-CoA。在一个实施方式中，所述细胞对3-羟基-(R)-丁酰基-CoA的活力高于3-羟基-(S)-丁酰基-CoA。在另一个具体的实施方式中，所述底物是4-羟基丁酰基-CoA。本发明还提供了包含本发明的细胞的培养基。
可以在将酰基-CoA转化为其相应的醛的途径中使用本发明的醛脱氢酶。已在(例如)WO 2010/127319、WO 2013/036764、美国专利No.9,017,983、US 2013/0066035中描述了包含醛脱氢酶的3-HBal和/或1,3-BDO的示例性途径，所述每篇专利作为参考并入本文。
在图1中显示并且在WO 2010/127319、WO 2013/036764、美国专利No.9,017,983和US 2013/0066035中描述了示例性3-HBal和/或1,3-BDO途径。包含醛脱氢酶的这些3-HBal和/或1,3-BDO途径包括(例如)(G)乙酰乙酰-CoA还原酶(酮还原)；(H)3-羟基丁酰基-CoA还原酶(醛形成)，在本文中也称为3-羟基丁醛脱氢酶、醛脱氢酶(ALD)；和(C)3-羟基丁醛还原酶，在本文中也称为1,3-BDO脱氢酶(参见图1)。可以通过使用硫解酶将两分子的乙酰-CoA转化为一分子的乙酰乙酰-CoA来形成乙酰乙酰-CoA。乙酰乙酰COA硫解酶将两分子的乙酰-CoA转化为一分子的乙酰乙酰-CoA和一分子的CoA(参见WO 2013/036764和US 2013/0066035)。
在WO 2010/127319的图2中显示了示例性的1,3-BDO途径。简要地，可以通过乙酰乙酰-CoA还原酶(酮还原)(EC 1.1.1.a)将乙酰乙酰-CoA转化为3-羟基丁酰基-CoA(图1的步骤G)。可以通过3-羟基丁酰基-CoA还原酶(醛形成)(EC 1.2.1.b)，在本文中也称为3-羟基丁醛脱氢酶，包括本发明的醛脱氢酶将3-羟基丁酰基-CoA转化为3-羟基丁醛(图1的步骤H)。可以通过3-羟基丁醛还原酶(EC 1.1.1.a)，在本文中也称为1,3-BDO脱氢酶将3-羟基丁醛转化为1,3-丁二醇(图1的步骤C)。
如本文所公开的，本发明的醛脱氢酶可以在途径中起作用以将3-羟基丁酰基-CoA转化为3-羟基丁醛。在如上所述的包含将3-羟基丁酰基-CoA转化为3-羟基丁醛的醛脱氢酶的途径中，所述途径将乙酰乙酰-CoA转化为3-羟基丁酰基-CoA(参见图1)。本发明的醛脱氢酶还可以用于在所述途径中包含3-羟基丁酰基-CoA作为底物/产品的其它3-HBal和/或1,3-BDO途径中。本领域技术人员可以在包含这种反应的任何所期望的途径中容易地使用本发明的醛脱氢酶将3-羟基丁酰基-CoA转化为3-羟基丁醛。
在图2中显示并且在WO 2008/115840、WO 2010/030711、WO 2010/141920、WO2011/047101、WO 2013/184602、WO 2014/176514、美国专利No.8,067,214、美国专利No.7,858,350、美国专利No.8,129,169、美国专利No.8,377,666、US 2013/0029381、US2014/0030779、US 2015/0148513和US 2014/0371417中描述了示例性的4-HBal和/或1,4-BDO途径。包含醛脱氢酶的这种4-HBal和/或1,4-BDO途径包括(例如)(1)丁二酰-CoA合成酶；(2)不依赖于CoA的琥珀酸半醛脱氢酶；(3)α-酮戊二醛脱氢酶；(4)谷氨酸：琥珀酸半醛转氨酶；(5)谷氨酸脱羧酶；(6)CoA-依赖性琥珀酸半醛脱氢酶；(7)4-羟基丁酸酯脱氢酶；(8)α-酮戊二酸脱羧酶；(9)4-羟基丁酰基CoA：乙酰-CoA转移酶；(10)丁酸酯激酶(也称为4-羟基丁酸酯激酶)；(11)磷酸转丁酰基酶(也称为磷酸-反式-4-羟基丁酰酶)；(12)醛脱氢酶(也称为4-羟基丁酰基-CoA还原酶)；(13)乙醇脱氢酶，如1,4-丁二醇脱氢酶(也称为4-羟基丁醛还原酶或4-羟基丁醛还原酶)(参见图2)。
与图2类似，在WO 2010/141920的图8A中显示了示例性的1,4-BDO途径。简要地，可以通过丁二酰-CoA还原酶(或者琥珀酸半醛脱氢酶)(EC 1.2.1.b)将丁二酰-CoA转化为琥珀酸半醛。可以通过4-羟基丁酸酯脱氢酶(EC 1.1.1.a)将琥珀酸半醛转化为4-羟基丁酸酯。作为另外一种选择，可以通过丁二酰-CoA还原酶(醇形成)(EC 1.1.1.c)将丁二酰-CoA转化为4-羟基丁酸酯。可以通过4-羟基丁酰基-CoA转移酶(EC 2.8.3.a)，通过4-羟基丁酰基-CoA水解酶(EC 3.1.2.a)或者通过4-羟基丁酰基-CoA连接酶(或者4-羟基丁酰基-CoA合成酶)(EC 6.2.1.a)将4-羟基丁酸酯转化为4-羟基丁酰基-CoA。作为另外一种选择，可以通过4-羟基丁酸酯激酶(EC 2.7.2.a)将4-羟基丁酸酯转化为4-羟基丁酰基-磷酸酯。可以通过磷酸转-4-羟基丁酰酶(EC 2.3.1.a)将4-羟基丁酰基-磷酸酯转化为4-羟基丁酰基-CoA。作为另外一种选择，可以通过4-羟基丁醛脱氢酶(磷酸化)(EC 1.2.1.d)将4-羟基丁酰基-磷酸酯转化为4-羟基丁醛。可以通过4-羟基丁酰基-CoA还原酶(或者4-羟基丁醛脱氢酶)(EC 1.2.1.b)，包括通过本发明的醛脱氢酶变体，将4-羟基丁酰基-CoA转化为4-羟基丁醛。作为另外一种选择，可以通过4-羟基丁酰基-CoA还原酶(醇形成)(EC 1.1.1.c)将4-羟基丁酰基-CoA转化为1,4-丁二醇。可以通过1,4-丁二醇脱氢酶(EC 1.1.1.a)将4-羟基丁醛转化为1,4-丁二醇。
WO 2010/141920的图8B也显示了示例性的1,4-BDO途径。简要地，可以通过α-酮戊二酸脱羧酶(EC 4.1.1.a)将α-酮戊二酸转化为琥珀酸半醛。作为另外一种选择，可以通过谷氨酸脱氢酶(EC 1.4.1.a)将α-酮戊二酸转化为谷氨酸。可以通过4-氨基丁酸酯氧化还原酶(脱氨基)(EC 1.4.1.a)或氨基丁酸酯转氨酶(EC 2.6.1.a)将4-氨基丁酸酯转化为琥珀酸半醛。可以通过谷氨酸脱羧酶(EC 4.1.1.a)将谷氨酸转化为4-氨基丁酸酯。可以通过4-羟基丁酸酯脱氢酶(EC 1.1.1.a)将琥珀酸半醛转化为4-羟基丁酸酯。可以通过4-羟基丁酰基-CoA转移酶(EC 2.8.3.a)，通过4-羟基丁酰基-CoA水解酶(EC 3.1.2.a)或者通过4-羟基丁酰基-CoA连接酶(或者4-羟基丁酰基-CoA合成酶)(EC 6.2.1.a)将4-羟基丁酸酯转化为4-羟基丁酰基-CoA。可以通过4-羟基丁酸酯激酶(EC 2.7.2.a)将4-羟基丁酸酯转化为4-羟基丁酰基-磷酸酯。可以通过磷酸转-4-羟基丁酰酶(EC 2.3.1.a)将4-羟基丁酰基-磷酸酯转化为4-羟基丁酰基-CoA。作为另外一种选择，可以通过4-羟基丁醛脱氢酶(磷酸化)(EC1.2.1.d)将4-羟基丁酰基-磷酸酯转化为4-羟基丁醛。可以通过4-羟基丁酰基-CoA还原酶(或者4-羟基丁醛脱氢酶)(EC1.2.1.b)，包括通过本发明的醛脱氢酶，将4-羟基丁酰基-CoA转化为4-羟基丁醛。可以通过4-羟基丁酰基-CoA还原酶(醇形成)(EC 1.1.1.c)将4-羟基丁酰基-CoA转化为1,4-丁二醇。可以通过1,4-丁二醇脱氢酶(EC 1.1.1.a)将4-羟基丁醛转化为1,4-丁二醇。
如本文所公开的，本发明的醛脱氢酶可以在途径中起作用以将4-羟基丁酰基-CoA转化为4-羟基丁醛。在包含将4-羟基丁酰基-CoA转化为4-羟基丁醛的醛脱氢酶的如上所述的途径中，所述途径将4-羟基丁酸酯转化为4-羟基丁酰基-CoA或者将4-羟基丁酰基磷酸酯转化为4-羟基丁酰基-CoA(参见图2)。本发明的醛脱氢酶还可以用于在所述途径中包含4-羟基丁酰基-CoA作为底物/产品的其它4-HBal和/或1,4-BDO途径中。本领域技术人员可以在包含这种反应的任何所期望的途径中容易地使用本发明的醛脱氢酶将4-羟基丁酰基-CoA转化为4-羟基丁醛。例如，如WO 2010/141290，图9A中所述和所示，可以将4-氧丁酰基-CoA转化为4-羟基丁酰基-CoA。另外，如WO 2010/141290，图10和11所述和所示，可以将5-羟基-2-氧戊酸转化为4-羟基丁酰基-CoA。另外，如WO 2010/141290，图12所述和所示，可以将乙酰乙酰-CoA、3-羟基丁酰基-CoA、巴豆酰-CoA和/或乙烯乙酰-CoA转化为4-羟基丁酰基-CoA。另外，如WO 2010/141290，图13中所述和所示，可以将4-羟基丁-2-烯酰基-CoA转化为4-羟基丁酰基-CoA。因此，本领域技术人员将容易地理解如何根据需要，在包含4-羟基丁酰基-CoA向4-羟基丁醛的转化的4-HBal和/或1,4-BDO途径中使用本发明的醛脱氢酶。
通过代表性的酶学委员会(EC)编号，以上显示了将常规中间代谢中间产品转化为1,3-BDO或1,4-BDO所需的酶的种类(还参见WO 2010/127319、WO 2013/036764、WO 2008/115840、WO 2010/030711、WO 2010/141920、WO 2011/047101、WO 2013/184602、WO 2014/176514、美国专利No.9,017,983、美国专利No.8,067,214、美国专利No.7,858,350、美国专利No.8,129,169、美国专利No.8,377,666、US 2013/0066035、US 2013/0029381、US 2014/0030779、US 2015/0148513和US 2014/0371417)。每个标签的前三个数字对应于前三个酶学委员会数字，其表示与底物特异性无关的一般转化类型。示例性的酶包括：1.1.1.a，氧化还原酶(酮向羟基或醛向醇)；1.1.1.c，氧化还原酶(2步，酰基-CoA向醇)；1.2.1.b，氧化还原酶(酰基-CoA向醛)；1.2.1.c，氧化还原酶(2-含氧酸向酰基-CoA，脱羧作用)；1.2.1.d，氧化还原酶(磷酸化/去磷酸化)；1.3.1.a，对CH-CH供体起作用的氧化还原酶；1.4.1.a，对氨基酸起作用的氧化还原酶(脱氨基)；2.3.1.a，酰基转移酶(转移磷酸基)；2.6.1.a，转氨酶；2.7.2.a，磷酸转移酶，羧基受体；2.8.3.a，辅酶-A转移酶；3.1.2.a，硫酯水解酶(辅酶A特异的)；4.1.1.a，羰基裂解酶；4.2.1.a，水解酶；4.3.1.a，解氨酶；5.3.3.a，异构酶；5.4.3.a，氨基变位酶；和6.2.1.a，酸-硫醇连接酶。
可以在细胞中或体外使用本发明的醛脱氢酶来将酰基-CoA转化为其相应的醛。如本文所公开的，本发明的醛脱氢酶具有有益和有用的性质，其包括(但不限于)对3-羟基丁酰基-CoA的R对映异构体高于S对映异构体的提高的特异性，对于3-羟基丁酰基-CoA和/或4-羟基丁酰基-CoA相较于乙酰-CoA的提高的特异性、提高的活力、减少的副产品产生、提高的kcat等。可以通过使用1,3-丁二醇脱氢酶将本发明的醛脱氢酶的产品，3-羟基-(R)-丁醛酶促转化为(R)-1,3-丁二醇，将本发明的醛脱氢酶用于产生1,3-丁二醇的R-形式(也称为(R)-1,3-丁二醇)。
1,3-丁二醇的生物来源的R-形式可以用于产生下游产品，对所述下游产品来说，R-形式是优选的。在一些实施方式中，所述R-形式可以用作药物和/或营养制剂(参见WO2014/190251)。例如，(R)-1,3-丁二醇可以用于产生(3R)-羟丁基(3R)-羟基丁酸酯，其可以具有有益效果，如提高血液中的酮体水平。提高酮体水平可以导致产生多种临床益处，包括身体和认知表现的增强以及心血管病况、糖尿病的治疗和线粒体功能障碍病症的治疗和肌肉疲劳和损伤的治疗(参见WO 2014/190251)。1,3-丁二醇的生物来源的R-形式可以用于生产下游产品，其中非石油基产品是所期望的，例如，通过用生物来源的R-形式替代石油-来源的外消旋物1,3-丁二醇，其S-形式或其R-形式。
在一个实施方式中，本发明提供了对所述化合物的R-形式对映体富集的3-HBal或1,3-BDO或与之有关的下游产品，如其酯或酰胺。在一些实施方式中，3-HBal或1,3-BDO是R-对映异构体富集的外消旋物，即与S-对映异构体相比，包括更多的R-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含55％或以上的R-对映异构体和45％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含60％或以上的R-对映异构体和40％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含65％或以上的R-对映异构体和35％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含70％或以上的R-对映异构体和30％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含75％或以上的R-对映异构体和25％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含80％或以上的R-对映异构体和20％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含85％或以上的R-对映异构体和15％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含90％或以上的R-对映异构体和10％或以下的S-对映异构体。例如，所述3-HBal或1,3-BDO外消旋物可以包含95％或以上的R-对映异构体和5％或以下的S-对映异构体。在一些实施方式中，所述3-HBal或1,3-BDO或与之有关的下游产品，如其酯或酰胺是大于90％的R形式，例如，大于95％、96％、97％、98％、99％或99.9％的R形式。在一个实施方式中，所述3-HBal和/或1,3-BDO或者与之有关的下游产品，如其酯或酰胺是≥55％的R-对映异构体，≥60％的R-对映异构体，≥65％的R-对映异构体，≥70％的R-对映异构体，≥75％的R-对映异构体，≥80％的R-对映异构体，≥85％的R-对映异构体，≥90％的R-对映异构体，或者≥95％的R-对映异构体，并且可以是高化学纯的，例如，≥99％，例如，≥95％，≥96％，≥97％，≥98％，≥99％，≥99.1％，≥99.2％，≥99.3％，≥99.4％，≥99.5％，≥99.6％，≥99.7％，≥99.8％或≥99.9％的R-对映异构体。
在一个实施方式中，将石油-来源的3-HBal和/或1,3-BDO前体的外消旋混合物，具体地3-羟基丁酰基-CoA的外消旋混合物用作本发明的醛脱氢酶的底物，其对R形式显示出优于S形式的提高的特异性，以产生对于R形式对映体富集的3-HBal或1,3-BDO或与之有关的下游产品，如其酯或酰胺。可以通过将石油-来源的前体进料至表达本发明的醛脱氢酶的细胞，具体地可以将前体转化为3-羟基丁酰基-CoA的细胞，来实施该反应，或者可以体外使用一种或多种酶将石油-来源的前体转化为3-羟基丁酰基-CoA来体外实施该反应，或者实施体内和体外反应的组合。可以类似地通过将石油-来源的前体进料至表达本发明的醛脱氢酶的细胞，具体地可以将前体转化为4-羟基丁酰基-CoA的细胞来实施使用本发明的醛脱氢酶来生产4-羟基丁酰基-CoA的反应，或者可以体外使用一种或多种酶将石油-来源的前体转化为4-羟基丁酰基-CoA来体外实施所述反应，或者实施体内和体外反应的组合。
尽管本文通常作为含有包含本发明的醛脱氢酶的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的细胞进行描述，但是应理解本发明还提供了包含编码本发明的醛脱氢酶的至少一种外源核酸的细胞。可以以足以产生所期望的产品，如3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的产品或与之有关的下游产品，如其酯或酰胺的量表达醛脱氢酶。图1和2中显示了并且本文描述了示例性的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径。
应理解可以根据需要使用本文所公开的任何途径，如实施例中所述的和图中举例说明的，包括图1和2的途径来产生细胞，所述细胞产生了任何途径中间体或产品，具体地，利用本发明的醛脱氢酶的途径的中间体或产品。如本文所公开的，产生中间体的这种细胞可以与表达一种或多种上游或下游途径酶的另一种细胞组合使用来产生所期望的产品。然而，应理解可以使用产生3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体的细胞来产生作为所期望的产品的中间体。
一般地参考代谢反应、其反应物或产品，或特定地参考编码与参考代谢反应、反应物或产品有关或催化它们的酶或与之有关的蛋白的一种或多种核酸或基因，描述了本发明。除非在本文中另外明确说明，否则本领域技术人员将理解对反应的提及也构成了对反应的反应物和产品的提及。类似地，除非在本文中另外明确说明，否则对反应物或产品的提及也提及了所述反应，并且对任何这些代谢组分的提及也提及了编码催化所提及的反应、反应物或产品的酶或参与所提及的反应、反应物或产品的蛋白的基因。同样地，考虑到熟知的代谢生物化学、酶学和基因组领域，在本文中对基因或编码核酸的提及也构成了对相应编码的酶和它所催化的反应或与所述反应有关的蛋白以及所述反应的反应物和产品的提及。
如本文所公开的，产品和作为羧酸的途径中间体可以以多种电离形式存在，包括完全质子化、部分质子化和完全去除质子化的形式。因此，后缀“酯(-ate)”或酸形式可以互换使用以描述游离酸形式以及任何去除质子化形式两者，具体地基于已知电离形式基于其中所述化合物所存在的pH。应理解羧酸酯产品或中间体包括羧酸酯产品或途径中间体的酯的形式，如O-羧酸酯和S-羧酸酯。O-和S-羧酸酯可以包括低级烷基，即C1至C6，支链或直链羧酸酯。一些这些O-或S-羧酸酯无限制地包括甲基、乙基、正丙基、正丁基、异丙基、仲丁基和叔丁基、戊基、己基O-或S-羧酸酯，任何这些还可以具有不饱和度，从而(例如)提供丙烯基、丁烯基、戊基和己烯基O-或S-羧酸酯。O-羧酸酯可以是生物合成途径的产品。其它生物合成可达到的O-羧酸酯可以包括中至长链基团，即C7-C22，来源于脂肪醇的O-羧酸酯，如庚基、辛基、壬基、癸基、十一基、月桂基、十三基、十四基、十五基、十六基、棕榈酰基、十七基、硬脂酰基、十九基、二十烷醇基、二十一烷基和二十二醇，任何这些可以任选地是支链的和/或含有不饱和度。还可以通过生物化学或化学方法，如游离羧酸产品的酯化或者O-或S-羧酸酯的转酯化来实现O-羧酸酯。通过CoA S-酯、半胱氨酰基S-酯、烷基硫酯和多种芳基和杂芳基硫酯举例说明了S-羧酸酯。
可以通过引入编码本发明的醛脱氢酶的可以表达的核酸，和任选地编码参与一种或多种3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径的一种或多种酶或蛋白的可以表达的核酸，和另外任选地编码产生与3-HBal、1,3-BDO、4-HBal或1,4-BDO有关的下游产品，如其酯或酰胺的酶的核酸来产生本发明的细胞。基于所选的宿主细胞，可以表达具体的3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径或下游产品的一些或全部的核酸。例如，如果对于所期望的生物合成途径，所选宿主缺乏一种或多种酶或蛋白，则将所缺乏的酶或蛋白的可表达核酸引入宿主以用于后续外源表达。作为另外一种选择，如果所选宿主显示出一些途径基因的内源表达，但是缺乏其它基因的表达，则包括所缺乏的酶或蛋白的编码核酸以实现3-HBal、1,3-BDO、4-HBal或1,4-BDO的生物合成，或者如果需要，可以提供内源表达的基因的外源表达以提高途径酶的表达。因此，可以通过引入本发明的醛脱氢酶和任选地外源酶或蛋白活性以获得所期望的生物合成途径，或者通过引入一种或多种外源酶或蛋白活性，包括本发明的醛脱氢酶，与一种或多种内源酶或蛋白一起产生所期望的产品，如3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，从而产生本发明的细胞。
宿主细胞可以选自(例如)细菌、酵母、真菌或适用于或适合于发酵过程的任何多种微生物，并且表达本发明的醛脱氢酶的非天然的细胞可以在它们中产生。示例性的细菌包括选自下列的任何种：肠杆菌目(Enterobacteriales)肠杆菌科(Enterobacteriaceae)，包括埃希氏菌属(Escherichia)和克雷伯氏菌属(Klebsiella)；气单胞菌目(Aeromonadales)琥珀酸弧菌科(Succinivibrionaceae)，包括厌氧螺菌属(Anaerobiospirillum)；巴斯德氏菌目(Pasteurellales)巴斯德氏菌科(Pasteurellaceae)，包括放线杆菌属(Actinobacillus)和曼海姆氏菌属(Mannheimia)；根瘤菌目(Rhizobiales)慢生根瘤菌科(Bradyrhizobiaceae)，包括根瘤菌属(Rhizobium)；芽孢杆菌目(Bacillales)芽孢杆菌科(Bacillaceae)，包括芽孢杆菌属(Bacillus)；放线菌目(Actinomycetales)棒杆菌科(Corynebacteriaceae)和链霉菌科(Streptomycetaceae)，分别包括棒状杆菌属(Corynebacterium)和链霉菌属(Streptomyces)；红螺菌目(Rhodospirillales)醋杆菌科(Acetobacteraceae)，包括葡糖杆菌属(Gluconobacter)；鞘脂单胞菌目(Sphingomonadales)鞘脂单胞菌科(Sphingomonadaceae)，包括发酵单胞菌属(Zymomonas)；乳杆菌目(Lactobacillales)乳杆菌科(Lactobacillaceae)和链球菌科(Streptococcaceae)，分别包括乳杆菌属(Lactobacillus)和乳球菌属(Lactococcus)；梭菌目(Clostridiales)梭菌科(Clostridiaceae)梭菌属(Clostridium)；和假单胞菌目(Pseudomonadales)假单胞菌科(Pseudomonadaceae)，包括假单胞菌属(Pseudomonas)。宿主细菌的非限制性种包括大肠杆菌(Escherichia coli)、产酸克雷伯氏菌(Klebsiellaoxytoca)、产琥珀酸厌氧螺菌(Anaerobiospirillum succiniciproducens)、产琥珀酸放线杆菌(Actinobacillus succinogenes)、产琥珀酸曼海姆氏菌(Mannheimiasucciniciproducens)、菜豆根瘤菌(Rhizobium etli)、枯草芽孢杆菌(Bacillussubtilis)、乳发酵短杆菌(Corynebacterium glutamicum)、氧化葡糖杆菌(Gluconobacteroxydans)、运动发酵单胞菌(Zymomonas mobilis)、乳酸乳球菌(Lactococcus lactis)、植物乳杆菌(Lactobacillus plantarum)、天蓝色链霉菌(Streptomyces coelicolor)、醋酪酸梭状芽孢杆菌(Clostridium acetobutylicum)、荧光假单胞菌(Pseudomonasfluorescens)和恶臭假单胞菌(Pseudomonas putida)。大肠杆菌(E.coli)是特别有用的宿主生物，因为它是适合于基因工程的良好鉴定的微生物。
类似地，示例性的酵母或真菌种包括选自下列的任何种：酵母目(Saccharomycetales)酵母菌科(Saccaromycetaceae)，包括酵母菌属(Saccharomyces)、克卢费氏酵母属(Kluyveromyces)和毕赤氏酵母属(Pichia)；酵母目(Saccharomycetales)耶罗威亚酵母科(Dipodascaceae)，包括耶罗威亚酵母属(Yarrowia)；裂殖酵母目(Schizosaccharomycetales)裂殖酵母科(Schizosaccaromycetaceae)，包括裂殖酵母属(Schizosaccharomyces)；散囊菌目(Eurotiales)发菌科(Trichocomaceae)，包括曲霉属(Aspergillus)；和毛霉菌目(Mucorales)毛霉菌科(Mucoraceae)，包括根霉属(Rhizopus)。宿主酵母或真菌的非限制性种包括酿酒酵母(Saccharomyces cerevisiae)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、土曲霉(Aspergillus terreus)、黑曲霉(Aspergillusniger)、巴斯德毕赤氏酵母(Pichia pastoris)、少根根霉(Rhizopus arrhizus)、米根霉(Rhizobus oryzae)、解脂耶罗威亚酵母(Yarrowia lipolytica)等。作为酵母的特别有用的宿主生物包括酿酒酵母(Saccharomyces cerevisiae)。
尽管本文一般地描述为使用微生物细胞作为宿主细胞，具体地用于产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，但是应理解宿主细胞可以是高等真核生物的细胞系，如哺乳动物细胞系或昆虫细胞系。因此，作为另外一种选择，应理解本文中对微生物宿主细胞的提及可以使用高等真核细胞系来产生所期望的产品。示例性的高等真核细胞系包括(但不限于)中国仓鼠卵巢(CHO)、人(Hela、人胚肾(HEK)293、Jurkat)、小鼠(3T3)、灵长类动物(Vero)，昆虫(Sf9)等。这些细胞系是可商购的(参见，例如，美国模式培养物保藏所(ATCC；Manassas VA)；Life Technologies,Carlsbad CA)。应理解任何适合的宿主细胞可以用于引入本发明的醛脱氢酶和任选地代谢和/或基因修饰以产生所期望的产品。
根据所选宿主细胞的3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径组成，本发明的非天然存在的细胞将包括至少一种外源表达的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径-编码核酸和一种或多种3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径或与之有关的下游产品，如其酯或酰胺，包括本发明的醛脱氢酶的多至全部编码核酸。例如，可以通过相应编码核酸，包括本发明的醛脱氢酶的外源表达，在缺乏途径酶或蛋白的宿主中建立3-HBal、1,3-BDO、4-HBal或1,4-BDO的生物合成。在缺乏3-HBal、1,3-BDO、4-HBal或1,4-BDO途径或与之有关的下游产品，如其酯或酰胺的所有的酶或蛋白的宿主中，可以包括所述途径中的所有酶或蛋白的外源表达，尽管应理解即使所述宿主含有所述途径酶或蛋白中的至少一种，但是仍可以表达所述途径的全部酶或蛋白。例如，可以包括用于产生3-HBal、1,3-BDO、4-HBal或1,4-BDO途径或与之有关的下游产品，如其酯或酰胺的途径中的所有酶或蛋白的外源表达，包括本发明的醛脱氢酶。
考虑到本文所提供的教导和指导，本领域技术人员将理解以可表达形式引入的编码核酸的数目将至少与所选宿主细胞的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径缺乏并列，如果将在细胞中包括3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的话。因此，基于具体途径，本发明的非天然存在的细胞可以具有1、2、3、4、5、6、7、8个等，以至多达全部编码构成本文所公开的3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径的酶或蛋白的核酸。在一些实施方式中，所述非天然存在的细胞还可以包括有利于或优化3-HBal、1,3-BDO、4-HBal或1,4-BDO的生物合成或者赋予宿主细胞其它有用功能的其它基因修饰。一种这种其它功能可以包括(例如)一种或多种3-HBal、1,3-BDO、4-HBal或1,4-BDO途径前体，如乙酰-CoA或乙酰乙酰-CoA的合成增强。
通常，选择宿主细胞，从而它可以在含有该途径的细胞中表达本发明的醛脱氢酶，并且任选地作为天然产生的分子或者作为提供所期望的前体的从头产生或者通过宿主细胞天然产生的前体的产生增加的工程产品产生3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的前体。如本文所公开的，宿主生物可以工程设计以提高前体的产量。另外，已工程设计以产生所期望的前体的细胞可以用作宿主生物，并且如果需要，可以进一步工程设计以表达3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的酶或蛋白，或与之有关的下游产品，如其酯或酰胺。
在一些实施方式中，从含有酶促合成3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的能力的宿主产生本发明的非天然存在的细胞。在该具体实施方式中，它对于提高3-HBal、1,3-BDO、4-HBal或1,4-BDO途径产品的合成或积累可以是有用的，以(例如)驱使3-HBal、1,3-BDO、4-HBal或1,4-BDO途径反应向3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生产的进行。可以通过(例如)编码一种或多种上述3-HBal、1,3-BDO、4-HBal或1,4-BDO途径酶或蛋白，包括本发明的醛脱氢酶的核酸的过表达来实现提高的合成或积累。可以(例如)通过内源基因的外源表达或者通过异源基因的外源表达，包括本发明的醛脱氢酶的外源表达来进行3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的酶和/或蛋白的过表达。因此，基于3-HBal、1,3-BDO、4-HBal或1,4-BDO途径，可以通过1、2、3、4、5、6、7、8或更多种，即多至全部编码3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径酶或蛋白或产生与之有关的下游产品，如其酯或酰胺的酶的核酸的过表达容易地将天然存在的生物转化为(例如)生产3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的本发明的非天然存在的细胞。另外，可以通过导致3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径或与之有关的下游产品，如其酯或酰胺中的酶活力提高的内源基因突变来产生非天然存在的生物。
在特别有用的实施方式中，使用了编码核酸的外源表达。外源表达赋予了定制宿主的表达和/或调控元件的能力和实现通过用户控制的所期望的表达水平的应用。然而，在其它实施方式中，也可以使用内源表达，如当与诱导型启动子或者其它调控元件连接时，通过除去负调控效应因子或者基因启动子的诱导。因此，可以通过提供适当的诱导剂来上调具有天然存在的诱导型启动子的内源基因，或者可以工程设计内源基因的调控区以引入诱导型调控元件，借此使得能够在所期望的时间调控内源基因表达的提高。类似地，可以作为引入非天然存在的细胞的外源基因的调控元件包含诱导型启动子。
应理解可以将任何一种或多种外源核酸引入细胞以产生本发明的非天然存在的细胞。可以引入核酸以(例如)赋予细胞3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺生物合成途径，包括引入编码本发明的醛脱氢酶的核酸。作为另外一种选择，可以引入编码核酸以产生细胞，所述细胞具有催化一些所需要的反应的生物合成能力，从而赋予了产生中间体的3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成能力。例如，具有3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径的非天然存在的细胞可以包含编码所期望的酶或蛋白，包括本发明的醛脱氢酶的至少两个外源核酸。因此，应理解可以在本发明的非天然存在的细胞中包含生物合成途径的两种或更多种酶或蛋白的任何组合，包括本发明的醛脱氢酶。类似地，应理解根据需要，可以在本发明的非天然存在的细胞中包含生物合成途径的3种或以上的酶或蛋白的任意组合，只要所期望的生物合成途径的酶和/或蛋白的组合导致相应所期望的产品的产生。类似地，根据需要，可以在本发明的非天然存在的细胞中包含如本文所公开的生物合成途径的4种或以上的酶或蛋白的任意组合，只要所期望的生物合成途径的酶和/或蛋白的组合导致相应所期望的产品的产生。
除如本文所述的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成外，还可以以彼此和/或与本领域中熟知的其它细胞和方法的不同组合使用本发明的非天然存在的细胞和方法以实现通过其它路线的产品生物合成。例如，除3-HBal、1,3-BDO、4-HBal或1,4-BDO生产菌的使用以外，生产3-HBal、1,3-BDO、4-HBal或1,4-BDO的一种替代方法是通过添加能够将3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品转化为3-HBal、1,3-BDO、4-HBal或1,4-BDO的另一种细胞。一种这种程序包括(例如)产生3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品的细胞的发酵。然后，3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品可以用作将3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品转化为3-HBal、1,3-BDO、4-HBal或1,4-BDO的第二细胞的底物。可以将3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品直接添加至第二生物的另一培养，或者可以通过(例如)细胞分离从3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品产生菌的原始培养中除去这些细胞，然后可以使用向发酵液中后续添加所述第二生物以产生最终产品，而不使用中间产品纯化步骤。可以任选地包括产生与3-HBal、1,3-BDO、4-HBal或1,4-BDO有关的下游产品，如其酯或酰胺的细胞以产生这种下游产品。
作为另外一种选择，可以使用导致底物向所期望的产品的转化的酶的组合或者底物对酶的顺序暴露来体外实施这些酶转化。作为另一种替代，如果需要，可以使用细胞-基转化和体外酶转化的组合。
在其它实施方式中，可以以多种子途径组合本发明的非天然存在的细胞和方法以实现(例如)3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成。在这些实施方式中，本发明的所期望的产品的生物合成途径可以分离成不同的细胞，并且所述不同的细胞可以共培养以产生最终产品。在该生物合成方案中，一种细胞的产品是第二细胞的底物，直至合成了最终产品。例如，可以通过构建含有用于一种途径中间产品向另一种途径中间产品或产品转化的生物合成途径的细胞来实现3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成。作为另外一种选择，还可以通过使用两种不同的细胞在相同容器中的共培养或共发酵从细胞生物合成产生3-HBal、1,3-BDO、4-HBal或1,4-BDO，其中所述第一细胞产生3-HBal、1,3-BDO、4-HBal或1,4-BDO中间体，而所述第二细胞将所述中间体转化为3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺。
考虑到本文所提供的教导和指导，本领域技术人员将理解对于本发明的非天然存在的细胞和方法，与其它细胞，与具有子途径的其它非天然存在的细胞的共培养以及与在本领域中熟知的产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的其它化学和/或生物化学程序组合一起存在多种组合和排列。
3-HBal、1,3-BDO、4-HBal或1,4-BDO途径酶或蛋白，或与之有关的下游产品，如其酯或酰胺的编码核酸源可以包括(例如)其中编码的基因产品能够催化参考反应的任何种。这些种包括原核和真核生物两者，其包括(但不限于)细菌，包括古细菌和真细菌，和真核生物，包括酵母、植物、昆虫、动物和哺乳动物，包括人。这些来源的示例性种包括(例如)大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)、克氏酵母菌(Saccharomyces kluyveri)、克氏梭菌(Clostridium kluyveri)、丙酮丁醇梭菌(Clostridium acetobutylicum)、拜氏梭菌(Clostridium beijerinckii)、糖乙酸多丁醇梭菌(Clostridium saccharoperbutylacetonicum)、产气荚膜梭菌(Clostridiuumperfringens)、难辨梭菌(Clostridium difficile)、肉毒梭菌(Clostridium botulinum)、酪丁酸梭菌(Clostridium tyrobutyricum)、假破伤风梭菌(Clostridiumtetanomorphum)、破伤风梭菌(Clostridium tetani)、丙酸梭菌(Clostridiumpropionicum)、氨基丁酸梭菌(Clostridium aminobutyricum)、近端梭菌(Clostridiumsubterminale)、史迪克兰梭菌(Clostridium sticklandii)、富养产碱菌(Ralstoniaeutropha)、牛分枝杆菌(Mycobacterium bovis)、结核分枝杆菌(Mycobacteriumtuberculosis)、牙龈卟啉单胞菌(Porphyromonas gingivalis)、拟南芥(Arabidopsisthaliana)、嗜热栖热菌(Thermus thermophilus)、假单胞菌(Pseudom onasspecies)(包括铜绿假单胞菌(Pseudomonas aeruginosa)、恶臭假单胞菌(Pseudomonas putida)、施氏假单胞菌(Pseudomonas stutzeri)、荧光假单胞菌(Pseudomonas fluorescens))、智人(Homosapiens)、穴兔(Oryctolagus cuniculus)、类球红杆菌(Rhodobacter spaeroides)、布氏嗜热厌氧性杆菌(Thermoanaerobacter brockii)、瑟杜生金属球菌(Metallosphaerasedula)、肠膜样明串珠菌(Leuconostoc mesenteroides)、嗜热光合绿曲菌(Chloroflexusaurantiacus)、凯斯特玫瑰弯菌(Roseiflexus castenholzii)、赤杆菌属(Erythrobacter)、蜡黄杨(Simmondsia chinensis)、不动杆菌(Acinetobacter species)(包括乙酸钙不动杆菌(Acinetobacter calcoaceticus)和贝氏不动杆菌(Acinetobacterbaylyi))、牙龈卟啉单胞菌(Porphyromonas gingivalis)、托克达硫化叶菌(Sulfolobustokodaii)、硫磺矿硫化叶菌(Sulfolobus solfataricus)、嗜酸热硫化叶菌(Sulfolobusacidocaldarius)、枯草芽胞杆菌(Bacillus subtilis)、蜡样芽孢杆菌(Bacilluscereus)、巨大芽胞杆菌(Bacillus megaterium)、短芽胞杆菌(Bacillus brevis)、短小芽胞杆菌(Bacillus pumilus)、褐鼠(Rattus norvegicus)、肺炎克雷伯氏菌(Klebsiellapneumonia)、产酸克雷伯氏菌(Klebsiella oxytoca)、纤细裸藻(Euglena gracilis)、齿垢密螺旋体(Treponema denticola)、热醋穆尔氏菌(Moorella thermoacetica)、海栖热袍菌(Thermotoga maritima)、嗜盐杆菌(Halobacterium salinarum)、嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)、敏捷气热菌(Aeropyrum pernix)、野猪(Susscrofa)、秀丽隐杆线虫(Caenorhabditis elegans)、谷氨酸棒杆菌(Corynebacteriumglutamicum)、发酵氨基酸球菌(Acidaminococcus fermentans)、乳酸乳球菌(Lactococcuslactis)、胚芽乳酸杆菌(Lactobacillus plantarum)、嗜热链球菌(Streptococcusthermophilus)、产气肠杆菌(Enterobacter aerogenes)、假丝酵母菌属(Candida)、土曲霉(Aspergillus terreus)、戊糖片球菌(Pediococcus pentosaceus)、运动发酵单胞菌(Zymomonas mobilis)、巴斯德醋酸杆菌(Acetobacter pasteurians)、乳酸克氏酵母菌(Kluyveromyces lactis)、巴氏真杆菌(Eubacterium barkeri)、多毛拟杆菌(Bacteroidescapillosus)、科氏厌氧性躯干菌(Anaerotruncus colihominis)、嗜热盐碱厌氧性菌(Natranaerobius thermophilus)、空肠曲杆菌(Campylobacter jejuni)、流感嗜血杆菌(Haemophilus influenzae)、粘质沙雷氏菌(Serratia marcescens)、无丙二酸柠檬酸杆菌(Citrobacter amalonaticus)、黄色粘球菌(Myxococcus xanthus)、具核梭杆菌(Fusobacterium nucleatum)、产黄青霉(Penicillium chrysogenum)、海洋γ变形菌(marine gamma proteobacterium)、丁酸产生菌(butyrate-producing bacterium)、伊文思奴卡菌(Nocardia iowensis)、皮疽奴卡菌(Nocardia farcinica)、灰色链霉菌(Streptomyces griseus)、粟酒裂殖酵母菌(Schizosaccharomyces pombe)、热葡糖苷酶地芽孢杆菌(Geobacillus thermoglucosidasius)、鼠伤寒沙门氏菌(Salmonellatyphimurium)、霍乱弧菌(Vibrio cholera)、幽门螺旋杆菌(Helicobacter pylori)、烟草(Nicotiana tabacum)、水稻(Oryza sativa)、地中海极嗜酯菌(Haloferaxmediterranei)、根癌农杆菌(Agrobacterium tumefaciens)、反硝化无色杆菌(Achromobacter denitrificans)、具核梭杆菌(Fusobacterium nucleatum)、棒状链霉菌(Streptomyces clavuligerus)、鲍氏不动杆菌(Acinetobacter baumanii)、小家鼠(Musmusculus)、克氏酵母菌(Lachancea kluyveri)、阴道滴虫(Trichomonas vaginalis)、布氏锥虫(Trypanosoma brucei)、施氏假单胞菌(Pseudomonas stutzeri)、大豆根瘤菌(Bradyrhizobium japonicum)、百脉根中慢生根瘤菌(Mesorhizobium loti)、欧洲牛(Bostaurus)、粘性烟草(Nicotiana glutinosa)、创伤弧菌(Vibrio vulnificus)、反刍月形单胞菌(Selenomonas ruminantium)、肠炎弧菌(Vibrio parahaemolyticus)、闪烁古球菌(Archaeoglobus fulgidus)、死海嗜盐古细菌(Haloarcula marismortui)、嗜气热棒菌(Pyrobaculum aerophilun)、耻垢分枝杆菌(Mycobacterium smegmatis)MC2 155、鸟分枝杆菌副结核亚种(Mycobacterium avium subsp.paratuberculosis)K-10、海栖分枝杆菌(Mycobacterium marinum)M、微变冢村氏菌DSM 20162(Tsukamurella paurometabola DSM20162)、蓝菌属PCC7001(Cyanobium PCC7001)、盘基网柄菌AX4(Dictyosteliumdiscoideum AX4)、发酵氨基酸球菌(Acidaminococcus fermentans)、贝氏不动杆菌(Acinetobacter baylyi)、乙酸钙不动杆菌(Acinetobacter calcoaceticus)、风产液菌(Aquifex aeolicus)、拟南芥(Arabidopsis thaliana)、闪烁古生球菌(Archaeoglobusfulgidus)、黑曲霉(Aspergillus niger)、土曲霉(Aspergillus terreus)、枯草芽孢杆菌(Bacillus subtilis)、黄牛(Bos taurus)、白假丝酵母(Candida albicans)、热带假丝酵母(Candida tropicalis)、莱茵衣藻(Chlamydomonas reinhardtii)、绿硫菌(Chlorobiumtepidum)、克氏柠檬酸杆菌(Citrobacter koseri)、柚子(Citrus junos)、丙酮丁醇梭菌(Clostridium acetobutylicum)、克氏梭菌(Clostridium kluyveri)、糖乙酸多丁醇梭菌(Clostridium saccharoperbutylacetonicum)、蓝菌属(Cyanobium)PCC7001、食烯烃脱硫杆菌(Desulfatibacillum alkenivorans)、盘基网柄菌(Dictyostelium discoideum)、具核梭杆菌(Fusobacterium nucleatum)、死海嗜盐古细菌(Haloarcula marismortui)、智人(Homo sapiens)、嗜热氢杆菌(Hydrogenobacter thermophilus)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、乳酸克鲁维酵母(Kluyveromyces lactis)、短乳酸杆菌(Lactobacillus brevis)、肠膜明串珠菌(Leuconostoc mesenteroides)、鸟型结核分枝杆菌(Mycobacterium avium)、牛型结核分枝杆菌(Mycobacterium bovis)、海栖分枝杆菌(Mycobacterium marinum)、耻垢分枝杆菌(Mycobacterium smegmatis)、烟草(Nicotianatabacum)、伊文思奴卡菌(Nocardia iowensis)、穴兔(Oryctolagus cuniculus)、产黄青霉(Penicilliumchrysogenum)、巴斯德毕赤氏酵母(Pichia pastoris)、牙龈红棕色单胞菌(Porphyromonas gingivalis)、牙龈红棕色单胞菌(Porphyromonas gingivalis)、铜绿假单胞菌(Pseudomonas aeruginos)、恶臭假单胞菌(Pseudomonas putida)、耐超高温热棒菌(Pyrobaculum aerophilum)、富养产碱菌(Ralstonia eutropha)、褐家鼠(Rattusnorvegicus)、球形红细菌(Rhodobacter sphaeroides)、酿酒酵母(Saccharomycescerevisiae)、肠沙门氏菌(Salmonella enteric)、鼠伤寒沙门氏菌(Salmonellatyphimurium)、粟酒裂殖酵母(Schizosaccharomyces pombe)、嗜酸热硫化叶菌(Sulfolobus acidocaldarius)、硫磺矿硫化叶菌(Sulfolobus solfataricus)、托克达硫化叶菌(Sulfolobus tokodaii)、腾冲嗜热杆菌(Thermoanaerobacter tengcongensis)、嗜热栖热菌(Thermus thermophilus)、布氏锥虫(Trypanosoma brucei)、微变冢村氏菌(Tsukamurella paurometabola)、解脂耶罗威亚酵母(Yarrowia lipolytica)、生枝动胶菌(Zoogloea ramigera)和运动发酵单胞菌(Zymomonas mobilis)、梭菌(Clostridumspecies)，包括(但不限于)糖乙酸多丁醇梭菌(Clostridiumsaccharoperbutylacetonicum)、拜氏梭菌(Clostridium beijerinckii)、糖丁酸梭菌(Clostridium saccharobutylicum)、肉毒梭菌(Clostridium botulinum)、甲基戊糖梭菌(Clostridium methylpentosum)、史迪克兰梭菌(Clostridium sticklandii)、植物发酵梭菌(Clostridium phytofermentans)、解糖梭菌(Clostridium saccharolyticum)、阿斯巴伐梭菌(Clostridium asparagiforme)、隐藏梭菌(Clostridium celatum)、食一氧化碳梭菌(Clostridium carboxidivorans)、梭状梭菌(Clostridium clostridioforme)、鲍氏梭菌(Clostridium bolteae)、温泉热碱芽胞杆菌(Caldalkalibacillus thermarum)、肉毒梭菌(Clostridium botulinum)、发酵酸酐菌(Pelosinus fermentans)、热解糖梭菌(Thermoanaerobacterium thermosaccharolyticum)、脱硫芽孢弯曲菌(Desulfosporosinus speices)、热厌氧杆菌(Thermoanaerobacterium species)，包括(但不限于)解糖热厌氧杆菌(Thermoanaerobacterium saccharolyticum)、解木聚糖热厌氧杆菌(Thermoanaerobacterium xylanolyticum)、长醋丝菌(Acetonema longum)、地芽孢杆菌(Geobacillus species)，包括(但不限于)热葡萄糖苷酶地芽孢杆菌(Geobacillusthermoglucosidans)、产氮芽孢杆菌(Bacillus azotoformans)、潜能栖热泉菌(Thermincola potens)、梭杆菌(Fusobacterium species)，包括(但不限于)具核梭杆菌(Fusobacterium nucleatum)、溃疡性梭杆菌(Fusobacterium ulcerans)、可变梭杆菌(Fusobacterium varium)、瘤胃球菌(Ruminococcus species)，包括(但不限于)活泼胃球菌(Ruminococcus gnavus)、卵胃球菌(Ruminococcus obeum)、毛螺科菌(Lachnospiraceaebacterium)、普氏梭杆菌(Flavonifractor plautii)、食葡糖罗斯氏菌(Roseburiainulinivorans)、伍氏醋酸杆菌(Acetobacterium woodii)、真杆菌(Eubacteriumspecies)，包括(但不限于)绳尾真杆菌(Eubacterium plexicaudatum)、霍氏真杆菌(Eubacterium hallii)、粘液真杆菌(Eubacterium limosum)、尤氏真杆菌(Eubacteriumyurii)、真杆菌科细菌(Eubacteriaceae bacterium)、海洋热沉积杆菌(Thermosediminibacter oceani)、多营养型泥杆菌(Ilyobacter polytropus)、沙棘(Shuttleworthia satelles)、解糖盐厌氧菌(Halanaerobium saccharolyticum)、产乙醇热厌氧杆菌(Thermoanaerobacter ethanolicus)、深红螺菌(Rhodospirillum rubrum)、弧菌(Vibrio)、丙酸丙酸杆菌(Propionibacterium propionicum)以及本文所公开的或者作为相应基因的来源生物，包括表4中所述的醛脱氢酶的来源生物可得的其它示例性种。然而，使用目前可得的超过550个种的完整基因组序列(这些的超过一半可在公开的数据库，如NCBI上获得)，包括395种微生物基因组以及多种酵母、真菌、植物和哺乳动物基因组，在相关或远源种的一种或更多种基因中鉴定编码3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成活性的基因，包括(例如)已知基因的同源物、直系同源体、旁系同源物和非直系同源基因置换，以及生物之间遗传学改变的互换是本领域中常规的和熟知的。因此，在本文中参考具体生物，如大肠杆菌(E.coli)所描述的，容许3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成的代谢改变，包括本发明的醛脱氢酶的表达，同样可以容易地应用于其它细胞，如微生物，包括原核生物和真核生物。考虑到本文所提供的教导和指导，本领域技术人员将知晓一种生物中举例说明的代谢改变可以等同地应用于其它生物。
在一些情况下，如当替代性的3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径存在于无关物种中时，可以通过(例如)来自催化类似，但不相同的代谢反应的无关物种的旁系同源物的外源表达为宿主物种赋予3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成以替代参考反应。因为在不同生物之间在代谢网络中存在某些差异，因此本领域技术人员将理解不同生物之间的真实基因使用可以不同。然而，考虑到本文所提供的教导和指导，本领域技术人员还将理解可以使用对本文中所举例说明的那些的同源代谢改变，将本发明的教导和方法应用于所有细胞以在所关心的物种中构建将合成3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之相关的下游产品，如其酯或酰胺的细胞，如果需要，包括引入本发明的醛脱氢酶。
可以(例如)通过在本领域中熟知的重组和检测方法来实施用于构建和测试产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，包括本发明的醛脱氢酶的非天然存在的宿主的表达水平的方法。这些方法可见于(例如)Sambrook等人,Molecular Cloning:A Laboratory Manual,第3版,Cold Spring Harbor Laboratory,NewYork(2001)；和Ausubel等人,Current Protocols in Molecular Biology,John Wileyand Sons,Baltimore,MD(1999)。
可以使用本领域中熟知的技术，包括(但不限于)缀合、电穿孔、化学转化、转导、转染和超声转化，将编码本发明的醛脱氢酶的外源核酸和任选地参与生产3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的途径的外源核酸序列稳定或短暂引入宿主细胞。对于大肠杆菌(E.coli)或其它原核细胞中的外源表达，真核核酸的基因或cDNA中的一些核酸序列可以编码靶向信号，如N末端线粒体或其它靶向信号，如果需要，其可以在转化至原核宿主细胞中之前除去。例如，线粒体前导序列的除去导致大肠杆菌(E.coli)中的表达提高(Hoffmeister等人,J.Biol.Chem.280:4329-4338(2005))。对于酵母或其它真核细胞中的外源表达，基因可以在胞液中表达而不添加前导序列，或者通过添加适合的靶向序列，如适合于宿主细胞的线粒体靶向或分泌信号，可以靶向线粒体或其它细胞器，或者靶向用于分泌。因此，应理解可以将除去或包括靶向序列的核酸序列的适当修饰引入外源核酸序列以赋予所期望的性质。此外，可以使用在本领域中熟知的技术对基因进行密码子优化以实现所述蛋白优化的表达。
可以构建表达载体以包括编码本发明的醛脱氢酶的核酸和/或任选地一种或多种3-HBal、1,3-BDO、4-HBal或1,4-BDO生物合成途径编码核酸或者编码生产与3-HBal、1,3-BDO、4-HBal或1,4-BDO有关的下游产品，如其酯或酰胺的酶的核酸，如本文中举例说明的可操作地连接至在宿主生物中起作用的表达控制序列。适合在本发明所述的宿主细胞中使用的表达载体包括(例如)质粒、噬菌体载体、病毒载体、附加体和人造染色体，包括对于向宿主染色体稳定整合可操作的载体和选择序列或标志物。另外，所述表达载体可以包括一个或多个可选择标志物基因和适当的表达控制序列。还可以包括(例如)提供抗生素或毒素抗性、补充营养缺陷或者提供培养基中不存在的关键营养物的可选择标志物基因。表达控制序列可以包括在本领域中熟知的组成型和诱导型启动子、转录增强子、转录终止子等。当共表达两种或更多种外源编码核酸时，两种核酸可以插入(例如)到单一表达载体或者不同的表达载体中。对于单一载体表达，所述编码核酸可以操作性地连接至一个公共表达控制序列或者连接至不同的表达控制序列，如一个诱导型启动子和一个组成型启动子。可以使用本领域中熟知的方法确认编码本发明的醛脱氢酶或编码参与代谢或合成途径的多肽的外源核酸序列的转化。这些方法包括(例如)核酸分析，如mRNA的RNA印迹或聚合酶链反应(PCR)扩增，或者用于基因产品表达的免疫印迹法，或者其它适合的分析方法以测试所引入的核酸序列或其相应基因产品的表达。本领域的那些技术人员应理解所述外源核酸以足够的量表达以产生所期望的产品，并且还应理解可以使用本领域中熟知的和如本文所公开的方法优化表达水平以获得足够的表达。
载体或表达载体还可以用于表达编码核酸以通过体外转录和翻译产生所编码的多肽。这些载体或表达载体将至少包括启动子，并且包括本文以上所述的载体。用于体外转录和翻译的这种载体通常是双链DNA。体外转录和翻译方法对于本领域技术人员来说是熟知的(参见Sambrook等人,Molecular Cloning:A Laboratory Manual,第3版,Cold SpringHarbor Laboratory,New York(2001)；和Ausubel等人,Current Protocols in MolecularBiology,John Wiley and Sons,Baltimore,MD(1999))。用于体外转录和翻译的试剂盒也是可商购的(参见，例如，Promega,Madison,WI；New England Biolabs,Ipswich,MA；ThermoFisher Scientific,Carlsbad,CA)。
在一个实施方式中，本发明提供了用于生产3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或其酯或酰胺的方法，其包括培养本发明的细胞以产生3-HBal和/或1,3-BDO或其酯或酰胺。这种细胞表达本发明的多肽。在一个实施方式中，本发明提供了用于生产4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)或其酯或酰胺的方法，其包括培养本发明的细胞以产生4-HBal和/或1,4-BDO或其酯或酰胺。在一个实施方式中，所述细胞处于基本厌氧的培养基中。在一个实施方式中，所述方法还可以包括分离或纯化3-HBal和/或1,3-BDO，或者4-HBal和/或1,4-BDO或其酯或酰胺。在具体的实施方式中，所述分离或纯化包括蒸馏。
在一个实施方式中，本发明提供了用于生产本发明的产品的方法，其包括使3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO与自身或另一种化合物在生产产品的反应中化学反应。
在一个实施方式中，本发明提供了生产3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)，或其酯或酰胺的方法，其包括向本发明的多肽提供底物并将所述底物转化为3-HBal和/或1,3-BDO，其中所述底物是1,3-羟基丁酰基-CoA的外消旋混合物。在一个实施方式中，3-HBal和/或1,3-BDO是对R形式对映体富集的。在一个实施方式中，本发明提供了生产4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)，或其酯或酰胺的方法，其包括向本发明的多肽提供底物并将所述底物转化为4-HBal和/或1,4-BDO，其中所述底物是1,4-羟基丁酰基-CoA。在一个实施方式中，多肽存在于细胞中，细胞裂解液中，或者分离自细胞或细胞裂解液。
在一个实施方式中，本发明提供了用于生产3-HBal和/或1,3-BDO，或者4-HBal和/或1,4-BDO的方法，其包括培育本发明的细胞裂解液以生产3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO。在一个实施方式中，将细胞裂解液与第二细胞裂解液混合，其中所述第二细胞裂解液包含酶活力以产生本发明的多肽的底物，或3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的下游产品。
本发明还提供了用于生产本发明的多肽的方法，其包括在细胞中表达所述多肽。本发明另外提供了用于生产本发明的多肽的方法，其包括体外转录和翻译本发明的核酸或者本发明的载体以生产所述多肽。
如本文所述，细胞可以用于表达本发明的醛脱氢酶，并且任选地所述细胞可以包括利用本发明的醛脱氢酶来生产所期望的产品，如3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的代谢途径。本文描述了用于表达所期望的产品的这些方法。作为另外一种选择，如本文所述，可以在细胞裂解液，例如，表达本发明的醛脱氢酶的细胞或者表达本发明的醛脱氢酶和代谢途径以生产所期望的产品的细胞的细胞裂解液中表达本发明的醛脱氢酶和/或产生所期望的产品。在另一个实施方式中，可以通过体外转录和翻译表达本发明的醛脱氢酶，其中在无细胞系统中生产醛脱氢酶。通过体外转录和翻译表达的醛脱氢酶可以用于体外实施反应。任选地，其它酶，或者含有这些酶的细胞裂解液可以用于将醛脱氢酶酶促反应产品体外转化为所期望的下游产品。
可以使用熟知的方法实施测试醛脱氢酶的表达或者3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的产生的适合的纯化和/或测定，包括测试醛脱氢酶活力的测定(还参见实施例)。对要测试的每个工程株，可以进行适当的重复生长，如重复三次培养。例如，可以监测工程生产宿主中的产品和副产品的形成。可以使用在本领域中熟知的常规程序，通过方法，如HPLC(高效液相色谱法)、GC-MS(气相色谱-质谱法)和LC-MS(液相色谱-质谱法)或者其它适合的分析方法来分析最终产品和中间产品及其它有机化合物。还可以通过培养上清液测试发酵液中产品的释放。可以使用(例如)示差折光检测器(对于葡萄糖和醇)和UV检测器(对于有机酸)，通过HPLC或者通过本领域中熟知的其它适合的测定和检测方法定量副产品和残余葡萄糖(Lin等人,Biotechnol.Bioeng.90:775-779(2005))。还可以使用本领域中熟知的方法测定来自外源DNA序列的各个酶或蛋白的活力(还参见实施例)。
使用本领域中熟知的多种方法，3-HBal、1,3-BDO、4-HBal或1,4-BDO或其它所期望的产品，如与之相关的下游产品，如其酯或酰胺可以分离自培养中的其它组分。这些分离方法包括(例如)提取程序以及包括下列的方法：连续液-液提取、渗透气化、膜滤法、膜分离、反渗透、电渗析、蒸馏、结晶、离心、提取过滤、离子交换色谱、尺寸排阻色谱、吸附色谱和超滤。上述所有方法在本领域中是熟知的。
可以培养本文所述的表达本发明的醛脱氢酶的任何非天然存在的细胞以生产和/或分泌本发明的生物合成产品。例如，可以培养产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的细胞以用于生物合成生产3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺。因此，在一些实施方式中，本发明提供了含有3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，或本文所述的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品的培养基。在一些方面，所述培养基还可以分离自产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间产品的本发明的非天然存在的细胞。将细胞与培养基分离的方法在本领域中是熟知的。示例性方法包括过滤、絮凝、沉淀、离心、沉降等。
对于本发明的醛脱氢酶或者3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺在表达本发明的醛脱氢酶的细胞中的生产，在具有碳源及其它必需营养素的培养基中培养重组株。有时希望并且可以非常期望在发酵罐中维持厌氧条件以降低整个过程的成本。可以(例如)通过首先用氮气向培养基鼓泡，然后用隔膜和钳口盖密封烧瓶来获得这些条件。对于未观察到厌氧生长的菌株，可以通过用小孔使隔膜穿孔以用于有限通气来施加微氧或基本厌氧条件。先前已描述了示例性厌氧条件并且所述示例性厌氧条件在本领域中是熟知的。在(例如)2007年8月10日提交的美国专利公开2009/0047719中描述了示例性的好氧和厌氧条件。如本文所公开的，可以以分批、进料-分批或连续方式进行发酵。如果需要，还可以在两阶段中进行发酵。第一阶段可以是好氧的以允许高生长，并因此允许高产量，然后是所期望的产品，如3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的高得率的厌氧阶段。
如果需要，可以根据需要，通过添加碱，如NaOH或其它碱，或者酸将培养基的pH维持在所期望的pH，具体地中性pH，如约7的pH，以将培养基维持在所期望的pH。可以使用分光光度计通过测量光密度(600nm)来确定生长速率，并且可以通过监测碳源随时间的减少来确定葡萄糖的吸收速率。
所述生长培养基还可以包含(例如)任何碳水化合物源，其可以向非天然存在的细胞提供碳源。这些源包括(例如)糖，如葡萄糖、木糖、阿拉伯糖、半乳糖、甘露糖、果糖、蔗糖和淀粉；或者甘油，并且应理解碳源可以作为唯一碳源单独使用或者与本文所述的或本领域中已知的其它碳源组合使用。其它碳水化合物源包括(例如)可再生原料和生物质。在本发明所述的方法中可以用作原料的示例性生物质类型包括纤维素生物质、半纤维素生物质和木质素原料或原料部分。这些生物质原料含有(例如)作为碳源有用的碳水化合物基质，如葡萄糖、木糖、阿拉伯糖、半乳糖、甘露糖、果糖和淀粉。考虑到本文所提供的教导和指导，本领域技术人员将理解除以上举例说明的那些以外，可再生原料和生物质也可以用于培养本发明的细胞以用于表达本发明的醛脱氢酶并且任选地用于生产3-HBal、1,3-BDO、4-HBal或1,4-BDO或其下游产品，如其酯或酰胺。
除可再生原料，如以上举例说明的那些外，还可以修饰产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或其下游产品(如其酯或酰胺)的本发明的细胞以用于在作为其碳源的合成气上生长。在该具体实施方式中，在产生3-HBal、1,3-BDO、4-HBal或1,4-BDO的生物中表达一种或多种蛋白或酶以提供用于利用合成气或其它气态碳源的代谢途径。
合成气(Synthesis gas)，也称为合成气(syngas)或发生炉煤气，它是煤和含碳材料，如生物质材料，包括农作物和残余物气化的主要产品。合成气主要是H2和CO的混合物并且可以得自任何有机原料，包括(但不限于)煤、煤油、天然气、生物质和有机废物的气化。气化通常在高燃料比氧的比下进行。尽管基本上是H2和CO，合成气还可以包括较小量的CO2及其它气体。因此，合成气提供了气态碳，如CO，和另外，CO2的经济合算的来源。
Wood-Ljungdahl途径催化CO和H2向乙酰-CoA及其它产品，如乙酸盐的转化。能够使用CO和合成气的生物通常还具有通过相同的基础酶组和通过Wood-Ljungdahl途径所涵盖的转化使用CO2和CO2/H2的混合物的能力。早已认识到了通过微生物的CO2向乙酸盐的H2-依赖性转化，其显示还可以通过相同生物使用CO并且涉及相同途径。已显示多种产乙酸菌在存在CO2的情况下生长并且产生了化合物，如乙酸盐，只要存在氢气来通过必需的还原当量(参见，例如，Drake,Acetogenesis,3-60页Chapman and Hall,New York,(1994))。这可以通过以下方程总结：
2CO2+4H2+n ADP+n Pi→CH3COOH+2H2O+n ATP
因此，具有Wood-Ljungdahl途径的非天然存在的微生物可以使用CO2和H2混合物并用于乙酰-CoA及其它所期望的产品的生产。
Wood-Ljungdahl途径在本领域中是熟知的并且包括12个反应，其可以分成两个分支：(1)甲基分支和(2)羰基分支。甲基分支将合成气转化为甲基-四氢叶酸(甲基-THF)，而羰基分支将甲基-THF转化为乙酰-CoA。按照以下酶或蛋白的顺序：铁氧化还原蛋白氧化还原酶、甲酸脱氢酶、甲酰四氢叶酸合成酶、甲川四氢叶酸环化脱水酶、亚甲基四氢叶酸脱氢酶和亚甲基四氢叶酸还原酶催化甲基分支中的反应。按照以下酶或蛋白的顺序：甲基四氢叶酸:类咕啉蛋白甲基转移酶(例如，AcsE)、类咕啉铁硫蛋白、镍-蛋白组装蛋白(例如，AcsF)、铁氧化还原蛋白、乙酰-CoA合酶、一氧化碳脱氢酶和镍-蛋白组装蛋白(例如，CooC)催化羰基分支中的反应(参见WO2009/094485)。按照关于引入足够数目的编码核酸以产生3-HBal、1,3-BDO、4-HBal或1,4-BDO途径或与之有关的下游产品，如其酯或酰胺，包括编码本发明的醛脱氢酶的核酸的本文所提供的教导和指导，本领域技术人员将理解还可以对引入至少在宿主生物中缺少的编码Wood-Ljungdahl酶或蛋白的核酸进行相同的工程设计。因此，一个或多个编码核酸向本发明的细胞的引入使得修饰的生物含有完整的Wood-Ljungdahl途径，这将赋予合成气利用能力。
另外，还原性(逆向)三羧酸循环结合一氧化碳脱氢酶和/或氢化酶活力还可以用于CO、CO2和/或H2向乙酰-CoA及其它产品，如乙酸盐的转化。能够通过还原性TCA途径固定碳的生物可以使用以下酶中的一种或多种：ATP柠檬酸盐-分解酶、柠檬酸裂解酶、乌头酸酶、异柠檬酸脱氢酶、α-酮戊二酸:铁氧还蛋白氧化还原酶、丁二酰-CoA合成酶、丁二酰-CoA转移酶、延胡索酸盐还原酶、富马酸酶、苹果酸脱氢酶、NAD(P)H:铁氧还蛋白氧化还原酶、一氧化碳脱氢酶和氢化酶。具体地，使用通过一氧化碳脱氢酶和氢化酶从CO和/或H2提取的还原当量来通过还原性TCA循环将CO2固定至乙酰-CoA或乙酸盐。可以通过酶，如乙酰-CoA转移酶、乙酸盐激酶/磷酸转乙酰酶和乙酰-CoA合成酶将乙酸盐转化为乙酰-CoA。通过丙酮酸盐:铁氧还蛋白氧化还原酶和糖异生酶，可以将乙酰-CoA转化为甘油醛-3-磷酸、磷酸烯醇丙酮酸和丙酮酸盐。例如，通过乙酰乙酰-CoA硫解酶，可以将乙酰-CoA转化为乙酰乙酰-CoA以集中在1,3-BDO途径，如本文所公开的(参见图1)。按照对于引入足够数目的编码核酸以产生3-HBal、1,3-BDO、4-HBal或1,4-BDO途径或产生与之有关的下游产品，如其酯或酰胺的途径的本文所提供的教导和指导，本领域技术人员将理解还可以相对于引入至少在宿主生物中缺少的编码还原性TCA途径的酶或蛋白的核酸来进行相同的工程设计。因此，可以实施一个或多个编码核酸向本发明的细胞中的引入，从而修饰的生物含有还原性TCA途径。
因此，考虑到本文所提供的教导和指导，本领域技术人员将理解可以产生非天然存在的细胞，当在碳源，如碳水化合物上生长时，其生产和/或分泌生物合成的本发明的化合物。这些化合物包括(例如)3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺和3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的任何中间代谢物。所有所需要的是在一种或多种所需要的酶或蛋白活力中进行工程设计以实现所期望的化合物或中间产品的生物合成，包括(例如)用于3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，包括本发明的醛脱氢酶的一些或全部生物合成途径的包括。因此，本发明提供了当在碳水化合物或其它碳源上生长时，生产和/或分泌3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的非天然存在的细胞，和当在碳水化合物或其它碳源上生长时，生产和/或分泌3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中所示的任何中间代谢物的非天然存在的细胞。本发明的产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的细胞可以引起从3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的中间体的合成。
使用如本文举例说明的本领域中熟知的方法构建本发明的非天然存在的细胞以外源表达本发明的醛脱氢酶和任选地至少一种编码3-HBal、1,3-BDO、4-HBal或1,4-BDO途径酶或蛋白或与之有关的下游产品，如其酯或酰胺的核酸。可以以足够的量表达酶或蛋白以产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺。应理解本发明的细胞在足以表达本发明的醛脱氢酶或产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的情况下培养。按照本文所提供的教导和指导，本发明的非天然存在的细胞可以实现3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成，从而导致产生约0.1-300mM或以上，例如，0.1-1.3M或以上的胞内浓度。通常，3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的胞内浓度在约3-150mM之间，具体地约5-125mM之间，并且更具体地约8-100mM之间，包括约10mM、20mM、50mM、80mM或以上。还可以从本发明的非天然存在的细胞实现每个这些示例性范围之间和以上的胞内浓度。例如，3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的胞内浓度可以在约100mM至1.3M之间，包括约100mM、200mM、500mM、800mM、1M、1.1M、1.2M、1.3M或以上。
使用熟知方法培养本发明的细胞。所述培养条件可以包括(例如)液体培养程序以及发酵及其它大规模培养程序。如本文所述，可以在厌氧或基本厌氧的培养条件下获得本发明的生物合成产品的特别有用的得率。
在一些实施方式中，培养条件包括厌氧或基本厌氧生长或者维持条件。先前已描述了示例性厌氧条件并且所述示例性厌氧条件在本领域中是熟知的。本文描述了并且在(例如)2007年8月10日提交的美国专利公开2009/0047719中描述了发酵过程的示例性厌氧条件。任何这些条件以及在本领域中熟知的其它厌氧条件可以用于非天然存在的细胞。在这些厌氧或基本厌氧条件下，3-HBal、1,3-BDO、4-HBal或1,4-BDO生产菌可以以5-10mM或以上以及在本文中举例说明的所有其它浓度的胞内浓度下合成3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺。应理解，尽管上述说明是指胞内浓度，但是3-HBal、1,3-BDO、4-HBal或1,4-BDO生产细胞可以胞内产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，和/或将产品分泌到培养基中。
如本文所述，实现3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成的一种示例性生长条件包括厌氧培养或发酵条件。在某些实施方式中，本发明的非天然存在的细胞可以在厌氧或基本厌氧条件下维持、培养或发酵。简要地，厌氧条件是指缺少氧的环境。基本厌氧条件包括(例如)培养、分批发酵或连续发酵，从而培养基中的溶氧浓度保持在0至10％饱和之间。基本厌氧条件还包括将细胞在维持在小于1％的氧的气氛的密封盒内的液体培养基或固体琼脂上生长或休眠。可以通过(例如)用N2/CO2的混合物或者其它适合的非氧气或气体向培养中鼓泡来维持氧的百分比。
本文所述的培养条件可以放大并连续生长以用于通过本发明的细胞生产3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺。示例性的生长程序包括(例如)进料-分批发酵和分批分离；进料-分批发酵和连续分离，或连续发酵和连续分离。所有这些方法在本领域中是熟知的。发酵程序对于商品化的量的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成生产是特别有用的。通常并且如非连续培养程序一样，3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的连续和/或近-连续生产将包括在充足的营养物和培养基中培养本发明的生产3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的非天然存在的细胞以保持和/或几乎保持指数期的生长。处于这些条件下的连续培养可以包括(例如)生长或培养1、2、3、4、5、6或7天或以上。另外，连续培养可以包括1周、2、3、4或5或更多周并且长达数月的更长的时间段。作为另外一种选择，如果适合于具体应用，可以将本发明的生物培养数小时。应理解连续和/或近-连续培养条件还可以包括这些示例性时间段之间的所有时间间隔。还应理解培养本发明的细胞的时间是出于所期望的目的，生产足够量的产品的足够的一段时间。
示例性发酵过程包括(但不限于)进料-分批发酵和分批分离；进料-分批发酵和连续分离；以及连续发酵和连续分离。在示例性分批发酵规程中，使生产生物在用适当气体鼓泡的适合尺寸的生物反应器中生长。在厌氧条件下，用惰性气体或气体，例如，氮气、N2/CO2混合物、氩气、氦等的组合对培养鼓泡。随着细胞生长和使用碳源，以大致平衡碳源和/或营养物的消耗的速度将其它碳源和/或其它营养物进料至生物反应器。将生物反应器的温度维持在所期望的温度，通常在22-37℃的范围内，但是基于生产生物的生长特征和/或发酵过程所需的条件，所述温度可以维持在更高或更低的温度。生长持续所期望的一段时间，从而在发酵罐中实现所期望的培养特征，例如，细胞密度、产品浓度等。在分批发酵过程中，基于所期望的培养条件，发酵的时间长度通常在几小时至几天的范围内，例如，8至24小时，或者1、2、3、4或5天，或者长达一周。根据需要，pH可以是控制或不控制的，在此情况下在运行结束时，其中pH不控制的培养通常将降低至pH 3-6。在培养期完成时，可以将发酵罐的内容物通过细胞分离单元，例如，离心机、过滤单元等以除去细胞和细胞碎片。在其中胞内表达所期望的产品的情况下，可以根据需要，在将细胞与发酵液分离之前或之后，使细胞酶促或化学裂解或破裂，以释放其它产品。可以将发酵液转移至产品分离单元。通过在本领域中使用的标准分离程序进行产品分离以将所期望的产品与稀释的水溶液分离。基于所述发酵过程的产品的化学特性，这些方法包括(但不限于)使用与水不混溶的有机溶剂(例如，甲苯或其它适合的溶剂，包括(但不限于)二乙醚、乙酸乙酯、四氢呋喃(THF)、二氯甲烷、氯仿、苯、戊烷、己烷、庚烷、石油醚、甲基叔丁基醚(MTBE)、二恶烷、二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)等)的液-液萃取法以提供所述产品的有机溶液，如果适当，标准蒸镏法等。
在示例性完全连续发酵规程中，生产生物通常首先以分批模式长成以实现所期望的细胞密度。当碳源和/或其它营养物耗尽时，以所期望的速率连续提供具有相同组成的进料培养基，并且以相同速率排出发酵液。在这些条件下，生物反应器中的产品浓度以及细胞密度通常保持恒定。如以上所讨论的，发酵罐的温度保持在所期望的温度。在连续发酵阶段，通常对于优化生产，期望维持适合的pH范围。可以使用常规方法监测和维持pH，其包括添加适合的酸或碱以维持所期望的pH范围。根据情况和需要，连续操作所述生物反应器延长的一段时间，通常至少一周至几周并且长达一个月或更长的时间。根据需要，定期监测发酵液和/或培养，包括多达每天取样，以确保产品浓度和/或细胞密度的一致性。以连续方式，随着新的进料培养基的提供，恒定除去发酵罐的内容物。根据需要，在除去或不除去细胞和细胞碎片的情况下，通常使含有细胞、培养基和产品的排出液流经受连续的产品分离程序。在本领域中使用的连续分离方法可以用于将所述产品与稀释的水溶液分离，其包括(但不限于)使用与水不混溶的有机溶剂(例如，甲苯或其它适合的溶剂，包括(但不限于)二乙醚、乙酸乙酯、四氢呋喃(THF)、二氯甲烷、氯仿、苯、戊烷、己烷、庚烷、石油醚、甲基叔丁基醚(MTBE)、二恶烷、二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)等)的连续液-液萃取法、标准连续蒸镏法等，或者本领域中熟知的其它方法。
发酵程序在本领域中是熟知的。简要地，可以在(例如)进料-分批发酵和分批分离；进料-分批发酵和连续分离，或连续发酵和连续分离中使用用于3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成生产的发酵。分批和连续发酵程序的实例在本领域中是熟知的并在本文中进行了描述。
除用于大量3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的连续生产的本文所述的使用3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生产菌的发酵程序之外，所述3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之相关的下游产品，如酯或酰胺的生产菌还可以(例如)同时经受化学合成和/或酶促过程以将所述产品转化为其它化合物，或者所述产品可以与发酵培养分离并且如果需要，顺序经受化学和/或酶转化以将所述产品转化为其它化合物。
除本文所公开的培养和发酵条件之外，实现本发明的醛脱氢酶的表达或者3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的生物合成的生长条件可以包括向所述培养条件中添加渗透保护剂。在某些实施方式中，本发明的非天然存在的细胞可以在存在渗透保护剂的情况下如本文所述进行维持、培养或发酵。简要地，渗透保护剂是指用作渗透剂并且帮助如本文所述的细胞在渗透应力下存活的化合物。渗透保护剂包括(但不限于)甜菜碱、氨基酸和糖海藻糖。这些的非限制性实例为甘氨酸甜菜碱、果仁糖甜菜碱、二甲基噻亭、二甲基锍基丙酸酯、3-二甲基锍基-2-甲基丙酸酯、哌啶酸、二甲基锍基乙酸酯、胆碱、L-卡尼汀和四氢嘧啶(ectoine)。在一个方面，所述渗透保护剂为甘氨酸甜菜碱。本领域的技术人员应理解适合于保护本文所述的细胞抵抗渗透应力的渗透保护剂的量和类型将取决于所使用的细胞。所述培养条件中渗透保护剂的量可以是(例如)不超过约0.1mM、不超过约0.5mM、不超过约1.0mM、不超过约1.5mM、不超过约2.0mM、不超过约2.5mM、不超过约3.0mM、不超过约5.0mM、不超过约7.0mM、不超过约10mM、不超过约50mM、不超过约100mM或者不超过约500mM。
在一些实施方式中，可以选择碳原料及其它细胞吸收源，如磷酸盐、氨、硫酸盐、氯化物及其它卤素以改变存在于3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者任何3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体中的原子的同位素分布。以上列举的多种碳原料及其它吸收源将在本文中统称为“吸收源”。吸收源可以提供对于存在于产品3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体中的任何原子，或者对于在背离3-HBal、1,3-BDO、4-HBal或1,4-BDO途径的反应中产生的副产品的同位素浓缩。可以对于任何靶原子，包括(例如)碳、氢、氧、氮、硫、磷、氯化物或其它卤素实现同位素富集。
在一些实施方式中，可以选择所述吸收源以改变碳-12、碳-13和碳-14的比。在一些实施方式中，可以选择所述吸收源以改变氧-16、氧-17和氧-18的比。在一些实施方式中，可以选择所述吸收源以改变氢、氘和氚的比。在一些实施方式中，可以选择所述吸收源以改变氮-14和氮-15的比。在一些实施方式中，可以选择所述吸收源以改变硫-32、硫-33、硫-34和硫-35的比。在一些实施方式中，可以选择所述吸收源以改变磷-31、磷-32和磷-33的比。在一些实施方式中，可以选择所述吸收源以改变氯-35、氯-36和氯-37的比。
在一些实施方式中，可以通过选择一种或多种吸收源将靶原子的同位素比改变至所期望的比。吸收源可以来源于天然来源，如自然界所存在的，或者来源于人为来源，并且本领域技术人员可以选择天然来源、人为来源或它们的组合以实现所期望的靶原子的同位素比。人为吸收源的实例包括(例如)至少部分来源于化学合成反应的吸收源。这些同位素富集的吸收源可以是商购的或者在实验室中制备的和/或任选地与所述吸收源的天然源混合以实现所期望的同位素比。在一些实施方式中，可以通过选择如自然界中所存在的所期望的吸收源来源来实现吸收源的靶原子同位素比。例如，如本文所讨论的，天然源可以是生物基源，其来源于生物或者通过生物合成，或者是如石油基产品或大气的来源。在一些这些实施方式中，碳源(例如)可以选自化石燃料-衍生的碳源，其可以相对不含碳-14，或者环境或大气碳源，如CO2，其可以比其石油-衍生的对应物具有更大量的碳-14。
在地球大气中，不稳定的碳同位素碳-14或放射性碳大致占1012个碳原子中的1个，并且其半衰期为约5700年。通过包括宇宙射线和常规氮(14N)的核反应，在上层大气中补充碳储量。由于其很早之前衰变，因此化石燃料不包含碳-14。化石燃料的燃烧降低了大气碳-14的占比，即所谓的“苏斯效应”。
确定化合物中原子的同位素比的方法对于本领域技术人员来说是熟知的。使用本领域中已知的技术，如加速质谱(AMS)、稳定同位素比质谱(SIRMS)和点特异性天然同位素分馏核磁共振技术(SNIF-NMR)，通过质谱容易地评价了同位素富集。这些质谱技术可以与分离技术，如液相色谱(LC)、高效液相色谱(HPLC)和/或气相色谱等集成。
就碳而言，在美国作为通过美国材料试验协会(ASTM)国际组织，使用放射性碳年代测定，用于确定固体、液体和气体样品的生物基含量的标准化分析方法开发了ASTMD6866。该标准基于用于确定产品的生物基含量的放射性碳年代测定的使用。ASTM D6866于2004年首次颁布，并且该标准当前有效的版本为ASTM D6866-11(2011年4月1日生效)。放射性碳年代测定技术对于本领域技术人员来说是熟知的，其包括本文所述的那些。
通过碳-14(14C)与碳-12(12C)的比值来估计化合物的生物基含量。具体地，根据下式计算现代分率(Fraction Modern，Fm)：Fm＝(S-B)/(M-B)，其中B、S和M分别代表空白、样品和现代参比的14C/12C比。现代分率是来自“现代”的样品的14C/12C比的偏差的测量。现代的定义是归一化至δ13CVPDB＝-19/1000的国家标准局(NBS)草酸I(即标准参考物质(SRM)4990b)的放射性碳浓度(公元1950年)的95％(Olsson,The use of Oxalic acid as aStandard.in,Radiocarbon Variations and Absolute Chronology,Nobel Symposium,12th Proc.,John Wiley&Sons,New York(1970))。使用国际上约定的定义：0.95乘以归一化至δ13CVPDB＝-19/1000的NBS草酸I(SRM 4990b)的比活，计算(例如)通过ASM所测量的质谱结果。这相当于1.176±0.010×10-12的绝对(公元1950年)14C/12C比(Karlen等人,ArkivGeofysik,4:465-471(1968))。标准计算考虑了一种同位素相对于另一种的差异吸收，例如，生物系统对12C的吸收优先于13C，然后优先于14C，并且这些修正反映为对于δ13所修正的Fm。
草酸标准品(SRM 4990b或HOx 1)是从一批1955年的甜菜所制备的。尽管制备了1000磅，但是这种草酸标准品不再是可商购的。草酸II标准品(HOx 2；N.I.S.T名称为SRM4990 C)是由一批1977年的法国甜菜糖蜜所制成的。在1980年代早期，12个实验室的小组测量了两种标准品的比值。草酸II与1的活力的比值为1.2933±0.001(加权平均数)。HOx II的同位素比为-17.8/1000。ASTM D6866-11建议使用可用的草酸II标准品SRM 4990 C(Hox2)作为现代标准品(参见Mann,Radiocarbon,25(2):519-527(1983)中有关原始vs.现用的草酸标准品的讨论)。Fm＝0％代表材料中完全缺少碳-14原子，因此表示是化石(例如，石油基)碳源。在对于1950年后来自核弹试验的向大气注入的碳-14进行修正之后，Fm＝100％表示完全现代的碳源。如本文所述，这种“现代”源包括生物基源。
如ASTM D6866中所述，由于1950年代的核试验项目的持续但逐渐减弱的影响，其导致了如ASTM D6866-11中所述的碳-14在大气中的大量富集，因此现代碳的百分比(pMC)可以大于100％。由于所有样品的碳-14活性是以“核弹前”的标准品为参考的，并且因为几乎所有的新的生物基产品是在核弹后的环境中产生的，因此所有pMC值(对于同位素份数修正后)必须乘以0.95(截止2010年)以更好地反映样品真正的生物基含量。大于103％的生物基含量表明要么是发生了分析误差，要么是生物基碳源超过数年。
ASTM D6866相对于材料的总有机物含量来定量生物基含量，但是不考虑所存在的无机碳及其它含有非碳的物质。例如，基于ASTM D6866，50％淀粉-基材料和50％水的产品将认为具有生物基含量＝100％(作为100％生物基的50％的有机物含量)。在另一个实例中，50％淀粉-基材料、25％石油基材料和25％的水的产品将具有生物基含量＝66.7％(75％有机物含量，但仅有50％的产品是生物基的)。在另一个实例中，作为50％有机碳并且是石油基产品的产品将认为具有生物基含量＝0％(50％有机碳，但是来自化石来源)。因此，基于用于确定化合物或材料的生物基含量的熟知方法和已知标准品，本领域技术人员可以容易地确定化合物或材料和/或使用具有所期望的生物基含量的化合物或材料所制备的下游产品的生物基含量。
应用碳-14年代测定技术来定量材料的生物基含量在本领域中是已知的(Currie等人,Nuclear Instruments and Methods in Physics Research B,172:281-287(2000))。例如，碳-14年代测定已用于定量含对苯二酸酯材料中的生物基内容物(Colonna等人,Green Chemistry,13:2543-2548(2011))。值得注意地，来源于可再生的1,3-丙二醇和石油-衍生的对苯二甲酸的聚丙烯对苯二甲酸酯(PPT)聚合物导致Fm值接近30％(即，因为3/11的所述聚合物碳衍生自可再生的1,3-丙二醇，8/11来自化石端成员对苯二甲酸)(Currie等人，如上，2000)。相反，衍生自可再生的1,4-丁二醇和可再生的对苯二甲酸两者的聚对苯二甲酸丁二醇酯聚合物导致生物基含量大于90％(Colonna等人，如上，2011)。
因此，在一些实施方式中，本发明提供了通过本发明的细胞所产生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之相关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体，其具有反映大气碳，也称为环境碳吸收源的碳-12、碳-13和碳-14比。例如，在一些方面，3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体可以具有至少10％，至少15％，至少20％，至少25％，至少30％，至少35％，至少40％，至少45％，至少50％，至少55％，至少60％，至少65％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少98％或多至100％的Fm值。在一些这些实施方式中，所述吸收源是CO2。在一些实施方式中，本文提供了具有反映石油基碳吸收源的碳-12、碳-13和碳-14比的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体。在该方面，3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体可以具有小于95％，小于90％，小于85％，小于80％，小于75％，小于70％，小于65％，小于60％，小于55％，小于50％，小于45％，小于40％，小于35％，小于30％，小于25％，小于20％，小于15％，小于10％，小于5％，小于2％或小于1％的Fm值。在一些实施方式中，本发明提供了具有通过大气碳吸收源与石油基吸收源的组合所获得的碳-12、碳-13和碳-14比的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体。使用这种吸收源的组合是可以改变碳-12、碳-13和碳-14比的一种方式，并且各个比将反映吸收源的比。
此外，本发明涉及如本文所公开的生物生产的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体，以及由此衍生的产品，其中3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体具有与环境中存在的CO2大致相同的值的碳-12、碳-13和碳-14同位素比。例如，在一些方面，本发明提供了生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或生物衍生的3-HBal、1,3-BDO、4-HBal、1,4-BDO中间体，其具有与环境中所存在的CO2大致相同的值的碳-12相对于碳-13相对于碳-14的同位素比或本文所公开的任何其它比值。如本文所公开的，应理解产品可以具有与环境中所存在的CO2大致相同的值的碳-12相对于碳-13相对于碳-14的同位素比或本文所公开的任何比值，其中所述产品是从如本文所公开的生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体产生的，其中对所述生物衍生的产品化学修饰以产生最终产品。如本文所述，化学修饰3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO的中间体的生物衍生产品以产生所期望的产品的方法对于本领域技术人员来说是熟知的。本发明还提供了塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯、尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品，其可以是基于3-HBal和/或1,3-BDO或与之有关的下游产品，如其酯或酰胺的，和塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM、尼龙等，其可以是基于4-HBal和/或1,4-BDO或与之有关的下游产品，如其酯或酰胺的，其具有与环境中存在的CO2大致相同的值的碳-12相对于碳-13相对于碳-14的同位素比，其中所述塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM、尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品是直接从或结合如本文所公开的生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体产生的。先前已描述了用于产生丁二烯和/或丁二烯-基产品的方法(参见，例如，WO 2010/127319、WO2013/036764、美国专利No.9,017,983、US 2013/0066035、WO/2012/018624、US 2012/0021478，以上每项专利作为参考并入本文)。使用(例如)脂肪酶，1,3-BDO可以与酸体内或体外反应以转化为酯。这些酯可以具有营养、药物和食品用途，并且当使用1,3-BDO的R-形式时，具有优势，因为这是动物和人作为能源的最佳利用形式(与S-形式或外消旋混合物相比)(例如，酮酯，如(R)-3-羟丁基-R-1,3-丁二醇单酯(其具有美国公认安全(GRAS)批准)和(R)-3-羟基丁酸酯甘油单酯或二酯)。所述酮酯可以口服递送并且所述酯释放通过身体使用的R-1,3-丁二醇(参见，例如，WO2013150153)。生产酰胺的方法在本领域中是熟知的(参见，例如，Goswami and Van Lanen,Mol.Biosyst.11(2):338-353(2015))。
因此，本发明对于提供改善的酶促路线和微生物以提供高度富集或基本对映体纯的并且相对于副产品还具有改善的纯度质量的改善的1,3-BDO，即R-1,3-丁二醇的组合物是特别有用的。1,3-BDO具有其它食品相关用途，包括直接作为食品源使用、食品成分、调味剂、调味剂的溶剂或增溶剂、稳定剂、乳化剂和抗-微生物剂和防腐剂。在制药工业中，1,3-BDO作为肠胃外药物溶剂使用。1,3-BDO在化妆品中作为以下成分使用：润肤剂、防止不溶性成分结晶的稀释剂、用于水溶性低的成分，如香料的增溶剂和作为抗-微生物剂和防腐剂。例如，它可以用作稀释剂，特别是在头发喷雾和卷发剂中；它降低了来自精油的香味的损失，防止通过微生物的腐败和用作苯甲酸酯的溶剂。1,3-BDO可以在0.1％至50％并且甚至小于0.1％并且甚至大于50％的浓度使用。它在头发和洗浴产品、眼部和面部化妆品、香料、个人清洁产品以及剃须和皮肤护理制剂中使用(参见，例如，化妆品成分审查委员会的报告："Final Report on the Safety Assessment of Butylene Glycol,Hexylene Glycol,Ethoxydiglycol,and Dipropylene Glycol",Journal of the American College ofToxicology,4卷,5期,1985，该文献作为参考并入本文)。该报告提供了1,3-BDO在化妆品中的具体使用和浓度；参见，例如，其中标题为“产品制剂数据(Product Formulation Data)”的报告的表2。
在一个实施方式中，本发明提供了包含生物衍生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的培养基，其中所述生物衍生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO具有反映大气二氧化碳吸收源的碳-12、碳-13和碳-14同位素比，并且其中生物衍生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO是通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的。在一个实施方式中，所述培养基分离自所述细胞。
在一个实施方式中，本发明提供了3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或者4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)，其具有反映大气二氧化碳吸收源的碳-12、碳-13和碳-14同位素比，其中所述3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO是通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的。在一个实施方式中，3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO具有至少80％，至少85％，至少90％，至少95％或至少98％的Fm值。
在一个实施方式中，本发明提供了通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或者4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)。在一个实施方式中，本发明提供了3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或者4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)，其具有反映大气二氧化碳吸收源的碳-12、碳-13和碳-14同位素比，其中所述3-HBal和/或1,3-BDO是通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的，其中所述3-HBal和/或1,3-BDO是对于R形式对映体富集的。在一个实施方式中，3-HBal和/或1,3-BDO具有至少80％，至少85％，至少90％，至少95％或至少98％的Fm值。
在一个实施方式中，本发明提供了通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)，其中所述3-HBal和/或1,3-BDO是对于R形式对映体富集的。在一个实施方式中，R形式是大于95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的3-HBal和/或1,3-BDO。在一个实施方式中，3-HBal和/或1,3-BDO是≥55％的R-对映异构体，≥60％的R-对映异构体，≥65％的R-对映异构体，≥70％的R对映异构体，≥75％的R-对映异构体，≥80％的R-对映异构体，≥85％的R-对映异构体，≥90％的R-对映异构体或者≥95％的R-对映异构体，并且可以是高度化学纯的，例如，≥99％，例如，≥95％，≥96％，≥97％，≥98％，≥99％，≥99.1％，≥99.2％，≥99.3％，≥99.4％，≥99.5％，≥99.6％，≥99.7％，≥99.8％或≥99.9％的R对映异构体。
在一个实施方式中，本发明提供了组合物，其包含通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO和分别除3-HBal和/或1,3-BDO或者4-HBal或1,4-BDO以外的化合物。在一个实施方式中，除3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO以外的化合物是分别产生3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO或者表达本发明的多肽的细胞的一部分。
在一个实施方式中，本发明提供了组合物，其包含通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO，或者产生3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的细胞裂解液或细胞培养上清液。
在一个实施方式中，本发明提供了包含通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO，其中所述产品是塑料、弹性纤维、聚氨脂、聚酯、聚羟基脂肪酸酯、聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)、聚对苯二甲酸丁二醇酯(PBT)、聚氨脂-聚脲共聚物、尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯或丁二烯-基产品。在一个实施方式中，所述产品是化妆品产品或食品添加剂。在一个实施方式中，所述产品包含至少0.1％、至少0.5％、至少1％、至少5％、至少10％、至少20％、至少30％、至少40％或至少50％的生物衍生的3-HBal和/或1,3-BDO，或者生物衍生的4-HBal和/或1,4-BDO。在一个实施方式中，所述产品作为重复单元包含产生的3-HBal和/或1,3-BDO，或者产生的4-HBal和/或1,4-BDO的部分。在一个实施方式中，本发明提供了通过模制通过本发明的细胞或在细胞裂解液中或者通过本发明所述的方法产生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO所制备的或者来源于它们的产品所获得的模制产品。
本发明还提供了组合物，其包含生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，和除了生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺以外的化合物。除所述生物衍生产品以外的化合物可以是细胞部分，例如，微量的细胞部分，或者可以是在存在具有产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的途径的本发明的非天然存在的细胞的情况下所产生的发酵液或培养基或其纯化或部分纯化的部分。如本文所公开的，所述组合物可以包含(例如)当通过具有减少的副产品形成的生物产生时的降低的副产品水平。所述组合物可以包含(例如)生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，和本发明的细胞裂解液或细胞培养上清液。
3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺是在商品化应用和工业应用中使用的化学品。这些应用的非限制性实例包括塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品的产生。此外，3-HBal、1,3-BDO、4-HBal或1,4-BDO也在广泛的产品的生产中用作原材料，所述产品包括塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品。因此，在一些实施方式中，本发明提供了生物基塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品，其包含通过本发明的非天然存在的细胞，例如，表达本发明的醛脱氢酶的细胞或者使用本文所公开的方法产生的细胞所产生的一种或多种生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体。
如本文所使用的，术语“生物衍生的”表示来源于生物或者通过生物合成并且可以被认为是可再生资源，因为它可以通过生物产生。这种生物，具体地本文所公开的本发明的细胞可以使用原料或生物质，如得自农业、植物、细菌或动物来源的糖或碳水化合物。作为另外一种选择，所述生物可以使用大气碳。如本文所使用的，术语“生物基的”是指完全或部分由本发明的生物衍生的化合物组成的如上所述的产品。生物基或生物衍生产品与石油衍生产品形成对比，其中所述产品来源于或合成自石油或石油化工原料。
在一些实施方式中，本发明提供了塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品，其包含生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体，其中所述生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体包括在塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品的生产中使用的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体的全部或部分。例如，最终的塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品可以含有生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体或其部分，它是生产塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品的结果。这种生产可以包括将生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体化学反应(例如，化学转化、化学官能化、化学偶联、氧化、还原、聚合、共聚合等)成最终的塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品。因此，在一些方面，本发明提供了生物基塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品，其包含至少2％，至少3％，至少5％，至少10％，至少15％，至少20％，至少25％，至少30％，至少35％，至少40％，至少50％，至少60％，至少70％，至少80％，至少90％，至少95％，至少98％或100％的如本文所公开的生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体。
另外，在一些实施方式中，本发明提供了组合物，其具有本文所公开的生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体和除所述生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体以外的化合物。例如，在一些方面，本发明提供了生物基塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品，其中在其生产中使用的所述3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体是生物衍生的和石油衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体的组合。例如，可以使用50％的生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺和50％的石油衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，或者生物衍生/石油衍生前体的其它所期望的比值，如60％/40％、70％/30％、80％/20％、90％/10％、95％/5％、100％/0％、40％/60％、30％/70％、20％/80％、10％/90％生产生物基塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品，只要所述产品的至少一部分包含通过本文所公开的细胞所产生的生物衍生产品。应理解，使用本发明的生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺或者生物衍生的3-HBal、1,3-BDO、4-HBal或1,4-BDO途径中间体生产塑料、弹性纤维、聚氨脂、聚酯，包括聚羟基脂肪酸酯，如聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)(也称为PTMO，聚环丁烷氧化物)、聚对苯二甲酸丁二醇酯(PBT)和聚氨脂-聚脲共聚物，称为氨纶(spandex)、elastane或LycraTM，尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品的方法在本领域中是熟知的。
为了产生更好的生产菌，可以利用代谢模型来优化生长条件。建模还可以用于设计另外优化所述途径的利用的基因敲除(参见，例如，美国专利公开US 2002/0012939、US2003/0224363、US 2004/0029149、US 2004/0072723、US 2003/0059792、US2002/0168654和US 2004/0009466，和美国专利No.7,127,379)。建模分析使得能够可靠地预测对于将代谢向3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的更有效的生产的方向转变对细胞生长的影响。
鉴定和设计有利于所期望的产品的生物合成的代谢改变的一种计算机方法是OptKnock计算框架(Burgard等人,Biotechnol.Bioeng.84:647-657(2003))。OptKnock是一种代谢建模和模拟程序，其建议基因缺失或破坏策略，从而导致产生了过量生产目标产品的遗传稳定的微生物。具体地，该框架检查微生物的完整代谢和/或生物化学网络，以表明遗传操作，其迫使所期望的生物化学品成为细胞生长的强制性副产品。通过策略性地布置基因缺失或其它功能基因破坏，通过将生物化学品的生产与细胞生长偶联，作为强迫性生长-偶联的生物化学品生产的结果，在生物反应中长时间向工程化菌株施加生长选择压力来引起性能的改善。最后，当构建基因缺失时，存在设计菌株回复为它们的野生型状态的可忽略的的可能性，因为OptKnock所选择的基因将从基因组中完全移除。因此，这种计算机方法可以用于鉴定替代途径，所述途径将引起所期望的产品的生物合成，或与非天然发生的细胞一起使用，以用于进一步优化所期望的产品的生物合成。
简要地，OptKnock是在本文所使用以表示用于细胞代谢建模的计算机方法和系统的术语。OptKnock程序涉及模型和方法的框架，其向流量平衡分析(flux balanceanalysis，FBA)模型中引入特定的约束。这些约束包括(例如)定性动力学信息、定性调控信息和/或DNA微阵列实验数据。通过(例如)收紧通过流量平衡模型所衍生的流量边界，并随后在存在基因添加或缺失的情况下探测代谢网络的性能限制，OptKnock还计算了多种代谢问题的解决方案。OptKnock计算框架容许构建模型方程，其容许有效查询代谢网络的性能限制，并提供用于解决所产生的混合-整数线性规划问题的方法。本文称作OptKnock的代谢建模和模拟方法描述于(例如)2002年1月10日提交的美国专利公开2002/0168654，2002年1月10日提交的国际专利No.PCT/US02/00660，以及2007年8月10日提交的美国专利公开2009/0047719。
鉴定和设计有利于产品的生物合成生产的代谢改变的另一种计算机方法是称为
Figure BDA0003082149040000481
的代谢模式和模拟系统。这种计算机方法和系统描述于(例如)2002年6月14日提交的美国专利公开2003/0233218，和2003年6月13日提交的国际专利申请No.PCT/US03/18838。
Figure BDA0003082149040000482
是一种计算机系统，其可以用于产生计算机网络模型并模拟物质、能量或电荷在生物系统的化学反应中的流量，从而定义包含系统中的化学反应的任何和所有可能的功能性的解空间，借此确定该生物系统所容许的活性范围。这种方法被称为基于约束的建模，因为所述解空间是由约束来定义的，如所包括的反应的已知的化学计量以及与通过反应的最大通量有关的反应热力学和容量约束。可以查询这些约束所定义的空间以确定生物系统或其生物化学组分的表型能力和行为。
这些计算机方法与生物学事实是一致的，因为生物系统是柔性的，并且可以以许多不同的方式实现相同的结果。通过进化机制设计生物系统，其受所有活体系统必须面对的基础性约束的限制。因而，基于约束的建模策略涵盖了这些一般性事实。此外，通过收紧约束对网络模型连续施加进一步的限制的能力导致了解空间大小的降低，借此提高了可以预测的生理学表现或表型的精确度。
考虑到本文所提供的教导和指导，本领域技术人员将能够应用代谢建模和模拟的多种计算机框架来设计和实施宿主细胞中所期望的化合物的生物合成。这些代谢建模和模拟方法包括(例如)以上作为
Figure BDA0003082149040000483
和OptKnock举例说明的计算机系统。为了说明本发明，对于建模和模拟，在本文中参考OptKnock计算框架描述了一些方法。本领域技术人员将知晓如何将OptKnock的代谢改变的鉴定、设计和实施应用于任何这些其它代谢建模和模拟计算机框架以及本领域中熟知的方法。
如上所述的方法将提供要破坏的一组代谢反应。所述组内的每个反应的去除或代谢修饰可以导致作为生物生长期期间的强制性产品产生所期望的产品。由于反应是已知的，因此双层OptKnock问题的解决方案还将提供编码一种或多种酶的相关基因，所述酶催化该反应组内的每个反应。鉴定一组反应以及编码参与每个反应的酶的它们的相应基因一般是自动过程，其通过将所述反应与具有酶与编码基因之间的关系的反应数据库相关联来实现。
一旦被鉴定，则通过功能性破坏编码该组内每个代谢反应的至少一个基因，在目标细胞或生物中实施要破坏以实现所期望的产品的产生的这组反应。实现所述反应组的功能性破坏的一种特别有用的方式是使每个编码基因缺失。然而，在一些情况下，通过其它遗传变异来破坏反应可以是有益的，其包括(例如)调控区的突变、缺失，如启动子或调控因子的顺式结合位点，或通过在任何一些位置的编码序列的截短。后者的这些变异导致基因集小于全部的缺失，这可以是有用的，例如，当期望快速评价产品的偶联时，或当不太可能发生遗传回复时。
为了鉴定对于上述双层OptKnock问题的其它生产性解决方案，其导致产生了要破坏或代谢修饰的其它反应组，这可以导致所期望的产品的生物合成，包括生长-偶联的生物合成，可以实施称为整体切割的优化方法。以每次迭代引入称为整体切割的附加约束，通过迭代求解以上举例说明的OptKnock问题来进行这种方法。整体切割约束有效防止了解算过程选择任何之前迭代中鉴定的完全相同的将产品生物合成与生长强制性偶联的反应组。例如，如果先前鉴定的生长-偶联代谢修饰指明了反应1、2和3进行破坏，则随后的约束防止在随后的解决方案中同时考虑相同反应。整体切割方法是本领域中是熟知的，并且可以在(例如)Burgard等人,Biotechnol.Prog.17:791-797(2001)中找到。对于代谢建模和模拟，如同本文参考它们与OptKnock计算框架组合使用所描述的所有方法，降低迭代计算分析中的冗余的整体切割方法也可以应用于本领域中熟知的其它计算框架，包括(例如)
Figure BDA0003082149040000491
本文举例说明的方法容许构建生物合成生产所期望的产品的细胞和生物，其包括目标生物化学产品的生产与工程化以具有所鉴定的遗传改变的细胞或生物的生长的强制性偶联。因此，本文所述的计算方法容许鉴别和实施由选自OptKnock或
Figure BDA0003082149040000492
的计算机方法所鉴别的代谢修饰。代谢修饰组可以包括(例如)添加一种或多种生物合成途径酶和/或一种或多种代谢反应的功能性破坏，包括(例如)通过基因缺失破坏。
如以上所讨论的，OptKnock方法的开发的前提是：突变的微生物网络在经过长期的生长选择后，可以朝着其计算预测的最大-生长表型进化。换言之，该方法利用了生物在选择压力下的自我优化能力。OptKnock框架允许对基因缺失组合进行穷尽列举，从而基于网络化学计量强迫生物化学生产与细胞生长之间偶联。最优基因/反应敲除的鉴别需要解决双层优化问题，该问题选择了一组活性反应，从而使得所产生的网络的最优生长解过量生产所关心的生物化学品(Burgard Biotechnol.Bioeng.84:647-657(2003))。
如先前举例说明的和在(例如)美国专利公开US 2002/0012939、US 2003/0224363、US 2004/0029149、US 2004/0072723、US 2003/0059792、US 2002/0168654和US2004/0009466，以及美国专利No.7,127,379中描述的，可以使用大肠杆菌(E.coli)代谢的计算机化学计量模型来鉴别代谢途径的关键基因。如本文公开的，OptKnock数学框架可以应用于精确的基因缺失，从而引起所期望的产品的生长-偶联生产。此外，双层OptKnock问题的解仅提供了一组缺失。为了列举所有有意义的解，即引起形成生长偶联的生产的所有敲除组，可以实施称为整体切割的优化技术。如以上所讨论的，这需要迭代求解OptKnock问题，其中每次迭代引入称为整体切割的附加约束。
如本文所公开的，本发明涉及醛脱氢酶变体(参见实施例)。在实施例中描述了这些变体的产生。任何多种方法可以用于产生醛脱氢酶变体，如本文所公开的醛脱氢酶变体。这些方法包括(但不限于)定点突变、随机诱变、组合文库和如下所述的其它诱变方法(参见Sambrook等人,Molecular Cloning:A Laboratory Manual,第3版,Cold Spring HarborLaboratory,New York(2001)；Ausubel等人,Current Protocols in Molecular Biology,John Wiley and Sons,Baltimore,MD(1999)；Gillman等人,Directed Evolution LibraryCreation:Methods and Protocols(Methods in Molecular Biology)Springer,第2版(2014)。
如本文所公开的，可以将编码3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的途径的所期望的活力的核酸引入宿主生物。在一些情况下，可以期望修饰3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺，途径酶或蛋白的活力以提高3-HBal、1,3-BDO、4-HBal或1,4-BDO或与之有关的下游产品，如其酯或酰胺的产生。例如，可以将已知提高蛋白或酶活力的突变引入编码核酸分子。另外，可以应用优化方法以提高酶或蛋白的活力和/或降低抑制活力，例如，降低负调节蛋白的活力。
一种这种优化方法是定向进化。定向进化是包括引入靶向特定基因的突变以改善和/或改变酶的性质的强有力的方法。可以通过开发和实施允许自动筛选多种酶变体(例如，>104种)的灵敏的高通量筛选测定来鉴别改善和/或改变的酶。通常进行突变和筛选的迭代循环以提供具有优化性质的酶。还已开发了可以帮助鉴别用于突变的基因区域的计算算法，并且所述计算算法可以显著减少需要产生和筛选的酶变体的数目。已发展了多种定向进化技术(有关综述，参见Hibbert等人,Biomol.Eng 22:11-19(2005)；Huisman andLalonde,In Biocatalysis in the pharmaceutical and biotechnology industries717-742页(2007),Patel(主编),CRC Press；Otten and Quax.Biomol.Eng 22:1-9(2005).；和Sen等人,Appl.Biochem.Biotechnol 143:212-223(2007))以有效产生多种变体文库，并且这些方法已成功应用于多种酶种类中广泛的性质的改善。已通过定向进化技术改善和/或改变的酶的特性包括，例如：对于非天然底物的转化，选择性/特异性；对于稳健的高温处理，温度稳定性；对于在较低或较高的pH条件下的生物加工，pH稳定性；底物或产品耐受性，从而可以实现高产品滴度；结合(Km)，包括拓宽底物结合以包括非天然底物；抑制(Ki)，以除去抑制副产品、底物或关键中间体；活力(kcat)，以提高酶促反应速率以实现所期望的流量；表达水平，以提高蛋白得率和整体途径流量；氧气稳定性，用于空气敏感性酶在好氧条件下的操作；和厌氧活力，用于好氧酶在不存在氧气的情况下的操作。
已发展了针对特定酶的所期望的性质，使基因突变和多样化的一些示例性方法。这些方法对于本领域技术人员来说是熟知的。任何这些可以用于改变和/或优化用于产生3-HBal、1,3-BDO、4-HBal或1,4-BDO或者其下游产品，如其酯或酰胺的途径酶或蛋白或本发明的醛脱氢酶的活力。这些方法包括(但不限于)EpPCR，其通过降低PCR反应中DNA聚合酶的保真度来引入随机点突变(Pritchard等人,J Theor.Biol.234:497-509(2005))；易错滚环扩增(epRCA)，其类似于epPCR，除了将整个环形质粒用作模板并且将在最后2个核苷酸上具有核酸外切酶耐受性硫代磷酸键的随机6-聚体用于扩增所述质粒，然后将其转化至细胞，并且在所述细胞中，所述质粒在串联重复序列再环化(Fujii等人,Nucleic Acids Res.32:e145(2004)；和Fujii等人,Nat.Protoc.1:2493-2497(2006))；DNA或家族重排，其通常包括用核酸酶，如DNA酶I或者EndoV消化两种或更多种变体基因以产生随机片段池，所述随机片段池在存在DNA聚合酶的情况下通过退火和延伸循环重新组装以产生嵌合基因文库(Stemmer,Proc Natl Acad Sci USA91:10747-10751(1994)；和Stemmer,Nature 370:389-391(1994))；交错延伸(StEP)，其需要模板引导(template priming)，然后是具有变性和非常短的持续时间的退火/延伸(短至5秒)的2步PCR的重复循环(Zhao等人,Nat.Biotechnol.16:258-261(1998))；随机引导重组(RPR)，其中使用随机序列引物来产生与模板的不同片段互补的多个短DNA片段(Shao等人,Nucleic Acids Res 26:681-683(1998))。
其它方法包括异源双链重组，其中将线性化质粒DNA用于形成通过错配修复所修复的异源双链(Volkov等人,Nucleic Acids Res.27:e18(1999)；和Volkov等人,MethodsEnzymol.328:456-463(2000))；临时模板随机嵌合(RACHITT)，其使用了DNA酶I断裂和单链DNA(ssDNA)的大小分级(Coco等人,Nat.Biotechnol.19:354-359(2001))；截短模板上的重组延伸(RETT)，其需要在存在用作模板池的单向ssDNA片段的情况下链从引物单向生长的模板转换(Lee等人,J.Molec.Catalysis 26:119-129(2003))；简并寡核苷酸基因重排(DOGS)，其中将简并引物用于控制分子之间的重组；(Bergquist and Gibbs,MethodsMol.Biol 352:191-204(2007)；Bergquist等人,Biomol.Eng22:63-72(2005)；Gibbs等人,Gene 271:13-20(2001))；用于产生杂合酶的增长截短法(ITCHY)，其产生了具有所关心的基因或基因片段的1个碱基对缺失的组合文库(Ostermeier等人,Proc.Natl.Acad.Sci.USA96:3562-3567(1999)；和Ostermeier等人,Nat.Biotechnol.17:1205-1209(1999))；用于产生杂合酶的硫代增长截短法(THIO-ITCHY)，其类似于ITCHY，除了将硫代磷酸酯dNTP用于产生截短(Lutz等人,Nucleic Acids Res 29:E16(2001))；SCRATCHY，其组合了用于重组基因的两种方法，ITCHY和DNA重排(Lutz等人,Proc.Natl.Acad.Sci.USA 98:11248-11253(2001))；随机漂变突变(RNDM)，其中通过epPCR进行突变，然后筛选/选择保留了可用活力的那些(Bergquist等人,Biomol.Eng.22:63-72(2005))；序列饱和突变(SeSaM)，随机突变方法，其使用硫代磷酸酯核苷酸的随机掺入和切割，产生随机长度片段池，所述随机长度片段池用作在存在“通用”碱基，如肌苷的情况下延伸的模板，并且含有肌苷的互补序列的复制提供了随机碱基掺入，并因此导致突变(Wong等人,Biotechnol.J.3:74-82(2008)；Wong等人,Nucleic Acids Res.32:e26(2004)；和Wong等人,Anal.Biochem.341:187-189(2005))；合成重排，其使用设计以编码“靶标中所有遗传多样性”的重叠寡核苷酸并且允许重排子代具有很高的多样性(Ness等人,Nat.Biotechnol.20:1251-1255(2002))；核苷酸交换和切割技术NexT，其利用了dUTP掺入与随后使用尿嘧啶DNA糖基化酶，然后用哌啶处理以进行终点DNA断裂的组合(Muller等人,Nucleic Acids Res.33:e117(2005))。
其它方法包括不依赖于序列同源性的蛋白质重组(SHIPREC)，其中将接头用于辅助两种远缘相关或不相关基因的融合，并且在所述两种基因之间产生一定范围的嵌合体，从而导致产生了单一混合杂交文库(Sieber等人,Nat.Biotechnol.19:456-460(2001))；基因位点饱和突变TM(GSSMTM)，其中起始材料包括在所期望的突变位点含有简并的插入和两个引物的超螺旋双链DNA(dsDNA)质粒(Kretz等人,Methods Enzymol.388:3-11(2004))；组合盒式突变(CCM)，其包括使用短寡核苷酸盒以替换具有大量可能的氨基酸序列变化的限制区(Reidhaar-Olson等人,Methods Enzymol.208:564-586(1991)；和Reidhaar-Olson等人Science 241:53-57(1988))；组合多级盒式突变(CMCM)，其本质上类似于CCM并且使用高突变率的epPCR来鉴别热点(hot spots)和热区(hot regions)，然后通过CMCM延伸以覆盖限定的蛋白序列空间区域(Reetz等人,Angew.Chem.Int.Ed Engl.40:3589-3591(2001))；致突变菌株技术，其中条件性ts致突变质粒使用了mutD5基因，其编码了DNA聚合酶III的突变体亚基，从而在选择期间和当不需要选择时，在阻断有害突变的积累期间，允许随机和自然突变频率提高20至4000-X(Selifonova等人,Appl.Environ.Microbiol.67:3645-3649(2001))；Low等人,J.Mol.Biol.260:359-3680(1996))。
其它示例性方法包括浏览突变(LTM)，它是评价和优化所选氨基酸的组合突变的多维突变方法(Rajpal等人,Proc.Natl.Acad.Sci.USA 102:8466-8471(2005))；基因重组装，它是可以一次应用于多个基因并且产生单个基因的大型嵌合体文库(多个突变)的DNA重排法(Verenium Corporation所提供的Tunable GeneReassemblyTM(TGRTM)技术)、计算机蛋白设计自动化(PDA)，它是锚定具有特定折叠的在结构上限定的蛋白主链，并且搜索可以稳定所述折叠和整体蛋白能学的用于氨基酸替换的序列空间的优化算法，并且它通常对具有已知三维结构的蛋白最有效(Hayes等人,Proc.Natl.Acad.Sci.USA 99:15926-15931(2002))；和迭代饱和突变(ISM)，其包括使用结构/功能的知识来选择用于酶改善的可能位点，使用突变法，如Stratagene QuikChange(Stratagene；San Diego CA)在所选位点进行饱和突变，对于所期望的性质进行筛选/选择，并且使用改善的克隆，在另一位点重复一次并继续重复直至实现所期望的活力(Reetz等人,Nat.Protoc.2:891-903(2007)；和Reetz等人,Angew.Chem.Int.Ed Engl.45:7745-7751(2006))。
可以单独或以任意组合使用任何上述突变方法。另外，所述定向进化方法中的任一种或组合可以结合如本文所述的适应进化技术使用。
应理解在本文所提供的本发明的定义内，还提供了不显著影响本发明的多个实施方式的活性的改变。因此，以下实施例旨在说明，但不限制本发明。
实施例
醛脱氢酶变体
本实施例描述了具有所期望的性质的醛脱氢酶变体的产生。
将诱变技术用于产生基于模板ALD-1的变体醛脱氢酶。使用易错PCR、定点突变和通过遗传选择期间的自发突变产生变体。模板ALD-1对应于以下所提供的醛脱氢酶：
MIKDTLVSITKDLKLKTNVENANLKNYKDDSSCFGVFENVENAISNAVHAQKILSLHYTKEQREKIITEIRKAALENKEILATMILEETHMGRYEDKILKHELVAKYTPGTEDLTTTAWSGDNGLTVVEMSPYGVIGAITPSTNPTETVICNSIGMIAAGNTVVFNGHPGAKKCVAFAVEMINKAIISCGGPENLVTTIKNPTMDSLDAIIKHPSIKLLCGTGGPGMVKTLLNSGKKAIGAGAGNPPVIVDDTADIEKAGKSIIEGCSFDNNLPCIAEKEVFVFENVADDLISNMLKNNAVIINEDQVSKLIDLVLQKNNETQEYSINKKWVGKDAKLFLDEIDVESPSSVKCIICEVSASHPFVMTELMMPILPIVRVKDIDEAIEYAKIAEQNRKHSAYIYSKNIDNLNRFEREIDTTIFVKNAKSFAGVGYEAEGFTTFTIAGSTGEGITSARNFTRQRRCVLAG(SEQ ID NO：1)。
以下提供了ALD-2和ALD-3的其它ALD序列：
ALD-2
MNTENIEQAIRKILSEELSNPQSSTATNTTVPGKNGIFKTVNEAIAATKAAQENYADQPISVRNKVIDAIREGFRPYIEDMAKRIHDETGMGTVSAKIAKLNNALYNTPGPEILQPEAETGDGGLVMYEYAPFGVIGAVGPSTNPSETVIANAIMMLAGGNTLFFGAHPGAKNITRWTIEKLNELVADATGLHNLVVSLETPSIESVQEVMQHPDVAMLSITGGPAVVHQALISGKKAVGAGAGNPPAMVDATANIALAAHNIVDSAAFDNNILCTAEKEVVVEAAVKDELIMRMQQEGAFLVTDSADIEKLAQMTIGPKGAPDRKFVGKDATYILDQAGISYTGTPTLIILEAAKDHPLVTTEMLMPILPVVCCPDFDSVLATATEVEGGLHHTASIHSENLPHINKAAHRLNTSIFVVNGPTYCGTGVATNGAHSGASALTIATPTGEGTATSKTYTRRRRLNSPEGFSLRTWEA(SEQ ID NO：2)
ALD-3
MTVNEQLVQDIIKNVVASMQLTQTNKTELGVFDDMNQAIEAAKEAQLVVKKMSMDQREKIISAIRKKTIEHAETLARMAVEETGMGNVGHKILKHQLVAEKTPGTEDITTTAWSGDRGLTLVEMGPFGVIGAITPCTNPSETIICNTIGMLAGGNTVVFNPHPAAIKTSNFAVQLINEASLSAGGPVNIACSVRKPTLDSSKIMMSHQDIPLIAATGGPGVVTAVLQSGKRGIGAGAGNPPVLVDETADIRKAAEDIINGCTFDNNLPCIAEKEVVAIDAIANELMNYMVKEQGCYAITKEQQEKLTNLVITPKGLNRNCVGKDARTLLGMIGIDVPSNIRCIIFEGEKEHPLISEELMMPILGIVRAKSFDDAVEKAVWLEHGNRHSAHIHSKNVDRITTYAKAIDTAILVKNAPSYAAIGFGGEGFCTFTIASRTGEGLTSASTFTKRRRCVMSDSLCIR(SEQ ID NO：3)
与S对映异构体相比，ALD-1对3-羟基丁酰基-CoA的R对映异构体稍微更特异。图3显示了ALD-1与ALD-2和ALD-3的序列对比。所述序列分别对应于SEQ ID NO：1、2和3。对于ALD-3还存在晶体结构(PDBID 4C3S)，并且ALD-2与ALD-3比ALD-1更密切相关。因此，将ALD-3用作模板。图3中的下划线为两个环区，第一个表示为A，第二个表示为B，两者均参与底物特异性和对映异构体特异性，如本文所确定的。ALD-1中的环A是序列LQKNNETQEYSINKKWVGKD(SEQ ID NO：124)，ALD-2中的是序列IGPKGAPDRKFVGKD(SEQ ID NO：125)并且ALD-3中的是序列ITPKGLNRNCVGKD(SEQ ID NO：126)。ALD-1中的环B是序列SFAGVGYEAEGFTTFTIA(SEQ ID NO：127)，ALD-2中的是序列TYCGTGVATNGAHSGASALTIA(SEQID NO：128)并且ALD-3中的是序列SYAAIGFGGEGFCTFTIA(SEQ ID NO：129)。来自ALD-2的底物特异性环A和B的序列和长度不同于ALD-1和ALD-3的那些；尽管如此，比对显示足够保守以有利于鉴别如本文所述的替换的相应位置，并且如果与图6所示的3D建模(显示两个环区相互作用以影响底物特异性和对映异构体特异性，特别是当用如本文所述的示例性替换修饰时)结合，则尤其是这样。ALD-1和ALD-3是51.9％相同的。ALD-1和ALD-2是35.9％相同的。ALD-3和ALD-2是40％相同的。产生了基于图3的比对的共有ALD序列。基于ALD-1、ALD-2和ALD-3的比对的环A的一致性是IXPKG-----XXNRKXVGKD(SEQ ID NO：5)。基于ALD-1、ALD-2和ALD-3的比对的环B的一致性是SYAGXGXXXE----GFXTFTIA(SEQ ID NO：6)。
实施了其它比对(图4)。图4A显示了与ALD-1相比，具有40-55％截止值的比对。图4B显示了与ALD-1相比，具有75-90％截止值的比对。图4C显示了与ALD-1相比，具有90％截止值的比对。图4A-4C中所示的示例性醛脱氢酶(ALD)的比对表明鉴别了对应于其中可以进行本发明的替换的代表性模板ALD序列中的位置的ALD中的位置。对两个重要环区域加下划线，第一个表示为A，第二个表示为B，两者均参与底物特异性和对映异构体特异性，如本文所确定的。图4A-4C表明可以在与ALD-1具有至少40％的同一性的ALD中鉴别本文所教导的替换的相应位置，特别是环A和B区，并且特别是非常保守的环B区。
相对于乙酰-CoA，提高变体45对于3HB-CoA的特异性的诱变导致产生了几种1,3BDO产量提高且乙醇减少的变体。相对于乙酰-CoA提高3-羟基丁酰基-CoA的特异性的突变提供了乙醇的减少，因为可以通过宿主细胞中天然的酶或者通过将3-羟基丁醛转化为1,3-丁二醇的途径酶，将从乙酰-CoA产生的乙醛转化为乙醇。通过提高经过产生将乙酰乙酰-CoA朝着1,3-丁二醇形成方向推动的1,3-丁二醇的酶促途径的通量，降低其对于通过天然酶或不太特异的途径酶的向4-羟基-2-丁酮的两步转化的可用性，提高醛脱氢酶的酶活力或者提高其对于3-羟基丁酰基-CoA的特异性的变体减少了4-羟基-2-丁酮。以下提供了变体45的序列：
MIKDTLVSITKDLKLKTNVENANLKNYKDDSSCFGVFENVENAISNAVHAQKILSLHYTKEQREKIITEIRKAALENKEILATMILEETHMGRYEDKILKHELVAKYTPGTEDLTTTAWSGDNGLTVVEMSPYGVIGAITPSTNPTETVICNSIGMIAAGNTVVFNGHPGAKKSVAFAVEMINKAIISCGGPENLVTTIKNPTRDSLDAIIKHPSIKLLVGTGGPGMVKTLLNSGKKAIGAGAGNPPVIVDDTADIEKAGKSIIEGASFDNNLPCIAEKEVFVFENVADDLISNMLKNNAVIINEDQVSKLIDLVLQKNNETQEYSINKKWVGKDAKLFLDEIDVESPSSVKCIITEVSASHPFVMTELMMPILPIVRVKDIDEAIEYAKIAEQNHKHSAYIYSKNIDNLNRFEREIDTTIFVKNAKSFAGVGYEAPGFTTFTIAGSTGEGITSARNFTRQRRIVLVG(SEQ ID NO：4)
所实施的测定是体外测定以通过监测随着NADH向NAD的转化的吸光值的降低来检验对3HB-CoA的活力。还使用乙酰-CoA(AcCoA)作为底物进行测定，并且将改善的酶鉴别为3HB-CoA vs.AcCoA的活力比的改善。相对于乙酰-CoA提高3-羟基丁酰基-CoA的特异性的突变提供了乙醇的减少，因为可以通过宿主细胞中天然的酶或者通过将3-羟基丁醛转化为1,3-丁二醇的途径酶，将从乙酰-CoA产生的乙醛转化为乙醇。
使用(R)和(S)3-羟基丁醛的对这些变体亚组的进一步研究表明与亲代酶(变体45)和野生型ALD-1(图5)相比，所测试的变体中有5种(952、955、957、959、961)对R对映异构体具有改善的选择性。图5A显示了ALD-2、ALD-1和ALD-1变体对3羟基-(R)-丁醛(柱组中的左侧柱)和3羟基-(S)-丁醛(柱组中的右侧柱)的比活。在35℃，在存在10mM R或者S 3-羟基丁醛的情况下，在IVI缓冲液pH 7.5，0.5mM NAD+，2mM CoA中测定纯化的抗生蛋白链菌素-标签化蛋白，并且通过NADH在340nm的吸光值变化监测活力。IVI缓冲液含有5mM磷酸二氢钾，20mM磷酸氢二钾，10mM谷氨酸钠一水合物和150mM氯化钾，pH 7.5。因此，以与图1中所示的相反方向进行测定中的酶反应，即所述反应测量3-羟基丁醛向3-羟基丁酰基-CoA的转化。如图5B所示，某些醛脱氢酶变体对于R-3-羟基丁醛(R-3HB-醛)显示出高于S-3-羟基丁醛(S-3HB-醛)的选择性。
使用ALD-1晶体结构的突变体959的计算机建模表明氨基酸替换F442N使得能够与R异构体，而不是(S)异构体的碳3上的羟基形成氢键网络(图6)。图6A-6C显示了醛脱氢酶959结构的飘带图。该图显示了3-羟基-(R)-丁醛(图6A)或3-羟基-(S)-丁醛(图6B)与959结构的对接。图6C显示当在对于3-羟基-(R)-丁醛的对接能量最有利的相同取向对接3-羟基-(S)-丁醛时，产生了不利的相互作用(圆形)，其中异亮氨酸位于活性位点。该模型表明突变F442N在蛋白和3-羟基-(R)-丁醛的羟基之间产生了氢键，这对于S对映异构体是不可能的。
表1A-1D中显示了示例性的醛脱氢酶变体。
表1A.示例性ALD变体
Figure BDA0003082149040000551
Figure BDA0003082149040000561
Figure BDA0003082149040000571
Figure BDA0003082149040000581
Figure BDA0003082149040000591
Figure BDA0003082149040000601
Figure BDA0003082149040000611
Figure BDA0003082149040000621
Figure BDA0003082149040000631
表1B.示例性ALD变体
Figure BDA0003082149040000641
Figure BDA0003082149040000651
Figure BDA0003082149040000661
Figure BDA0003082149040000671
Figure BDA0003082149040000681
表1C.示例性ALD变体
Figure BDA0003082149040000691
Figure BDA0003082149040000701
Figure BDA0003082149040000711
Figure BDA0003082149040000721
Figure BDA0003082149040000731
表1D.示例性ALD变体
Figure BDA0003082149040000741
Figure BDA0003082149040000751
Figure BDA0003082149040000761
Figure BDA0003082149040000771
Figure BDA0003082149040000781
Figure BDA0003082149040000791
Figure BDA0003082149040000801
Figure BDA0003082149040000811
Figure BDA0003082149040000821
Figure BDA0003082149040000831
确定并且在表2中显示了ALD变体的多种活性。
表2：示例性ALD变体的活性。
Figure BDA0003082149040000841
Figure BDA0003082149040000851
Figure BDA0003082149040000861
Figure BDA0003082149040000871
Figure BDA0003082149040000881
Figure BDA0003082149040000891
Figure BDA0003082149040000901
Figure BDA0003082149040000911
Figure BDA0003082149040000921
Figure BDA0003082149040000931
Figure BDA0003082149040000941
Figure BDA0003082149040000951
Figure BDA0003082149040000961
1*对于其它二醇有活性
2'-＝特异性<1'
'+＝特异性在1,0-2.0'之间
'++＝特异性在2.0-3.0'之间
'+++＝特异性>3.0
3'-＝相对活性<1'
'+＝相对活性>1'
表3显示了示例性ALD变体的其它活性。通过ALD变体在48小时获得了高达大于50g/升，大于60g/升，大于70g/升，大于80g/升和大于90g/升的1,3-BDO生产水平。
Figure BDA0003082149040000981
Figure BDA0003082149040000991
Figure BDA0003082149040001001
Figure BDA0003082149040001011
Figure BDA0003082149040001021
如上所述的这些醛脱氢酶变体可以作用于3-羟基丁醛的R形式，其可以用于生产R-3-羟基丁醛的立体异构体或者具有更高比例的R形式的R和S形式的混合物。可以使用这种立体异构体来制备下游产品的立体异构体，如R-1,3-丁二醇。这些立体异构体具有作为药物或营养制剂的有用性。
这些结果表明了具有所期望的性质的醛脱氢酶变体的产生，其对于3-羟基丁醛、1,3-丁二醇、4-羟基丁醛或1,4-丁二醇或者通过包含醛脱氢酶的代谢途径所产生的其它所期望的产品的大规模生产是有用的。
如上所述的变体基于ALD-1亲代序列。应理解如表1、2或3所示的变体氨基酸位置可以应用于同源醛脱氢酶序列。表4提供了基于同源性的示例性ALD序列。本领域技术人员将容易地理解可以通过用于比对序列的常规且熟知的方法来分析这些序列(例如BLAST,blast.ncbi.nlm.nih.gov；Altschul等人,"J.Mol.Biol.215:403-410(1990))。此外，可以使用BLAST，通过搜索公开可用的序列数据库，如在国家生物技术信息中心(NationalCenter for Biotechnology Information)(NCBI)GenBank数据库、欧洲分子生物学实验室(European Molecular Biology Laboratory，EMBL)、ExPasy Prosite查询的数据库或者其它公开可用的序列数据库鉴别其它同源ALD序列。这些比对可以提供有关保守残基的信息，所述信息可以用于鉴别保持酶活力的共有序列以及产生其它酶变体的位置。
Figure BDA0003082149040001041
Figure BDA0003082149040001051
Figure BDA0003082149040001061
Figure BDA0003082149040001071
Figure BDA0003082149040001081
Figure BDA0003082149040001091
Figure BDA0003082149040001101
Figure BDA0003082149040001111
Figure BDA0003082149040001121
Figure BDA0003082149040001131
Figure BDA0003082149040001141
Figure BDA0003082149040001151
Figure BDA0003082149040001161
Figure BDA0003082149040001171
Figure BDA0003082149040001181
Figure BDA0003082149040001191
Figure BDA0003082149040001201
应理解可以单独使用或可以与其它变体氨基酸位置组合使用各个ALD变体，如以上所述的那些，其包括2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个，即多至如本文所公开的全部变体氨基酸位置(参见表1-3)，以产生具有所期望的活性的其它变体。示例性的ALD变体包括(但不限于)在表1-3中的任一个中所公开的氨基酸位置，例如，在对应于ALD-1的氨基酸序列(SEQ ID NO：1)的氨基酸位置12、19、33、44、65、66、72、73、107、122、129、139、143、145、155、163、167、174、189、204、220、227、229、230、243、244、254、267、315、353、356、396、429、432、437、440、441、442、444、447、450、460、464或467处的单一替换或者一个或多个替换的组合(参见表1-3)。例如，所述ALD变体包括(但不限于)在对应于ALD-1的氨基酸序列(SEQ ID NO：1)的氨基酸位置D12、V19、C33、I44、K65、I66、K72、A73、Y107、D122、E129、I139、T143、P145、G155、V163、G167、C174、C189、M204、C220、M227、K229、T230、A243、G244、A254、C267、V315、C353、C356、R396、F429、V432、E437、T440、T441、F442、I444、S447、E450、R460、C464或A467处的氨基酸替换、单一替换或一个或多个替换的组合(参见表1-3)。应理解可以在一个或多个所期望的氨基酸位置进行其它19种氨基酸的任意替换。
在一个实施方式中，所述变体ALD包含位置12处的氨基酸替换，即D12A。在一个实施方式中，所述变体ALD包含位置19处的氨基酸替换，即V19I。在一个实施方式中，所述变体ALD包含位置33处的氨基酸替换，即C33R。在一个实施方式中，所述变体ALD包含位置44处的氨基酸替换，即I44L。在一个实施方式中，所述变体ALD包含位置65处的氨基酸替换，即K65A。在一个实施方式中，所述变体ALD包含位置66处的氨基酸替换，其选自I66M、I66Q、I66N、I66H、I66T和I66S。在一个实施方式中，所述变体ALD包含位置72处的氨基酸替换，即K72N。在一个实施方式中，所述变体ALD包含位置73处的氨基酸替换，其选自A73S、A73D、A73G、A73L、A73Q、A73F、A73E、A73W、A73R、A73C和A73M。在一个实施方式中，所述变体ALD包含位置107处的氨基酸替换，即Y107K。在一个实施方式中，所述变体ALD包含位置122处的氨基酸替换，即D122N。在一个实施方式中，所述变体ALD包含位置129处的氨基酸替换，即E129I。在一个实施方式中，所述变体乙醛包含位置139处的氨基酸替换，其选自I139S、I139V和I139L。在一个实施方式中，所述变体ALD包含位置143处的氨基酸替换，即T143N或T143S。在一个实施方式中，所述变体ALD包含位置163处的氨基酸替换，其选自V163C、V163G和V163T。在一个实施方式中，所述变体ALD包含位置167处的氨基酸替换，即G167S。在一个实施方式中，所述变体ALD包含位置174处的氨基酸替换，即C174S。在一个实施方式中，所述变体ALD包含位置189处的氨基酸替换，即C189A。在一个实施方式中，所述变体ALD包含位置204处的氨基酸替换，即M204R。在一个实施方式中，所述变体ALD包含位置220处的氨基酸替换，即C220V。在一个实施方式中，所述变体ALD包含位置227处的氨基酸替换，其选自M227K、M227Q、M227I、M227V、M227C、M227L和M227A。在一个实施方式中，所述变体ALD包含位置229处的氨基酸替换，即K229S。在一个实施方式中，所述变体ALD包含位置230处的氨基酸替换，其选自T230R、T230K、T230H、T230A、T230M、T230C、T230L、T230S、T230Y、T230G、T230T、T230I、T230W、T230N、T230V和T230Q。在一个实施方式中，所述变体ALD包含位置243处的氨基酸替换，其选自A243P、A243Q、A243E、A243S、A243N、A243K、A243L、A243C、A243M和A243I。在一个实施方式中，所述变体ALD包含位置254处的氨基酸替换，即A254T。在一个实施方式中，所述变体ALD包含位置267处的氨基酸替换，即C267A。在一个实施方式中，所述变体ALD包含位置315处的氨基酸替换，即V315A。在一个实施方式中，所述变体ALD包含位置353处的氨基酸替换，即C353A。在一个实施方式中，所述变体ALD包含位置356处的氨基酸替换，即C356T或C356L。在一个实施方式中，所述变体ALD包含位置396处的氨基酸替换，即R396H。在一个实施方式中，所述变体ALD包含位置429处的氨基酸替换，其选自F429Y、F429Q、F429H、F429M、F429D和F429L。在一个实施方式中，所述变体ALD包含位置432处的氨基酸替换，即V432V或V432N。在一个实施方式中，所述变体ALD包含位置437处的氨基酸替换，即E437P。在一个实施方式中，所述变体ALD包含位置440处的氨基酸替换，即T440H。在一个实施方式中，所述变体ALD包含位置441处的氨基酸替换，即T441G。在一个实施方式中，所述变体ALD包含位置442处的氨基酸替换，其选自F442T、F442Y、F442H、F442N、F442Q、F442M和F442F。在一个实施方式中，所述变体ALD包含位置444处的氨基酸替换，即I444V。在一个实施方式中，所述变体ALD包含位置447处的氨基酸替换，其选自S447M、S447P、S447H、S447K、S447R、S447T、S447E和S447S。在一个实施方式中，所述变体ALD包含位置460处的氨基酸替换，即R460K。在一个实施方式中，所述变体ALD包含位置464处的氨基酸替换，即C464V或C464I。在一个实施方式中，所述变体ALD包含位置467处的氨基酸替换，即A467V。任何上述氨基酸位置可以用于单一氨基酸替换，或者一个或多个替换的组合以产生本发明的ALD变体。
例如，ALD变体可以包含两个或更多个氨基酸替换，如D12和I139；K65和C174；M204和C220；C464和A467；R396和F442；C356和F442；C174和A243；K65和I66；I66和A73；I66和C174；I66和M204；I66和C220；I66和M227；I66和T230；I66和A243；I66和A243；I66和C267；I66和C356；I66和R396；I66和E437；I66和F442；I66和S447；I66和C464；I66和A467等。例如，ALD变体可以包含两个或更多个氨基酸替换，如D12A和I139L；K65A和C174S；M204R和C220V；C464I和A467V；R396H和F442N；C356T和F442M；C174S和A243Q；K65A和I66H；I66H和A73S；I66H和C174S；I66H和M204R；I66H和C220V；I66H和M227I；I66H和T230C；I66H和A243Q；I66H和A243P；I66H和C267A；I66H和C356T；I66H和R396H；I66H和E437P；I66H和F442N；I66H和S447P；I66H和C464I；I66H和A467V；K65A和I66T；I66M和A73S；I66T和C174S；I66T和M204R；I66T和C220V；I66T和M227I；I66T和T230C；I66T和A243Q；I66T和A243P；I66T和C267A；I66T和C356T；I66T和R396H；I66T和E437P；I66T和F442N；I66T和S447P；I66T和C464I；I66T和A467V；K65A和I66M；I66M和A73S；I66M和C174S；I66M和M204R；I66M和C220V；I66M和M227I；I66M和T230C；I66M和A243Q；I66M和A243P；I66M和C267A；I66M和C356T；I66M和R396H；I66M和E437P；I66M和F442N；I66M和S447P；I66M和C464I；I66M和A467V；K65A和I66N；I66N和A73S；I66N和C174S；I66N和M204R；I66N和C220V；I66N和M227I；I66N和T230C；I66N和A243Q；I66N和A243P；I66N和C267A；I66N和C356T；I66N和R396H；I66N和E437P；I66N和F442N；I66N和S447P；I66N和C464I；I66N和A467V、K65A和I66Q；I66Q和A73S；I66Q和C174S；I66Q和M204R；I66Q和C220V；I66Q和M227I；I66Q和T230C；I66Q和A243Q；I66Q和A243P；I66Q和C267A；I66Q和C356T；I66Q和R396H；I66Q和E437P；I66Q和F442N；I66Q和S447P；I66Q和C464I；I66Q和A467V；K65A和I66S；I66S和A73S；I66S和C174S；I66S和M204R；I66S和C220V；I66S和M227I；I66S和T230C；I66S和A243Q；I66S和A243P；I66S和C267A；I66S和C356T；I66S和R396H；I66S和E437P；I66S和F442N；I66S和S447P；I66S和C464I；I66S和A467V等。
ALD变体还可以包含3个或更多个氨基酸替换，如D12、I139和R396；K65、C174和C356；M204、C220和A243；C174、C464和A467；A243、R396和F442；C220、C356和F442；C174、A243和E437；K65、I66和A243；I66、A73和E437；I66、C174和F442；I66、M204和R396；I66、C220和S447；I66、M227和C267；I66、T230和A243；I66、A243和C464；I66、A243和A467；I66、M204和C267；I66、C356和R396；I66、R396和F442；I66、E437和A467；I66、C220和F442；I66、S447和C464；I66、M204和C464；I66、C174和A467。例如，ALD变体可以包含3个或更多个氨基酸替换，如D12A、I139L和R396H；K65A、C174S和C356T；M204R、C220V和A243Q；C174S、C464I和A467V；A243P、R396H和F442N；C220V、C356T和F442M；C174S、A243Q和E437P；K65A、I66H和A243Q；I66H、A73S和E437P；I66H、C174S和F442N；I66H、M204R和R396H；I66H、C220V和S447P；I66H、M227I和C267A；I66H、T230C和A243P；I66H、A243Q和C464I；I66H、A243P和A467V；I66H、M204R和C267A；I66H、C356T和R396M；I66H、R396H和F442N；I66H、E437P和A467V；I66H、C220V和F442N；I66H、S447P和C464I；I66H、M204R和C464I；I66H、C174S和A467V；K65A、I66T和A243Q；I66M、A73S和E437P；I66T、C174S和F442N；I66T、M204R和R396H；I66T、C220V和S447P；I66T、M227I和C267A；I66T、T230C和A243P；I66T、A243Q和C464I；I66T、A243P和A467V；I66T、M204R和C267A；I66T、C356T和R396M；I66T、R396H和F442N；I66T、E437P和A467V；I66T、C220V和F442N；I66T、S447P和C464I；I66T、M204R和C464I；I66T,和C174S和A467V；K65A、I66M和A243Q；I66M、A73S和A437P；I66M、C174S和F442N；I66M、M204R和R396H；I66M、C220V和F442N；I66M、M227I和C267A；I66M、T230C和A243P；I66M、A243Q和C464I；I66M、A243P和A467V；I66M、M204R和C267A；I66M、C356T和R396M；I66M、R396H和F442N；I66M、E437P和A467V；I66M、C220V和F442N；I66M、S447P和C464I；I66M、M204R和C464I；I66M、C174S和A467V；K65A、I66N和A243Q；I66N、A73S和M227I；I66N、C174S和E437P；I66N、M204R和R396H；I66N、C220V和S447P；I66N、C174S和M227I；I66N、T230C和C356T；I66N、M204R和A243Q；I66N、A243P和S447P；I66N、C267A和C356T；I66N、C220V和C356T；I66N、R396H和E437P；I66N、M227I和E437P；I66N、F442N和A467V；I66N、M227I和S447P；I66N、M227I和C464I；I66N、A73S和A467V、K65A、I66Q和C220V；I66Q、A73S和M227I；I66Q、C174S和R396H；I66Q、M204R和C220V；I66Q、C220V和E437P；I66Q、M227I和F442N；I66Q、C174S和T230C；I66Q、A243Q和C356T；I66Q、A243P和C267A；I66Q、C267A和C356T；I66Q、C220V和C356T；I66Q、R396H和E437P；I66Q、M204R和E437P；I66Q、M227I和F442N；I66Q、F442N和S447P；I66Q、C256A和C464I；I66Q、A73S和A467V；K65A、I66S和A73S；I66S、A73S和C220V；I66S、C174S和C267A；I66S、M204R和R396H；I66S、C220V和T230C；I66S、C220V和M227I；I66S、T230C和A243P；I66S、A243Q和C356T；I66S、M227I和A243P；I66S、C267A和F442N；I66S、M204R和C356T；I66S、T230C和R396H；I66S、M204R和E437P；I66S、C220V和F442N；I66S、A73S和S447P；I66S、C174S和C464I；I66S、C356T和A467V等。应理解如上所述的两个或更多个或者3个或更多个氨基酸替换的组合的这些组合仅是示例性的并且本领域技术人员可以容易地确定所期望的ALD的所期望的氨基酸替换的组合。
基于本文的教导内容，本领域技术人员可以容易地鉴别出对应于同源ALD序列中的对应于ALD-1的氨基酸序列(SEQ ID NO：1)的任何氨基酸位置12、19、33、44、65、66、72、73、107、122、129、139、143、145、155、163、167、174、189、204、220、227、229、230、243、244、254、267、315、353、356、396、429、432、437、440、441、442、444、447、450、460、464或467的氨基酸位置。例如，如图4A中的比对所示，ALD-1的氨基酸I139对应于SEQ ID NO：13和20的氨基酸I133。对于SEQ ID NO：24，对应的位置是V199。使用氨基酸序列比对的熟知方法，通常使用如本文所公开的缺省参数，本领域技术人员可以容易地确定另一ALD序列中对应于与ALD-1的氨基酸序列(SEQ ID NO：1)对应的任何氨基酸位置12、19、33、44、65、66、72、73、107、122、129、139、143、145、155、163、167、174、189、204、220、227、229、230、243、244、254、267、315、353、356、396、429、432、437、440、441、442、444、447、450、460、464或467的氨基酸位置。
还将理解ALD变体可以含有2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个，即多至如本文(例如)在表1-3中所公开的所有变体氨基酸位置。基于如本文所公开的任何单一氨基酸替换或其组合，如以上和表1-3中所述的氨基酸变体位置，本领域技术人员可以容易地产生ALD变体。在具体的实施方式中，所述ALD变体是表1-3中所公开的那些。
在整个本发明申请中，已参考了多个出版物。在本发明申请中，这些出版物的公开内容，包括GenBank登录号.版本符号(GenBank accession.version designations)和/或GI编号公开以其全部内容作为参考并入本文以更全面地描述本发明所属领域的技术现状。尽管已参考以上提供的实例描述了本发明，但是应理解可以在不背离本发明的精神的情况下，做出多种改变。
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<110> 基因组股份公司
<120> 醛脱氢酶变体及其使用方法
<130> 12956-462-228
<140>
<141>
<150> 62/740,830
<151> 2018-10-03
<150> 62/737,053
<151> 2018-09-26
<160> 129
<170> PatentIn version 3.5
<210> 1
<211> 468
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
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His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
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Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile
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Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
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Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
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Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
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Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
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Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
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Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
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Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
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Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Asp Ser Leu Asp
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Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
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Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
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Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
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Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
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Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
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Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
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<213> 短乳酸杆菌（Lactobacillus brevis）
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Ala Thr Glu Val Glu Gly Gly Leu His His Thr Ala Ser Ile His Ser
385 390 395 400
Glu Asn Leu Pro His Ile Asn Lys Ala Ala His Arg Leu Asn Thr Ser
405 410 415
Ile Phe Val Val Asn Gly Pro Thr Tyr Cys Gly Thr Gly Val Ala Thr
420 425 430
Asn Gly Ala His Ser Gly Ala Ser Ala Leu Thr Ile Ala Thr Pro Thr
435 440 445
Gly Glu Gly Thr Ala Thr Ser Lys Thr Tyr Thr Arg Arg Arg Arg Leu
450 455 460
Asn Ser Pro Glu Gly Phe Ser Leu Arg Thr Trp Glu Ala
465 470 475
<210> 3
<211> 462
<212> PRT
<213> 植物发酵梭菌（Clostridium phytofermentans）
<400> 3
Met Thr Val Asn Glu Gln Leu Val Gln Asp Ile Ile Lys Asn Val Val
1 5 10 15
Ala Ser Met Gln Leu Thr Gln Thr Asn Lys Thr Glu Leu Gly Val Phe
20 25 30
Asp Asp Met Asn Gln Ala Ile Glu Ala Ala Lys Glu Ala Gln Leu Val
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Ser Ala Ile
50 55 60
Arg Lys Lys Thr Ile Glu His Ala Glu Thr Leu Ala Arg Met Ala Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Val Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Ile
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Asn Phe Ala Val Gln Leu Ile
165 170 175
Asn Glu Ala Ser Leu Ser Ala Gly Gly Pro Val Asn Ile Ala Cys Ser
180 185 190
Val Arg Lys Pro Thr Leu Asp Ser Ser Lys Ile Met Met Ser His Gln
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Gln Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Val Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Ile Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Val Val Ala Ile Asp Ala Ile Ala Asn Glu Leu Met Asn Tyr
275 280 285
Met Val Lys Glu Gln Gly Cys Tyr Ala Ile Thr Lys Glu Gln Gln Glu
290 295 300
Lys Leu Thr Asn Leu Val Ile Thr Pro Lys Gly Leu Asn Arg Asn Cys
305 310 315 320
Val Gly Lys Asp Ala Arg Thr Leu Leu Gly Met Ile Gly Ile Asp Val
325 330 335
Pro Ser Asn Ile Arg Cys Ile Ile Phe Glu Gly Glu Lys Glu His Pro
340 345 350
Leu Ile Ser Glu Glu Leu Met Met Pro Ile Leu Gly Ile Val Arg Ala
355 360 365
Lys Ser Phe Asp Asp Ala Val Glu Lys Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Arg Ile Thr
385 390 395 400
Thr Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Ala Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 4
<211> 468
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /备注="人工序列描述: 合成
多肽"
<400> 4
Met Ile Lys Asp Thr Leu Val Ser Ile Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ala Asn Leu Lys Asn Tyr Lys Asp Asp Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Asn Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Ser Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Arg Asp Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Val Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Ala Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Ser Val Lys
340 345 350
Cys Ile Ile Thr Glu Val Ser Ala Ser His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn His Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Pro Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Ile
450 455 460
Val Leu Val Gly
465
<210> 5
<211> 15
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /备注="人工序列描述: 合成
肽"
<220>
<221> MOD_RES
<222> (2)..(2)
<223> 任何氨基酸
<220>
<221> MOD_RES
<222> (6)..(7)
<223> 任何氨基酸
<220>
<221> MOD_RES
<222> (11)..(11)
<223> 任何氨基酸
<400> 5
Ile Xaa Pro Lys Gly Xaa Xaa Asn Arg Lys Xaa Val Gly Lys Asp
1 5 10 15
<210> 6
<211> 18
<212> PRT
<213> 人工序列
<220>
<221> 来源
<223> /备注="人工序列描述: 合成
肽"
<220>
<221> MOD_RES
<222> (5)..(5)
<223> 任何氨基酸
<220>
<221> MOD_RES
<222> (7)..(9)
<223> 任何氨基酸
<220>
<221> MOD_RES
<222> (13)..(13)
<223> 任何氨基酸
<400> 6
Ser Tyr Ala Gly Xaa Gly Xaa Xaa Xaa Glu Gly Phe Xaa Thr Phe Thr
1 5 10 15
Ile Ala
<210> 7
<211> 468
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 7
Met Ile Lys Asp Thr Leu Val Ser Ile Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ala Asn Leu Lys Asn Tyr Lys Asp Asp Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Asn Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Asp Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Ser Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Ser Ala Arg His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 8
<211> 386
<212> PRT
<213> 食葡糖罗斯氏菌（Roseburia inulinivorans）
<400> 8
Met Gly Val Asn Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu
1 5 10 15
Lys His His Leu Thr Ala Asp Lys Val Pro Gly Thr Glu Asp Ile Ser
20 25 30
Thr Ile Ala Trp Ser Gly Asp Arg Gly Leu Thr Leu Val Glu Met Gly
35 40 45
Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Ala Thr Asn Pro Ser Glu
50 55 60
Thr Val Ile Cys Asn Cys Ile Gly Met Leu Ala Gly Gly Asn Thr Val
65 70 75 80
Val Phe Asn Pro His Pro Asn Ala Lys Lys Thr Thr Ile Tyr Thr Ile
85 90 95
Asn Met Ile Asn Glu Ala Ser Ile Glu Ala Gly Gly Pro Asp Asn Ile
100 105 110
Ala Val Thr Val Glu Ala Pro Thr Leu Asp Thr Ser Ala Ile Met Met
115 120 125
Lys His Pro Ser Ile His Leu Leu Val Ala Thr Gly Gly Pro Gly Val
130 135 140
Val Thr Ala Val Leu Ser Ser Gly Lys Arg Ala Ile Gly Ala Gly Ala
145 150 155 160
Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala
165 170 175
Ala Gln Asp Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys
180 185 190
Ile Ala Glu Lys Glu Ile Val Ala Val Asp Ser Val Ala Asp Glu Leu
195 200 205
Met Asn Tyr Met Ile Ser Glu Asn Gly Cys Tyr Leu Ala Ser Lys Glu
210 215 220
Ile Gln Asp Lys Leu Val Gln Thr Val Phe Thr Pro Lys Gly Ala Leu
225 230 235 240
Asn Arg Lys Cys Val Gly Arg Ser Ala Gln Thr Leu Leu Ala Met Val
245 250 255
Gly Val Asn Val Gly Pro Glu Ile Arg Cys Ile Val Phe Glu Gly Gln
260 265 270
Lys Glu His Pro Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly
275 280 285
Met Val Arg Val Lys Ser Phe Glu Glu Gly Val Glu Thr Ala Val Trp
290 295 300
Leu Glu His Gly Asn Arg His Ser Ala His Ile His Ser Lys Asn Val
305 310 315 320
Asp His Ile Thr Thr Tyr Ala Arg Ala Leu Asp Thr Ala Ile Leu Val
325 330 335
Lys Asn Gly Pro Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Tyr
340 345 350
Cys Thr Phe Thr Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ala Ala
355 360 365
His Ser Phe Thr Lys Ser Arg Arg Cys Thr Met Ser Asp Ser Leu Cys
370 375 380
Ile Arg
385
<210> 9
<211> 467
<212> PRT
<213> 芽孢杆菌属（Bacillus sp.）
<400> 9
Met Asn Pro Ala Glu Leu Pro His Gln Val His Glu Ser Gly Ala Asn
1 5 10 15
Gly Val Phe Asp Arg Ile Glu Asp Ala Ile Glu Ala Gly Tyr Ile Ala
20 25 30
Gln Leu Asn Tyr Val Lys Gln Phe Gln Leu Lys Asp Arg Glu Lys Ile
35 40 45
Ile Thr Ala Ile Arg Glu Ala Val Ile Glu Asn Lys Glu Lys Leu Ala
50 55 60
Gln Met Val Phe Glu Glu Thr Lys Leu Gly Arg Tyr Glu Asp Lys Ile
65 70 75 80
Ala Lys His Glu Leu Val Ala Arg Lys Thr Pro Gly Thr Glu Asp Ile
85 90 95
Thr Thr Ala Ala Phe Ser Gly Asp Glu Gly Leu Thr Ile Ile Glu Gln
100 105 110
Ala Pro Phe Gly Leu Val Gly Ala Val Thr Pro Val Thr Asn Pro Thr
115 120 125
Glu Thr Ile Ile Asn Asn Ser Ile Ser Leu Leu Ala Ala Gly Asn Ala
130 135 140
Val Val Leu Asn Val His Pro Ser Ser Lys Ala Ser Cys Ala Phe Val
145 150 155 160
Val Asn Leu Ile Asn Gln Ala Ile Lys Asp Thr Gly Gly Pro Glu Asn
165 170 175
Leu Val Ser Met Val Lys Asp Pro Thr Leu Glu Thr Leu Asn Arg Ile
180 185 190
Ile Glu Ser Pro Lys Val Lys Leu Leu Val Gly Thr Gly Gly Leu Gly
195 200 205
Met Val Lys Thr Leu Leu Lys Ser Gly Lys Lys Ala Ile Gly Ala Gly
210 215 220
Ala Gly Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Leu Lys Gln
225 230 235 240
Ala Ala Lys Ser Ile Ile Glu Gly Ala Ser Phe Asp Asn Asn Leu Leu
245 250 255
Cys Ile Ala Glu Lys Glu Leu Phe Val Ile Asp Ser Val Ala Asp Asp
260 265 270
Leu Ile Phe His Met Leu Asn Glu Gly Ala Tyr Met Leu Asp Gln Gln
275 280 285
Gln Leu Ser Lys Leu Met Ser Phe Ala Leu Glu Glu Asn Val His Gln
290 295 300
Glu Ala Gly Gly Cys Ser Leu Asp Asn Lys Arg Glu Tyr His Val Ser
305 310 315 320
Lys Asp Trp Val Gly Lys Asp Ala Val Ser Phe Leu Arg Gln Leu Gly
325 330 335
Ile Ala His Glu Glu Asp Ile Lys Leu Leu Ile Cys Glu Val Asp Phe
340 345 350
Asp His Pro Phe Val Gln Leu Glu Gln Met Met Pro Val Phe Pro Ile
355 360 365
Val Arg Val Gly Asn Leu Asp Glu Ala Ile Glu Met Ala Leu Leu Ala
370 375 380
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys Asn Val Asp
385 390 395 400
His Leu Thr Lys Phe Ala Arg Ala Ile Glu Thr Thr Ile Phe Val Lys
405 410 415
Asn Ala Ser Ser Leu Ala Gly Val Gly Phe Gly Gly Glu Gly His Thr
420 425 430
Thr Met Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser Ala Lys
435 440 445
Thr Phe Thr Arg Gln Arg Arg Cys Val Leu Ala Glu Gly Gly Phe Arg
450 455 460
Ile Ile Gly
465
<210> 10
<211> 467
<212> PRT
<213> 番茄芽孢杆菌（Bacillus solani）
<400> 10
Met Asn Pro Ala Glu Leu Pro His Gln Val His Glu Ser Gly Ala Asn
1 5 10 15
Gly Val Phe Asp Arg Ile Glu Asp Ala Ile Glu Ala Gly Tyr Ile Ala
20 25 30
Gln Leu Asn Tyr Val Lys Gln Phe Gln Leu Lys Asp Arg Glu Lys Ile
35 40 45
Ile Thr Ala Ile Arg Glu Ala Val Ile Glu Asn Lys Glu Lys Leu Ala
50 55 60
Gln Met Val Phe Glu Glu Thr Lys Leu Gly Arg Tyr Glu Asp Lys Ile
65 70 75 80
Ala Lys His Glu Leu Val Ala Arg Lys Thr Pro Gly Thr Glu Asp Ile
85 90 95
Thr Thr Ala Ala Phe Ser Gly Asp Glu Gly Leu Thr Ile Ile Glu Gln
100 105 110
Ala Pro Phe Gly Leu Val Gly Ala Val Thr Pro Val Thr Asn Pro Thr
115 120 125
Glu Thr Ile Ile Asn Asn Ser Ile Ser Leu Leu Ala Ala Gly Asn Ala
130 135 140
Val Val Leu Asn Val His Pro Ser Ser Lys Val Ser Cys Ala Phe Val
145 150 155 160
Val Asn Leu Ile Asn Gln Ala Ile Lys Asp Thr Gly Gly Pro Glu Asn
165 170 175
Leu Val Ser Met Val Lys Asp Pro Thr Leu Glu Thr Leu Asn Arg Ile
180 185 190
Ile Glu Ser Pro Lys Val Lys Leu Leu Val Gly Thr Gly Gly Pro Gly
195 200 205
Met Val Lys Thr Leu Leu Lys Ser Gly Lys Lys Ala Ile Gly Ala Gly
210 215 220
Ala Gly Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Leu Lys Gln
225 230 235 240
Ala Ala Lys Ser Ile Ile Glu Gly Ala Ser Phe Asp Asn Asn Leu Leu
245 250 255
Cys Ile Ala Glu Lys Glu Leu Phe Val Ile Asp Ser Val Ala Asp Asp
260 265 270
Leu Ile Phe His Met Leu Asn Glu Gly Ala Tyr Met Leu Asp Gln Gln
275 280 285
Gln Leu Ser Lys Leu Met Ser Phe Ala Leu Glu Glu Asn Val His Gln
290 295 300
Glu Ala Gly Gly Cys Ser Leu Asp Asn Lys Arg Glu Tyr His Val Ser
305 310 315 320
Lys Asp Trp Val Gly Lys Asp Ala Val Ser Phe Leu Arg Gln Leu Gly
325 330 335
Ile Ala His Glu Glu Asp Ile Lys Leu Leu Ile Cys Glu Val Asp Phe
340 345 350
Asp His Pro Phe Val Gln Leu Glu Gln Met Met Pro Val Phe Pro Ile
355 360 365
Val Arg Val Gly Asn Leu Asp Glu Ala Ile Glu Met Ala Leu Leu Ala
370 375 380
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys Asn Val Asp
385 390 395 400
His Leu Thr Lys Phe Ala Arg Ala Ile Glu Thr Thr Ile Phe Val Lys
405 410 415
Asn Ala Ser Ser Leu Ala Gly Val Gly Phe Gly Gly Glu Gly His Thr
420 425 430
Thr Met Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser Ala Lys
435 440 445
Thr Phe Thr Arg Gln Arg Arg Cys Val Leu Ala Glu Gly Gly Phe Arg
450 455 460
Ile Ile Gly
465
<210> 11
<211> 473
<212> PRT
<213> 醛脱氢酶（Terrisporobacter othiniensis）
<400> 11
Met Asp Ile Asp Val Lys Leu Ile Glu Lys Met Val Ser Asp Ala Leu
1 5 10 15
Lys Glu Ile Lys Ile Glu Asn Ile Thr Gln Glu Val Glu Lys Asn Ser
20 25 30
Ile Glu Asp Asn Tyr Gly Val Phe Lys Thr Ile Glu Gly Ala Ile Asp
35 40 45
Ala Ser Tyr Val Ala Gln Lys Glu Leu Leu Phe Ser Lys Ile Ser Asp
50 55 60
Arg Gln Lys Tyr Val Asp Ala Ile Arg Ser Ala Ile Leu Asn Gln Glu
65 70 75 80
Asn Leu Glu Leu Ile Ser Lys Leu Ala Val Asp Glu Thr Glu Ile Gly
85 90 95
Cys Tyr Glu His Lys Leu Ile Lys Asn Arg Leu Ala Ala Glu Lys Thr
100 105 110
Pro Gly Thr Glu Asp Leu Ile Ser Ser Val Lys Thr Gly Asp Asp Gly
115 120 125
Leu Thr Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr
130 135 140
Pro Thr Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Gly Met
145 150 155 160
Ile Ala Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Thr
165 170 175
Lys Val Ser Gln Thr Ile Ile Lys Ile Leu Asn Lys Ala Leu Glu Glu
180 185 190
Val Gly Ala Pro Lys Asn Leu Ile Thr Met Val Glu Lys Pro Ser Ile
195 200 205
Glu Asn Thr Asn Lys Met Ile Glu Asn Pro Lys Val Arg Phe Leu Val
210 215 220
Ala Thr Gly Gly Pro Ser Ile Val Lys Lys Val Leu Ser Ser Gly Lys
225 230 235 240
Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu
245 250 255
Thr Ala Asp Ile Arg Lys Ala Ala Lys Asp Ile Ile Asp Gly Cys Ser
260 265 270
Phe Asp Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val
275 280 285
Ala Ser Ile Cys Asp Ser Leu Ile Glu Asn Met Lys Leu Asn Gly Ala
290 295 300
Tyr Leu Val Lys Asp Lys Lys Val Ile Glu Gln Leu Leu Ser Val Val
305 310 315 320
Ala Lys Glu Asn Gly Ala Pro Lys Thr Asn Phe Val Gly Lys Ser Ala
325 330 335
Lys Tyr Ile Leu Asp Lys Ile Gly Val Thr Val Asp Asp Asn Ile Lys
340 345 350
Ala Ile Ile Met Glu Val Asp Lys Asp His Thr Phe Val Gln Glu Glu
355 360 365
Met Met Met Pro Ile Leu Pro Ile Val Arg Val Glu Asp Val Asp Lys
370 375 380
Ala Ile Glu Tyr Ala Gln Glu Ala Glu His Gly Asn Arg His Thr Ala
385 390 395 400
Ile Met His Ser Lys Asn Ile Asp Lys Leu Ser Lys Met Ser Lys Ile
405 410 415
Met Glu Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile
420 425 430
Gly Val Gly Gly Glu Gly His Ser Thr Phe Thr Ile Ala Gly Pro Thr
435 440 445
Gly Glu Gly Leu Thr Ser Pro Lys Ser Phe Cys Arg Ile Arg Arg Cys
450 455 460
Val Val Ser Asp Ser Phe Ser Ile Arg
465 470
<210> 12
<211> 457
<212> PRT
<213> 食葡糖罗斯氏菌（Roseburia inulinivorans）
<400> 12
Met Val His Asp Ile Val Gln Lys Val Met Ala Asn Met Gln Ile Ser
1 5 10 15
Gly Ser Val Ser Gly Met His Gly Val Phe Lys Asp Met Asn Asp Ala
20 25 30
Ile Asn Ala Ser Ile Glu Ala Gln Lys Lys Val Cys Thr Met Thr Leu
35 40 45
Asp Gln Arg Glu Gln Ile Ile Ser Leu Ile Arg Lys Lys Thr His Glu
50 55 60
Asn Ala Glu Ile Leu Ala Asn Met Gly Val Asn Glu Thr Gly Met Gly
65 70 75 80
Asn Val Gly Asp Lys Ile Leu Lys His His Leu Thr Ala Asp Lys Val
85 90 95
Pro Gly Thr Glu Asp Ile Ser Thr Ile Ala Trp Ser Gly Asp Arg Gly
100 105 110
Leu Thr Leu Val Glu Met Gly Pro Phe Gly Val Ile Gly Ala Ile Thr
115 120 125
Pro Ala Thr Asn Pro Ser Glu Thr Val Ile Cys Asn Cys Ile Gly Met
130 135 140
Leu Ala Gly Gly Asn Thr Val Val Phe Asn Pro His Pro Asn Ala Lys
145 150 155 160
Lys Thr Thr Ile Tyr Thr Ile Asn Met Ile Asn Glu Ala Ser Ile Glu
165 170 175
Ala Gly Gly Pro Asp Asn Ile Ala Val Thr Val Glu Ala Pro Thr Leu
180 185 190
Asp Thr Ser Ala Ile Met Met Lys His Pro Ser Ile His Leu Leu Val
195 200 205
Ala Thr Gly Gly Pro Gly Val Val Thr Ala Val Leu Ser Ser Gly Lys
210 215 220
Arg Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu
225 230 235 240
Thr Ala Asp Ile Arg Lys Ala Ala Gln Asp Ile Val Asn Gly Cys Thr
245 250 255
Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu Lys Glu Ile Val Ala Val
260 265 270
Asp Ser Val Ala Asp Glu Leu Met Asn Tyr Met Ile Ser Glu Asn Gly
275 280 285
Cys Tyr Leu Ala Ser Lys Glu Ile Gln Asp Lys Leu Val Gln Thr Val
290 295 300
Phe Thr Pro Lys Gly Ala Leu Asn Arg Lys Cys Val Gly Arg Ser Ala
305 310 315 320
Gln Thr Leu Leu Ala Met Val Gly Val Asn Val Gly Pro Glu Ile Arg
325 330 335
Cys Ile Val Phe Glu Gly Gln Lys Glu His Pro Leu Ile Ala Glu Glu
340 345 350
Leu Met Met Pro Ile Leu Gly Met Val Arg Val Lys Ser Phe Glu Glu
355 360 365
Gly Val Glu Thr Ala Val Trp Leu Glu His Gly Asn Arg His Ser Ala
370 375 380
His Ile His Ser Lys Asn Val Asp His Ile Thr Thr Tyr Ala Arg Ala
385 390 395 400
Leu Asp Thr Ala Ile Leu Val Lys Asn Gly Pro Ser Tyr Ala Ala Leu
405 410 415
Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr Ile Ala Ser Arg Thr
420 425 430
Gly Glu Gly Leu Thr Ala Ala His Ser Phe Thr Lys Ser Arg Arg Cys
435 440 445
Thr Met Ser Asp Ser Leu Cys Ile Arg
450 455
<210> 13
<211> 462
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 13
Met Ser Val Asn Glu Arg Met Val Gln Asp Ile Val Gln Glu Val Val
1 5 10 15
Ala Lys Met Gln Ile Ala Ser Asp Val Thr Gly Asn His Gly Val Phe
20 25 30
Gln Asp Met Asn Ala Ala Ile Glu Ala Ala Lys Lys Thr Gln Lys Val
35 40 45
Val Ala Arg Met Ser Met Asp Gln Arg Glu Lys Ile Ile Ser Asn Ile
50 55 60
Arg Ala Lys Ile Lys Glu His Ala Glu Ile Phe Ala Arg Met Gly Val
65 70 75 80
Gln Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Gln Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Tyr Ala Val Asn Leu Ile
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr
180 185 190
Val Glu Asn Pro Thr Leu Glu Ser Ser Asn Ile Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Met His Tyr
275 280 285
Met Ile Ser Glu Gln Gly Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Ala Leu Thr Glu Val Val Leu Lys Gly Gly Arg Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Val Thr Val
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Ala Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Cys Asp Ser Leu Cys Ile Arg
450 455 460
<210> 14
<211> 509
<212> PRT
<213> 芽孢杆菌属还原硒酸盐芽孢杆菌（Bacillus selenitireducens)
<400> 14
Met Ser Ile Ser Glu Asp Met Leu Lys Gln Ile Val Lys Ser Val Met
1 5 10 15
Asn Asn Val Glu Lys Glu Leu Gly Glu Ser Pro Lys Pro Gln Pro Arg
20 25 30
Thr Ile Pro Val Thr Val Leu Asn Glu Val Thr Pro Val Lys Glu Ser
35 40 45
Arg Asp Pro Ser Pro Val His Gln His Val Leu Gly Val Phe Pro Asp
50 55 60
Val Asp Gln Ala Val His Ala Ala Ala Gly Ser Gln Lys Glu Trp Val
65 70 75 80
Lys Arg Pro Val Ser Glu Arg Arg Val Ile Leu Glu Ala Met Lys Gln
85 90 95
Thr Val Asp Ser Gln Lys Glu Arg Tyr Ser Glu Leu Ala Val Glu Glu
100 105 110
Thr Gly Leu Gly Asn Val Ala Asp Lys Ile Ala Lys His Glu Leu Ile
115 120 125
Ile Thr Lys Thr Pro Gly Val Glu Asp Leu Arg Thr Asp Ala Val Ser
130 135 140
Gly Asp His Gly Leu Thr Ile Glu Glu Asp Ala Pro Phe Gly Val Ile
145 150 155 160
Gly Ala Val Thr Pro Val Thr Asn Pro Thr Thr Thr Val Ile His Asn
165 170 175
Ser Leu Val Met Leu Ala Ala Gly Asn Ala Val Val Phe Asn Val His
180 185 190
Pro Ser Ser Lys Ala Thr Cys Gln Arg Val Val Ser Asp Leu Asn Ala
195 200 205
Ala Ile Lys Asp Ala Gly Gly Pro Gln Asn Leu Ile Thr Met Ile Ala
210 215 220
Glu Pro Thr Leu Asp Thr Leu Asn Gln Leu Ala Gly His Gln Glu Ile
225 230 235 240
Arg Leu Leu Val Gly Thr Gly Gly Gln Gly Leu Val Arg Ser Leu Leu
245 250 255
Gln Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val
260 265 270
Ile Val Asp Glu Thr Ala Asp Ile Glu Ala Ala Ala Lys Ala Ile Ile
275 280 285
Leu Gly Ala Ser Phe Asp Asn Asn Ile Leu Cys Ile Ala Glu Lys Glu
290 295 300
Val Phe Ala Leu Asp Val Ile Tyr Asp Asp Leu Ile Tyr His Leu Leu
305 310 315 320
Gln Glu Gly Ala Tyr Met Leu Ser Glu Ser Glu Leu Ser Gln Val Met
325 330 335
Lys Thr Val Leu Val Gly Asp Ala Pro Ile Glu Ala Ala Lys Ser Cys
340 345 350
Ser Val Ser Val Arg Pro Asp Leu His Ile Ala Lys Ala Trp Val Gly
355 360 365
Lys Glu Ala Ser Ala Ile Leu Lys Ala Ala Thr Gly Lys Asp Leu Pro
370 375 380
Val Lys Leu Leu Ile Cys Asp Val Glu Ala Thr His Pro Phe Val Gln
385 390 395 400
Leu Glu Gln Met Met Pro Val Leu Pro Ile Val Arg Met Pro Asp Phe
405 410 415
Asp Ala Ala Val Glu Ala Ala Val Lys Ala Glu Lys Gly Asn Arg His
420 425 430
Thr Ala Val Ile His Ser Lys Asn Val Asp Arg Leu Thr Gln Phe Ala
435 440 445
Arg Arg Ile Glu Thr Thr Ile Phe Val Lys Asn Ala Ser Ser Leu Ala
450 455 460
Gly Val Gly Phe Gly Gly Glu Gly Tyr Ala Thr Met Thr Ile Ala Gly
465 470 475 480
Pro Thr Gly Glu Gly Ile Thr Ser Pro Arg Thr Phe Thr Arg Lys Arg
485 490 495
Arg Cys Val Leu Ala Glu Gly Gly Phe Arg Ile Ile Gly
500 505
<210> 15
<211> 462
<212> PRT
<213> 卵形布劳特菌（Blautia obeum）
<400> 15
Met Pro Ile Ser Glu Ser Met Val Gln Asp Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Asp Ala Pro Thr Gly Lys His Gly Ile Phe
20 25 30
Lys Asp Met Asn Asp Ala Ile Glu Ala Ala Lys Lys Ser Glu Leu Ile
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Thr Cys Ile
50 55 60
Arg Lys Lys Ile Lys Glu Asn Ala Glu Val Met Ala Arg Met Gly Val
65 70 75 80
Asp Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His His
85 90 95
Leu Val Ala Asp Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Leu
130 135 140
Cys Asn Thr Met Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Phe Ala Ile Asn Leu Val
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Val Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Thr Ser Asn Ile Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Val Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Gln Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Val Val Ala Val Ser Ser Val Val Asp Glu Leu Met His Tyr
275 280 285
Met Leu Thr Glu Asn Asp Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Lys Leu Thr Glu Val Val Leu Ala Gly Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Arg Thr Leu Leu Ser Met Ile Gly Val Asn Ala
325 330 335
Pro Ala Asn Ile Arg Cys Ile Ile Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Ile Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ser Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 16
<211> 478
<212> PRT
<213> 鲍氏梭菌（Clostridium bolteae）
<400> 16
Met Lys Glu Gly Val Ile Arg Leu Asp Met Asp Ile Lys Val Ile Glu
1 5 10 15
Gln Leu Val Glu Gln Ala Leu Lys Glu Ile Lys Ala Glu Gln Pro Leu
20 25 30
Lys Phe Thr Ala Pro Lys Leu Glu Arg Tyr Gly Val Phe Lys Thr Met
35 40 45
Asp Glu Ala Ile Ala Ala Ser Glu Glu Ala Gln Lys Lys Leu Leu Phe
50 55 60
Ser Lys Ile Ser Asp Arg Gln Lys Tyr Val Asp Val Ile Arg Ser Thr
65 70 75 80
Ile Ile Lys Arg Glu Asn Leu Glu Leu Ile Ser Arg Leu Ser Val Glu
85 90 95
Glu Thr Glu Ile Gly Asp Tyr Glu His Lys Leu Ile Lys Asn Arg Leu
100 105 110
Ala Ala Glu Lys Thr Pro Gly Thr Glu Asp Leu Leu Thr Glu Ala Ile
115 120 125
Thr Gly Asp Asn Gly Leu Thr Leu Val Glu Tyr Cys Pro Phe Gly Val
130 135 140
Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu Thr Ile Ile Asn
145 150 155 160
Asn Ser Ile Ser Met Ile Ala Gly Gly Asn Thr Val Val Phe Ser Pro
165 170 175
His Pro Arg Ala Lys Lys Val Ser Gln Met Thr Val Lys Met Leu Asn
180 185 190
Lys Ala Leu Ile Asp Asn Gly Ala Pro Pro Asn Leu Ile Thr Met Val
195 200 205
Glu Glu Pro Ser Ile Glu Asn Thr Asn Lys Met Ile Asp Asn Pro Ser
210 215 220
Val Arg Leu Leu Val Ala Thr Gly Gly Pro Ser Ile Val Lys Lys Val
225 230 235 240
Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro
245 250 255
Val Val Val Asp Glu Thr Ala Asp Ile Asp Lys Ala Ala Lys Asp Ile
260 265 270
Val Asp Gly Cys Ser Phe Asp Asn Asn Val Pro Cys Ile Ala Glu Lys
275 280 285
Glu Val Phe Ala Val Asp Ser Ile Cys Asp Tyr Leu Ile His His Met
290 295 300
Lys Glu Asn Gly Ala Tyr Gln Ile Thr Asp Pro Met Leu Leu Glu Gln
305 310 315 320
Leu Val Ala Leu Val Thr Thr Glu Lys Gly Gly Pro Lys Thr Ser Phe
325 330 335
Val Gly Lys Ser Ala Arg Tyr Ile Leu Asp Lys Leu Gly Ile Thr Val
340 345 350
Asp Ala Ser Val Arg Val Ile Ile Met Glu Val Pro Lys Asp His Leu
355 360 365
Leu Val Gln Glu Glu Met Met Met Pro Ile Leu Pro Val Val Arg Val
370 375 380
Ser Asp Val Asp Thr Ala Ile Glu Tyr Ala His Gln Ala Glu His Gly
385 390 395 400
Asn Arg His Thr Ala Met Met His Ser Lys Asn Val Glu Lys Leu Ser
405 410 415
Lys Met Ala Lys Ile Met Glu Thr Thr Ile Phe Val Lys Asn Ala Pro
420 425 430
Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Tyr Thr Thr Phe Thr
435 440 445
Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Pro Arg Thr Phe Cys
450 455 460
Arg Lys Arg Lys Cys Val Met Thr Asp Ala Phe Ser Ile Arg
465 470 475
<210> 17
<211> 515
<212> PRT
<213> 消化咸海鲜芽孢杆菌（Jeotgalibacillus alimentarius）
<400> 17
Met Ser Ile Ser Glu Glu Thr Leu Gln Gln Ile Ile Lys Ser Val Val
1 5 10 15
Thr Gln Val Glu Ser Glu Leu Gly His Lys His Ser Ala Pro Ala Thr
20 25 30
Gly Ser Gln Ser Ala Thr Pro Val Ala Pro Val Lys Met Lys Ala Val
35 40 45
Thr Asn Lys Pro Val Phe Lys Glu His Thr Tyr Arg Ser Ser Gly Glu
50 55 60
Gly Ile Tyr Thr Thr Val Asp Glu Ala Val Ser Arg Ser Ala Ala Ala
65 70 75 80
Gln Lys Lys Tyr Val Lys His Phe Thr Met Asn Asp Arg Val Thr Val
85 90 95
Leu Asn Ala Ile Lys Gln Thr Val Leu Ser Ser Lys Asp Thr Leu Ser
100 105 110
Lys Met Ala Val Glu Glu Thr Gly Ile Gly Cys Tyr Glu Asp Lys Ile
115 120 125
Gln Lys His Glu Leu Val Cys Lys Lys Thr Pro Gly Ile Glu Asp Leu
130 135 140
Lys Thr Glu Ala Met Ser Gly Asp Asp Gly Leu Thr Ile Ile Glu Glu
145 150 155 160
Ala Pro Phe Gly Val Ile Gly Ala Val Thr Pro Val Thr Asn Pro Thr
165 170 175
Thr Thr Ile Ile Asn Asn Ser Leu Ser Met Leu Ala Ala Gly Asn Thr
180 185 190
Val Val Phe Asn Val His Pro Ser Ser Lys Lys Val Cys Ser Tyr Leu
195 200 205
Ile Arg Glu Leu His Gln Ser Ile Val Gln Ala Gly Gly Pro Ala Asp
210 215 220
Leu Ile Thr Met Val Ala Asp Pro Thr Leu Asp Thr Leu Asn Glu Leu
225 230 235 240
Ala Ala His Pro Asp Ile Arg Leu Leu Val Gly Thr Gly Gly Pro Gly
245 250 255
Leu Val Lys Ser Leu Leu Gln Ser Gly Lys Lys Ala Ile Gly Ala Gly
260 265 270
Ala Gly Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Leu Val Asn
275 280 285
Ala Ala Lys Ser Ile Ile Leu Gly Ala Ser Phe Asp His Asn Leu Leu
290 295 300
Cys Ile Ala Glu Lys Glu Val Phe Val Leu Glu Glu Ala Ala Asn Glu
305 310 315 320
Leu Ile Tyr Gln Met Leu Asp Gln Gly Ala Tyr Met Leu Asn Asn Glu
325 330 335
Glu Leu Ser Arg Val Met Ser Leu Val Leu Thr Glu Asp Ser Ser Ser
340 345 350
Pro Val Ala Gly Gly Cys Thr Gly Lys Pro Ser Lys Lys Tyr His Val
355 360 365
Lys Lys Glu Trp Ile Gly Gln Ser Ala Ala Ala Ile Ala Arg Ala Ala
370 375 380
Gly Ile Asn Lys Glu Asn Ile Lys Leu Leu Ile Cys Glu Thr Asp Pro
385 390 395 400
Asp His Pro Phe Val Val Leu Glu Gln Met Met Pro Val Leu Pro Ile
405 410 415
Val Lys Thr Gln Ser Phe Glu Glu Ala Val Glu Trp Ala Val Ala Ala
420 425 430
Glu Lys Gly Asn Arg His Thr Ala Val Ile His Ser Thr Asn Val Asp
435 440 445
Arg Met Thr Ala Phe Ala Arg Ala Ile Glu Thr Thr Ile Phe Val Lys
450 455 460
Asn Ala Ser Ser Leu Ala Gly Val Gly Phe Gly Gly Glu Gly His Thr
465 470 475 480
Thr Met Thr Ile Ala Gly Pro Thr Gly Glu Gly Val Thr Ser Ala Arg
485 490 495
Ser Phe Thr Arg Lys Arg Arg Cys Val Leu Ala Glu Gly Gly Phe Arg
500 505 510
Ile Ile Gly
515
<210> 18
<211> 470
<212> PRT
<213> 平野梭菌（Clostridium hiranonis）
<400> 18
Met Lys Met Glu Leu Asp Leu Ile Gln Glu Met Ile Lys Gln Val Leu
1 5 10 15
Glu Glu Ile Lys Glu Glu Gly Val Glu Val Ser Ser Lys Glu Glu Tyr
20 25 30
Gly Tyr Gly Val Phe Asp Ser Met Val Glu Ala Ile Asp Ala Ser Glu
35 40 45
Lys Ala Gln Lys Glu Leu Phe Glu Cys Ser Val Gln Gln Arg Asp Lys
50 55 60
Phe Val Asp Ala Ile Arg Ala Glu Ile Leu Lys Lys Glu Asn Leu Glu
65 70 75 80
Met Ile Ser Tyr Asp Ala Val Glu Glu Thr Lys Ile Gly Arg Val Glu
85 90 95
Asp Lys Ile Ile Lys Asn Arg Val Ala Ala Glu Asn Thr Pro Gly Thr
100 105 110
Glu Asp Leu Lys Thr Arg Ala Ile Thr Gly Glu Asp Gly Leu Thr Ile
115 120 125
Glu Glu Tyr Cys Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr Thr
130 135 140
Asn Pro Thr Glu Thr Leu Ile Asn Asn Ser Ile Ser Met Ile Ala Gly
145 150 155 160
Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Lys Val Ser
165 170 175
Ile Lys Leu Val Lys Met Met Asn Lys Ala Leu Glu Glu Ala Gly Ala
180 185 190
Pro Arg Asn Leu Ile Thr Met Val Lys Glu Pro Ser Ile Glu Asn Ser
195 200 205
Lys Ile Met Met Glu Ser Pro Lys Val Arg Leu Leu Val Ala Thr Gly
210 215 220
Gly Pro Ala Ile Val Lys Gln Val Leu Ser Ala Gly Lys Lys Ala Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp
245 250 255
Ile Glu Lys Ala Ala Lys Asp Ile Val Ser Gly Ala Ser Phe Asp Asn
260 265 270
Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Glu Ser Val
275 280 285
Val Asp Gln Leu Ile Tyr Tyr Met Lys Lys Asn Gly Ala Tyr Glu Ile
290 295 300
Thr Ser Pro Glu Val Leu Glu Gln Leu Asp Lys Ala Val Ser Lys Glu
305 310 315 320
Asn Gly Lys Pro Asn Pro Ser Leu Val Gly Lys Ser Ala Lys Glu Leu
325 330 335
Leu Ala Leu Val Gly Ile Asn Val Asp Asp Asp Val Lys Leu Val Ile
340 345 350
Ala Arg Thr Asn Lys Asp His His Leu Val Thr Glu Glu Met Leu Met
355 360 365
Pro Ile Leu Pro Ile Val Ser Val Ser Asp Val Asp Thr Ala Ile Asp
370 375 380
Trp Ala Tyr Glu Ala Glu Ala Gly Asn Arg His Thr Ala Ile Met His
385 390 395 400
Ser Lys Asn Val Asp Lys Leu Thr Lys Met Ala Lys Lys Leu Glu Ala
405 410 415
Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val Gly
420 425 430
Gly Glu Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
435 440 445
Ile Thr Ser Ala Lys Ser Phe Cys Arg Ile Arg Arg Cys Val Met Ser
450 455 460
Glu Ala Leu Ser Ile Arg
465 470
<210> 19
<211> 466
<212> PRT
<213> 嗜热厌氧杆菌属（Thermoanaerobacter sp.）
<400> 19
Met Ile Asp Glu Asn Leu Val Val Thr Ile Thr Lys Lys Ile Leu Asn
1 5 10 15
Glu Ile Asn Leu Lys Glu Ala Glu Glu Lys Lys Glu Lys Asp Asn Pro
20 25 30
Asp Leu Gly Ile Phe Asn Asp Val Asn Glu Ala Val Glu Cys Ala Lys
35 40 45
Glu Ala Gln Lys Lys Phe Ala Leu Met Asp Leu Glu Lys Arg Glu Glu
50 55 60
Ile Ile Ala Ala Ile Arg Glu Ala Cys Val Asn Asn Ala Arg Leu Leu
65 70 75 80
Ala Glu Ile Ala Cys Ser Glu Thr Gly Arg Gly Arg Val Glu Asp Lys
85 90 95
Val Ala Lys Asn Ile Leu Ala Ala Lys Lys Thr Pro Gly Thr Glu Asp
100 105 110
Leu Lys Pro Thr Ala Trp Thr Gly Asp Arg Gly Leu Thr Leu Val Glu
115 120 125
Met Ala Pro Val Gly Val Ile Ala Ser Ile Thr Pro Val Thr Asn Pro
130 135 140
Thr Ala Thr Ile Ile Asn Asn Thr Ile Ser Met Leu Ala Ala Gly Asn
145 150 155 160
Ala Val Val Phe Asn Pro His Pro Ser Ala Lys Lys Thr Ser Asn Lys
165 170 175
Ala Val Glu Ile Ile Asn Glu Ala Ile Leu Lys Val Gly Ala Pro Asn
180 185 190
Gly Leu Val Cys Ser Ile Asn Asn Pro Thr Ile Gln Thr Ala Gln Lys
195 200 205
Leu Met Glu His Pro Glu Val Asn Met Val Val Val Thr Gly Gly Lys
210 215 220
Ala Val Val Gln Thr Ala Leu Arg Cys Gly Lys Lys Val Ile Gly Ala
225 230 235 240
Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Val
245 250 255
Lys Ala Ala His Asp Ile Ala Cys Gly Ala Ser Phe Asp Asn Asn Leu
260 265 270
Pro Cys Ile Ala Glu Lys Glu Ile Ile Ala Val Glu Arg Ile Ala Asp
275 280 285
Thr Leu Leu Glu Arg Met Lys Arg Glu Gly Ala Tyr Val Leu His Gly
290 295 300
Lys Asp Ile Asp Arg Met Thr Glu Leu Ile Phe Gln Gly Gly Ala Ile
305 310 315 320
Asn Lys Asp Leu Ile Gly Arg Asp Ala His Phe Ile Leu Ser Gln Ile
325 330 335
Gly Ile Glu Thr Gly Lys Asp Ile Arg Leu Val Val Met Pro Val Asp
340 345 350
Val Ser His Pro Leu Val Tyr His Glu Gln Leu Met Pro Val Ile Pro
355 360 365
Phe Val Thr Val Pro Thr Val Glu Glu Ala Ile Asn Leu Ala Val Lys
370 375 380
Ala Glu Gly Gly Asn Arg His Thr Ala Met Met His Ser Lys Asn Val
385 390 395 400
Glu Asn Met Thr Ala Phe Ala Arg Ala Ile Gln Thr Thr Ile Phe Val
405 410 415
Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly Glu Gly Tyr
420 425 430
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala
435 440 445
Arg Thr Phe Thr Arg Gln Arg Arg Cys Val Leu Val Asp Ala Phe Arg
450 455 460
Ile Val
465
<210> 20
<211> 462
<212> PRT
<213> 梭菌目 (Clostridiales sp.)
<400> 20
Met Pro Ile Asn Glu Asn Met Val Gln Glu Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Asp Ala Pro Thr Gly Lys His Gly Ile Phe
20 25 30
Lys Glu Met Asn Asp Ala Ile Glu Ala Ala Lys Lys Ser Gln Leu Ile
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Thr Cys Ile
50 55 60
Arg Lys Lys Ile Lys Glu Asn Ala Glu Val Met Ala Arg Met Gly Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His His
85 90 95
Leu Val Ala Asp Lys Thr Pro Gly Thr Glu Val Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Leu
130 135 140
Cys Asn Thr Met Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Tyr Ala Ile Asn Leu Leu
165 170 175
Asn Glu Ala Ser Leu Glu Ser Gly Gly Pro Asp Asn Ile Ala Val Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Thr Ser Asn Val Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Thr Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Ser Ser Ile Val Asp Glu Leu Met His Tyr
275 280 285
Leu Val Thr Glu Asn Asp Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Lys Leu Thr Glu Val Val Leu Ala Gly Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Arg Thr Leu Leu Ser Met Ile Gly Val Asn Ala
325 330 335
Pro Ala Asn Ile Arg Cys Ile Val Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Arg Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Ile Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Ala Pro
405 410 415
Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Cys Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ala Asp Ser Leu Cys Ile Arg
450 455 460
<210> 21
<211> 468
<212> PRT
<213> 白蚁塞巴鲁德菌（Sebaldella termitidis）
<400> 21
Met Leu Asp Gly Leu Gln Leu Glu Asp Ile Ile Lys Lys Val Ile Asn
1 5 10 15
Asp Val Lys Asn Glu Lys Asp Ile Asn Ile Thr Asn Lys Glu Asn Ser
20 25 30
Cys Gly His Gly Ile Phe Thr Asn Ile Glu Thr Ala Val Asp Lys Ala
35 40 45
Tyr Glu Ala Gln Gln Thr Tyr Asn Ser Arg Ser Leu Glu Glu Arg Arg
50 55 60
Asn Ile Ile Ser Asn Ile Arg Lys Glu Leu Leu Lys Tyr Thr Glu Glu
65 70 75 80
Met Ala Glu Lys Thr Val Ala Glu Thr Lys Met Gly Arg Ile Lys Asp
85 90 95
Lys Ile Leu Lys Asn Lys Leu Ala Ile Glu Lys Thr Pro Gly Val Glu
100 105 110
Asp Leu Gly Thr Glu Val Phe Thr Gly Asp Asp Gly Leu Thr Leu Val
115 120 125
Glu Leu Ser Ala Phe Gly Val Leu Gly Ser Val Thr Pro Val Thr Asn
130 135 140
Pro Thr Glu Thr Ile Ile Asn Asn Thr Ile Gly Ala Leu Ala Gly Gly
145 150 155 160
Asn Ser Ile Val Phe Cys Pro His Pro Ser Ala Lys Asn Ile Cys Leu
165 170 175
Trp Leu Ile Lys Lys Leu Asn Gly Ile Ile Thr Glu Ala Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Ser Ala Ser Glu Ala Lys Lys Glu Asn Val Asp
195 200 205
Ile Leu Phe Ser His Glu Lys Ile Asn Met Leu Val Ile Thr Gly Gly
210 215 220
Thr Glu Ile Val Lys Leu Ala Leu Lys Ser Gly Lys Lys Val Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Ile
245 250 255
Glu Lys Ala Ala Lys Asp Ile Val Asn Gly Ala Gly Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Leu Val Leu Glu Ser Val Ala
275 280 285
Asp Tyr Leu Ile Phe Asn Met Glu Lys Ala Gly Ala Phe His Ile Thr
290 295 300
Asp Lys Glu Asp Ile Lys Lys Leu Glu Asp Thr Val Tyr Lys Asn Gly
305 310 315 320
Met Val Asn Lys Glu Phe Ile Gly Lys Asp Ala Gly Phe Ile Leu Glu
325 330 335
Lys Ser Gly Ile Lys Cys Ser Phe Asp Pro Ala Leu Ile Thr Leu Glu
340 345 350
Thr Asp Ile Asn His Val Phe Val Gln Lys Glu Leu Met Met Pro Val
355 360 365
Leu Ala Val Val Arg Gln Lys Asn Phe Glu Glu Ala Leu Lys Asn Ala
370 375 380
Ile Leu Thr Glu His Gly Leu Lys His Thr Ala Val Met His Ser Gln
385 390 395 400
Asn Val Thr Arg Leu Ser Ile Ala Ala Arg Glu Met Gln Thr Thr Ile
405 410 415
Phe Val Lys Asn Ala Pro Ser Tyr Ala Ala Leu Gly Phe Gln Gly Glu
420 425 430
Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr
435 440 445
Ser Ala Arg Asn Phe Thr Arg Lys Arg Arg Cys Val Leu Gly Gly Ser
450 455 460
Phe Ser Ile Arg
465
<210> 22
<211> 462
<212> PRT
<213> 绳尾真杆菌（Eubacterium plexicaudatum）
<400> 22
Met Ser Val Asn Asp Gln Met Val Gln Asp Ile Val Arg Gln Val Leu
1 5 10 15
Ala Asn Met Arg Ile Ser Ser Asp Ala Ser Gly Ser Arg Gly Val Phe
20 25 30
Ser Asp Met Asn Glu Ala Val Glu Ala Ala Lys Lys Ala Gln Ala Val
35 40 45
Ile Gly Lys Met Pro Met Asp His Arg Glu Lys Ile Ile Ser Ser Ile
50 55 60
Arg Ala Lys Ile Met Glu Asn Ala Glu Ile Leu Ala Arg Met Gly Val
65 70 75 80
Lys Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Lys Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Leu
130 135 140
Cys Asn Thr Ile Gly Met Val Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Phe Ala Val Asn Leu Val
165 170 175
Asn Glu Ala Ser Val Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr
180 185 190
Val Glu His Pro Thr Leu Asp Thr Ser Ala Ile Met Met Lys His Lys
195 200 205
Asp Ile His Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Met His Tyr
275 280 285
Met Ile Ser Glu Gln Gly Cys Tyr Leu Ala Ser Ala Lys Glu Gln Glu
290 295 300
Ala Leu Ile Ser Val Val Leu Lys Gly Gly Gln Leu Asn Arg Asp Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Val Gln Ala
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Asp Ser Phe Glu Asp Ala Val Glu Lys Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp His Ile Thr
385 390 395 400
Thr Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Ala Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Cys Asp Ser Leu Cys Ile Arg
450 455 460
<210> 23
<211> 505
<212> PRT
<213> 埃希氏杆菌（Escherichia sp.）
<400> 23
Met Asn Asp Ile Glu Ile Ala Gln Ala Val Ser Thr Ile Leu Ser Lys
1 5 10 15
Phe Thr Lys Ala Thr Pro Asp Glu Ala Pro Ala Thr Ser Glu Ala Ala
20 25 30
Arg Val Asp Gly Leu Asp Glu Ile Val Ala Lys Ala Leu Ala Gln His
35 40 45
Ser Ser Val Arg Asp Ala Ser Ala Ile Ser Gln Val Ala Lys Val Ala
50 55 60
Asn Ala Ser Thr Gly Ala Phe Asp Thr Met Asp Glu Ala Ile Ser Ala
65 70 75 80
Ala Val Leu Ala Gln Val Gln Tyr Arg His Cys Ser Met Gln Asp Arg
85 90 95
Ala Ser Phe Ile Asn Gly Ile Arg Asp Val Phe Leu Gln Glu Asp Val
100 105 110
Leu Cys Ala Leu Ser Arg Met Ala Val Glu Glu Thr Gly Met Gly Asn
115 120 125
Tyr Glu Asp Lys Leu Ile Lys Asn Arg Val Ala Ala Leu Lys Thr Pro
130 135 140
Gly Ile Glu Asp Leu Thr Thr Ser Ala Val Ser Gly Asp Gly Gly Leu
145 150 155 160
Thr Leu Ile Glu Tyr Ser Ala Phe Gly Val Ile Gly Ser Ile Thr Pro
165 170 175
Thr Thr Asn Pro Thr Glu Thr Ile Ile Asn Asn Ser Ile Gly Met Leu
180 185 190
Ala Ala Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ser Arg Lys
195 200 205
Val Ser Leu Tyr Ala Val Glu Leu Ile Asn Asn Lys Leu Ala Gln Leu
210 215 220
Gly Ala Pro Ala Asn Met Val Val Thr Val Thr Lys Pro Ser Ile Asp
225 230 235 240
Asn Thr Asn Val Leu Ile Asn Asp Pro Arg Ile Asn Met Leu Val Ala
245 250 255
Thr Gly Gly Pro Ala Ile Val Lys Thr Val Met Ser Ser Gly Lys Lys
260 265 270
Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Ala Val Val Asp Glu Thr
275 280 285
Ala Asp Ile Glu Lys Ala Ala Arg Asp Ile Ile Lys Gly Cys Ser Phe
290 295 300
Asp Asn Asn Leu Pro Cys Val Ala Glu Lys Glu Val Ile Val Val Asn
305 310 315 320
Gln Val Ala Asp Tyr Leu Ile His Cys Met Lys Lys Ser Gly Ala Tyr
325 330 335
Leu Leu Cys Asp Lys Lys Leu Ser Gln Gln Leu Gln Ser Leu Val Leu
340 345 350
Asn Glu Lys Gly Thr Gly Pro Asn Thr Ala Phe Val Gly Lys Asp Ala
355 360 365
Arg Tyr Ile Leu Gln Gln Leu Gly Ile Gln Val Gly Asp Asp Ile Lys
370 375 380
Val Ile Leu Ile Glu Ala Glu Lys Thr His Pro Phe Val Val His Glu
385 390 395 400
Leu Met Met Pro Val Leu Pro Val Val Arg Val Asp Asn Val Asp Glu
405 410 415
Ala Ile Glu Leu Ala Val Lys Val Glu His Gly Asn Arg His Thr Ala
420 425 430
Val Met His Ser Thr Asn Val Glu Lys Leu Thr Lys Met Ala Arg Leu
435 440 445
Ile Gln Thr Thr Ile Phe Val Lys Asn Gly Pro Ser Tyr Ala Gly Leu
450 455 460
Gly Val Gly Gly Glu Gly His Ala Thr Phe Thr Ile Ala Gly Pro Thr
465 470 475 480
Gly Glu Gly Leu Thr Ser Ala Arg Ser Phe Ala Arg Arg Arg Arg Cys
485 490 495
Val Met Val Glu Ala Leu Asn Ile Arg
500 505
<210> 24
<211> 529
<212> PRT
<213> 深红螺菌（Rhodospirillum rubrum）
<400> 24
Met Asn Asp Gly Gln Ile Ala Ala Ala Val Ala Lys Val Leu Glu Ala
1 5 10 15
Tyr Gly Val Pro Ala Asp Pro Ser Ala Ala Ala Pro Ala Pro Ala Ala
20 25 30
Pro Val Ala Pro Ala Ala Pro Thr Ala Gly Ser Val Ser Glu Met Ile
35 40 45
Ala Arg Gly Ile Ala Lys Ala Ser Ser Asp Asp Gln Ile Ala Gln Ile
50 55 60
Val Ala Lys Val Val Gly Asp Tyr Ser Ala Gln Ala Ala Lys Pro Ala
65 70 75 80
Val Val Pro Gly Ala Ala Ala Ser Thr Glu Ala Gly Asp Gly Val Phe
85 90 95
Asp Thr Met Asp Ala Ala Val Asp Ala Ala Val Leu Ala Gln Gln Gln
100 105 110
Tyr Leu Leu Cys Ser Met Thr Asp Arg Gln Arg Phe Val Asp Gly Ile
115 120 125
Arg Glu Val Ile Leu Gln Lys Asp Thr Leu Glu Leu Ile Ser Arg Met
130 135 140
Ala Ala Glu Glu Thr Gly Met Gly Asn Tyr Glu His Lys Leu Ile Lys
145 150 155 160
Asn Arg Leu Ala Ala Glu Lys Thr Pro Gly Thr Glu Asp Leu Thr Thr
165 170 175
Glu Ala Phe Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Tyr Ser Pro
180 185 190
Phe Gly Ala Ile Gly Ala Val Ala Pro Thr Thr Asn Pro Thr Glu Thr
195 200 205
Ile Ile Cys Asn Ser Ile Gly Met Leu Ala Ala Gly Asn Ser Val Ile
210 215 220
Phe Ser Pro His Pro Arg Ala Thr Lys Val Ser Leu Leu Thr Val Lys
225 230 235 240
Leu Ile Asn Gln Lys Leu Ala Cys Leu Gly Ala Pro Ala Asn Leu Val
245 250 255
Val Thr Val Ser Lys Pro Ser Val Glu Asn Thr Asn Ala Met Met Ala
260 265 270
His Pro Lys Ile Arg Met Leu Val Ala Thr Gly Gly Pro Gly Ile Val
275 280 285
Lys Ala Val Met Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly
290 295 300
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala
305 310 315 320
Leu Asp Ile Ile Asn Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile
325 330 335
Ala Glu Lys Glu Ile Ile Ala Val Ala Gln Ile Ala Asp Tyr Leu Ile
340 345 350
Phe Ser Met Lys Lys Gln Gly Ala Tyr Gln Ile Thr Asp Pro Ala Val
355 360 365
Leu Arg Lys Leu Gln Asp Leu Val Leu Thr Ala Lys Gly Gly Pro Gln
370 375 380
Thr Ser Cys Val Gly Lys Ser Ala Val Trp Leu Leu Asn Lys Ile Gly
385 390 395 400
Ile Glu Val Asp Ser Ser Val Lys Val Ile Leu Met Glu Val Pro Lys
405 410 415
Glu His Pro Phe Val Gln Glu Glu Leu Met Met Pro Ile Leu Pro Leu
420 425 430
Val Arg Val Ser Asp Val Asp Glu Ala Ile Ala Val Ala Ile Glu Val
435 440 445
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Thr Asn Val Arg
450 455 460
Lys Leu Thr Lys Met Ala Lys Leu Ile Gln Thr Thr Ile Phe Val Lys
465 470 475 480
Asn Gly Pro Ser Tyr Ala Gly Leu Gly Val Gly Gly Glu Gly Tyr Thr
485 490 495
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys
500 505 510
Ser Phe Ala Arg Lys Arg Lys Cys Val Met Val Glu Ala Leu Asn Ile
515 520 525
Arg
<210> 25
<211> 472
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 25
Met Asp Val Asp Val Val Leu Val Glu Lys Leu Val Arg Gln Ala Ile
1 5 10 15
Glu Glu Val Lys Asn Lys Asn Leu Leu Asn Leu Asp Lys Phe Glu Ser
20 25 30
Val Lys Asn Tyr Gly Ile Phe Gly Thr Met Asp Ala Ala Val Glu Ala
35 40 45
Ser Phe Val Ala Gln Lys Gln Leu Leu Asn Ala Ser Met Thr Asp Lys
50 55 60
Gln Lys Tyr Val Asp Thr Ile Lys Ala Thr Ile Leu Lys Lys Glu Asn
65 70 75 80
Leu Glu Leu Ile Ser Arg Met Ser Val Glu Glu Thr Glu Ile Gly Lys
85 90 95
Tyr Glu His Lys Leu Ile Lys Asn Arg Val Ala Ala Glu Lys Thr Pro
100 105 110
Gly Ile Glu Asp Leu Thr Thr Glu Ala Met Thr Gly Asp Asn Gly Leu
115 120 125
Thr Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro
130 135 140
Thr Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Ser Met Ile
145 150 155 160
Ala Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Asn
165 170 175
Val Ser Ile Lys Leu Val Thr Met Leu Asn Lys Ala Leu Glu Glu Ala
180 185 190
Gly Ala Pro Asp Asn Leu Ile Ala Thr Val Lys Glu Pro Ser Ile Glu
195 200 205
Asn Thr Asn Ile Met Met Glu His Pro Lys Ile Arg Met Leu Val Ala
210 215 220
Thr Gly Gly Pro Ala Ile Val Asn Lys Val Met Ser Thr Gly Lys Lys
225 230 235 240
Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr
245 250 255
Ala Asp Ile Glu Lys Ala Ala Ile Asp Ile Val Asn Gly Cys Ser Phe
260 265 270
Asp Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp
275 280 285
Gln Ile Cys Asp Tyr Leu Ile His Tyr Met Lys Leu Asn Gly Ala Tyr
290 295 300
Glu Ile Lys Asp Arg Asp Leu Ile Gln Lys Leu Leu Asp Leu Val Thr
305 310 315 320
Asn Glu Asn Gly Gly Pro Lys Val Ser Phe Val Gly Lys Ser Ala Pro
325 330 335
Tyr Ile Leu Asn Lys Leu Gly Ile Ser Val Asp Glu Asn Ile Lys Val
340 345 350
Ile Ile Met Glu Val Glu Lys Asn His His Phe Val Leu Glu Glu Met
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Thr Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Tyr Val Ala Glu His Gly Asn Arg His Thr Ala Ile
385 390 395 400
Met His Ser Lys Asn Val Asp Lys Leu Thr Lys Met Ala Arg Leu Leu
405 410 415
Glu Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly
420 425 430
Val Gly Gly Glu Gly Thr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Leu Thr Thr Ala Arg Ser Phe Cys Arg Lys Arg Arg Cys Val
450 455 460
Met Val Asp Ala Phe Asn Ile Arg
465 470
<210> 26
<211> 468
<212> PRT
<213> 霍氏真杆菌（Eubacterium hallii）
<400> 26
Met Asn Ile Asp Val Glu Leu Ile Glu Lys Val Val Lys Lys Val Leu
1 5 10 15
Asn Asp Val Glu Thr Gly Ser Ser Glu Ser Glu Tyr Gly Tyr Gly Ile
20 25 30
Phe Asp Thr Met Asp Glu Ala Ile Glu Ala Ser Ala Lys Ala Gln Lys
35 40 45
Glu Tyr Met Asn His Ser Met Ala Asp Arg Gln Arg Tyr Val Glu Gly
50 55 60
Ile Arg Glu Val Val Cys Thr Lys Glu Asn Leu Glu Tyr Met Ser Lys
65 70 75 80
Leu Ala Val Glu Glu Ser Gly Met Gly Ala Tyr Glu Tyr Lys Val Ile
85 90 95
Lys Asn Arg Leu Ala Ala Val Lys Ser Pro Gly Val Glu Asp Leu Thr
100 105 110
Thr Glu Ala Leu Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Tyr Cys
115 120 125
Pro Phe Gly Val Ile Gly Ala Ile Ala Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Val Ile Cys Asn Ser Ile Ala Met Leu Ala Gly Gly Asn Thr Val
145 150 155 160
Val Phe Ser Pro His Pro Arg Ser Lys Gly Val Ser Ile Trp Leu Ile
165 170 175
Lys Lys Leu Asn Ala Lys Leu Glu Glu Leu Gly Ala Pro Arg Asn Leu
180 185 190
Ile Val Thr Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Ile Met Met
195 200 205
Asn His Pro Lys Val Arg Met Leu Val Ala Thr Gly Gly Pro Gly Ile
210 215 220
Val Lys Ala Val Met Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Val Asn Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Ile Ala Glu Lys Glu Val Ile Ala Val Asp Gln Ile Ala Asp Tyr Leu
275 280 285
Ile Phe Asn Met Lys Asn Asn Gly Ala Tyr Glu Val Lys Asp Pro Glu
290 295 300
Ile Ile Glu Lys Met Val Asp Leu Val Thr Lys Asp Arg Lys Lys Pro
305 310 315 320
Ala Val Asn Phe Val Gly Lys Ser Ala Gln Tyr Ile Leu Asp Lys Val
325 330 335
Gly Ile Lys Val Gly Pro Glu Val Lys Cys Ile Ile Met Glu Ala Pro
340 345 350
Lys Asp His Pro Phe Val Gln Ile Glu Leu Met Met Pro Ile Leu Pro
355 360 365
Ile Val Arg Val Pro Asn Val Asp Glu Ala Ile Asp Phe Ala Val Glu
370 375 380
Val Glu His Gly Asn Arg His Thr Ala Met Met His Ser Lys Asn Val
385 390 395 400
Asp Lys Leu Thr Lys Met Ala Lys Glu Ile Glu Thr Thr Ile Phe Val
405 410 415
Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Met Gly Tyr
420 425 430
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala
435 440 445
Lys Ser Phe Cys Arg Lys Arg Arg Cys Val Leu Gln Asp Gly Leu His
450 455 460
Ile Arg Met Lys
465
<210> 27
<211> 532
<212> PRT
<213> 弧菌属（Vibrio sp.）
<400> 27
Met Asn Glu Gln Glu Ile Ala His Ala Val Glu Asn Val Leu Ser Lys
1 5 10 15
Tyr Thr Asn Val Thr Ala Gln Asn Ala Glu Pro Val Ser Tyr Ser Ser
20 25 30
Asn Ala Ser Leu Glu Asn Ile Val Ser Gln Ala Leu Ala Gly Asn Met
35 40 45
Val Lys Gln Pro Glu Thr Gln Thr Ala Pro Asp Leu Asn Ser Asn Ile
50 55 60
Glu Asn Ile Val Ser Gln Ile Leu Ala Glu Asn Gln Ala Lys Pro Gln
65 70 75 80
Ser Val Gln Cys Gln Ser Ala Asn His Gly Thr Thr Glu Tyr Leu Gly
85 90 95
Cys Phe Ala Ser Met Glu Glu Ala Ile Ser Ala Ala Ser His Ala Gln
100 105 110
Val Gln Tyr Arg His Cys Thr Met Gly Asp Arg Ala Ser Phe Val Lys
115 120 125
Gly Ile Arg Glu Val Phe Thr Gln Asp Asp Val Leu Glu Lys Ile Ser
130 135 140
Arg Met Ala Val Glu Glu Thr Gly Met Gly Asn Tyr Ala Asp Lys Leu
145 150 155 160
Thr Lys Asn Arg Ile Ala Ala Thr Lys Thr Pro Gly Ile Glu Asp Leu
165 170 175
Thr Thr Ser Ala Leu Ser Gly Asp Ser Gly Leu Thr Leu Thr Glu Phe
180 185 190
Ser Ala Tyr Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr
195 200 205
Glu Thr Ile Ile Asn Asn Ser Ile Gly Met Leu Ala Ala Gly Asn Thr
210 215 220
Val Val Tyr Ser Pro His Pro Arg Ser Arg Asn Val Ser Leu Val Ala
225 230 235 240
Val Asp Leu Ile Asn Arg Lys Leu Ala Glu Leu Gly Ala Pro Ala Asn
245 250 255
Leu Val Val Thr Val Leu Glu Pro Ser Ile Asp Asn Thr Asn Ala Met
260 265 270
Met Asn Asp Pro Arg Val Asn Met Leu Val Ala Thr Gly Gly Pro Ser
275 280 285
Ile Val Lys Thr Val Met Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly
290 295 300
Ala Gly Asn Pro Pro Ala Val Val Asp Glu Thr Ala Asn Ile Glu Lys
305 310 315 320
Ala Ala Lys Asp Ile Ile Asn Gly Cys Ala Phe Asp Asn Asn Leu Pro
325 330 335
Cys Ile Ala Glu Lys Glu Val Ile Val Val Asn Glu Val Ala Asp Tyr
340 345 350
Leu Ile His Cys Met Lys Lys Ser Gly Ala Tyr Leu Leu Cys Asp Lys
355 360 365
Gln Lys Ile Gln Gln Leu Gln Ser Leu Val Leu Asn Glu Lys Gly Thr
370 375 380
Gly Pro Asn Thr Ser Phe Val Gly Lys Gly Ala Arg Tyr Ile Leu Asp
385 390 395 400
Lys Leu Asn Ile Gln Val Ser Asp Asp Ile Lys Val Ile Leu Ile Glu
405 410 415
Thr Glu Arg Asn His Pro Phe Val Val His Glu Leu Met Met Pro Ile
420 425 430
Leu Pro Val Val Arg Val Glu Asn Val Asp Glu Ala Ile Asp Leu Ala
435 440 445
Ile Lys Val Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Thr
450 455 460
Asn Val Glu Lys Leu Ser Lys Met Ala Arg Leu Ile Gln Thr Thr Ile
465 470 475 480
Phe Val Lys Asn Gly Pro Ser Tyr Ser Gly Ile Gly Val Gly Gly Glu
485 490 495
Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr
500 505 510
Ser Ala Arg Ser Phe Ala Arg Tyr Arg Arg Cys Val Met Val Glu Ala
515 520 525
Leu Asn Ile Arg
530
<210> 28
<211> 464
<212> PRT
<213> 沼泽红假单胞菌（Rhodopseudomonas palustris）
<400> 28
Met Val Ala Lys Ala Ile Arg Asp His Ala Gly Thr Ala Gln Pro Ser
1 5 10 15
Gly Asn Ala Ala Thr Ser Ser Ala Ala Val Ser Asp Gly Val Phe Glu
20 25 30
Thr Met Asp Ala Ala Val Glu Ala Ala Ala Leu Ala Gln Gln Gln Tyr
35 40 45
Leu Leu Cys Ser Met Ser Asp Arg Ala Arg Phe Val Gln Gly Ile Arg
50 55 60
Asp Val Ile Leu Asn Gln Asp Thr Leu Glu Lys Met Ser Arg Met Ala
65 70 75 80
Val Glu Glu Thr Gly Met Gly Asn Tyr Glu His Lys Leu Ile Lys Asn
85 90 95
Arg Leu Ala Gly Glu Lys Thr Pro Gly Ile Glu Asp Leu Thr Thr Asp
100 105 110
Ala Phe Ser Gly Asp Asn Gly Leu Thr Leu Val Glu Tyr Ser Pro Phe
115 120 125
Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu Thr Ile
130 135 140
Val Cys Asn Ser Ile Gly Met Leu Ala Ala Gly Asn Ser Val Val Phe
145 150 155 160
Ser Pro His Pro Arg Ala Arg Gln Val Ser Leu Leu Leu Val Arg Leu
165 170 175
Ile Asn Gln Lys Leu Ala Ala Leu Gly Ala Pro Glu Asn Leu Val Val
180 185 190
Thr Val Glu Lys Pro Ser Ile Glu Asn Thr Asn Ala Met Met Ala His
195 200 205
Pro Lys Val Arg Met Leu Val Ala Thr Gly Gly Pro Ala Ile Val Lys
210 215 220
Ala Val Leu Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Val Val Val Asp Glu Thr Ala Asn Ile Glu Lys Ala Ala Cys
245 250 255
Asp Ile Val Asn Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Val Ala
260 265 270
Glu Lys Glu Ile Ile Ala Val Ala Gln Ile Ala Asp Tyr Leu Ile Phe
275 280 285
Asn Leu Lys Lys Asn Gly Ala Tyr Glu Ile Lys Asp Pro Ala Val Leu
290 295 300
Gln Gln Leu Gln Asp Leu Val Leu Thr Ala Lys Gly Gly Pro Gln Thr
305 310 315 320
Lys Cys Val Gly Lys Ser Ala Val Trp Leu Leu Ser Gln Ile Gly Ile
325 330 335
Ser Val Asp Ala Ser Ile Lys Ile Ile Leu Met Glu Val Pro Arg Glu
340 345 350
His Pro Phe Val Gln Glu Glu Leu Met Met Pro Ile Leu Pro Leu Val
355 360 365
Arg Val Glu Thr Val Asp Asp Ala Ile Asp Leu Ala Ile Glu Val Glu
370 375 380
His Asp Asn Arg His Thr Ala Ile Met His Ser Thr Asp Val Arg Lys
385 390 395 400
Leu Thr Lys Met Ala Lys Leu Ile Gln Thr Thr Ile Phe Val Lys Asn
405 410 415
Gly Pro Ser Tyr Ala Gly Leu Gly Ala Gly Gly Glu Gly Tyr Ser Thr
420 425 430
Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys Ser
435 440 445
Phe Ala Arg Arg Arg Lys Cys Val Met Val Glu Ala Leu Asn Ile Arg
450 455 460
<210> 29
<211> 468
<212> PRT
<213> 食烯烃脱硫杆菌（Desulfatibacillum alkenivorans）
<400> 29
Met Ser Val Lys Glu Phe Ala Leu Glu Asp Met Val Ala Ser Val Ile
1 5 10 15
Met Glu Met Met Asn Lys Asp Asp Asp Ser Cys Gln Pro Thr Gly Asp
20 25 30
Gly Ile Tyr Glu Thr Ile Asp Glu Ala Val Ala Lys Ala Lys Ala Ala
35 40 45
Gln Pro Arg Leu Ile Ser Leu Ser Leu Glu Lys Arg Glu Ala Ile Leu
50 55 60
Thr Ala Ile Arg Lys Ile Ser Leu Glu Lys Asn Glu Glu Trp Ala Lys
65 70 75 80
Ala Thr Val Ala Glu Thr Gly Leu Gly Arg Val Glu Asp Lys Ile Ala
85 90 95
Glu Asn Ile Leu Ala Ala Thr Lys Thr Pro Gly Thr Glu Asp Leu Asp
100 105 110
Ala Lys Ala Leu Ser Gly Asp Ala Gly Leu Thr Leu Ile Glu Tyr Ala
115 120 125
Pro Phe Gly Val Ile Gly Ser Leu Thr Pro Val Thr Asn Ala Thr Gly
130 135 140
Thr Leu Ile Asn Asn Thr Ile Ser Met Leu Ala Gly Gly Asn Thr Val
145 150 155 160
Val Tyr Asn Val His Pro Ser Ala Leu Lys Ile Ser Thr Glu Val Ile
165 170 175
Arg Thr Phe His Lys Val Ile Val Glu Asn Gly Gly Pro Glu Gly Cys
180 185 190
Val Gly Met Val Ala Thr Pro Thr Met Glu Thr Ala Gly Glu Ile Met
195 200 205
Ala His Pro Asp Ile Asn Val Leu Val Ala Thr Gly Gly Ala Gly Val
210 215 220
Val Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Cys Ile Arg Lys Ala
245 250 255
Ala Glu Glu Ile Ile Ala Gly His Ser Ile Asn Asn Asn Ile Phe Cys
260 265 270
Ile Ser Glu Lys Glu Val Ile Ala Val Asp Glu Val Ala Asp Asn Leu
275 280 285
Leu Lys Phe Met Glu Glu Thr Gly Lys Ala Ala Ile Leu Thr Pro Glu
290 295 300
Glu Ala Gln Lys Val Thr Glu Thr Val Ile His Asp Asn His Val Val
305 310 315 320
Lys Asp Tyr Val Gly Lys Asn Ala Ser Val Ile Ile Glu Gly Ala Gly
325 330 335
Leu Thr Arg Leu Ala Gly Lys Lys Asp Leu Arg Cys Leu Val Phe Glu
340 345 350
Ala Asp Cys Lys His Pro Met Val Trp Ile Glu Gln Met Met Pro Val
355 360 365
Leu Pro Met Val Arg Val Lys Asp Val Trp Glu Gly Ile Asp Leu Ala
370 375 380
Val Lys Val Glu Gln Gly Asn Arg His Thr Ala Met Met His Ser Thr
385 390 395 400
Asn Val Glu His Leu Thr Ala Leu Ala Arg Ala Ile Gln Thr Thr Ile
405 410 415
Phe Val Lys Asn Gly Pro Ser Tyr Ser Gly Ile Gly Leu Asn Gly Glu
420 425 430
Gly His Ala Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr
435 440 445
Ser Ala Lys Ser Phe Cys Arg Gln Arg Arg Cys Val Leu Ile Asp Ser
450 455 460
Phe Arg Ile Val
465
<210> 30
<211> 469
<212> PRT
<213> 糖丁酸梭菌（Clostridium saccharobutylicum）
<400> 30
Met Asn Asn Asn Leu Phe Val Ser Pro Glu Thr Lys Asp Leu Lys Leu
1 5 10 15
Arg Thr Asn Val Glu Asn Leu Lys Phe Lys Gly Cys Glu Gly Gly Ser
20 25 30
Thr Tyr Ile Gly Val Phe Glu Asn Ala Glu Thr Ala Ile Asp Glu Ala
35 40 45
Val Asn Ala Gln Lys Arg Leu Ser Leu Tyr Tyr Thr Lys Glu Gln Arg
50 55 60
Glu Lys Ile Ile Thr Glu Ile Arg Lys Val Thr Leu Lys Asn Lys Glu
65 70 75 80
Ile Leu Ala Gln Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu
85 90 95
Asp Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr
100 105 110
Glu Asp Leu Ala Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val
115 120 125
Val Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr
130 135 140
Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Gly Met Ile Ala Ser
145 150 155 160
Gly Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val
165 170 175
Ala Phe Ala Val Asp Met Ile Asn Arg Ala Ile Ile Ser Cys Gly Gly
180 185 190
Pro Arg Asn Leu Val Thr Ala Ile Lys Asn Pro Thr Met Glu Ser Leu
195 200 205
Asp Ala Ile Ile Lys His Pro Ala Ile Lys Leu Leu Cys Gly Thr Gly
210 215 220
Gly Pro Gly Met Val Lys Thr Leu Leu Ser Ser Gly Lys Lys Ser Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp
245 250 255
Ile Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val
275 280 285
Ala Asp Asp Leu Ile Lys Asn Met Leu Lys Asn Asn Ala Val Ile Ile
290 295 300
Asn Lys Asp Gln Val Ser Arg Leu Val Asn Leu Val Leu Gln Lys Asn
305 310 315 320
Asn Glu Thr Ser Glu Tyr Thr Ile Asn Lys Lys Trp Val Gly Lys Asp
325 330 335
Ala Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Ser Ser Asp Val
340 345 350
Arg Cys Ile Ile Cys Glu Val Asp Ala Asp His Pro Phe Val Met Thr
355 360 365
Glu Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp
370 375 380
Glu Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser
385 390 395 400
Ala Tyr Ile Tyr Ser Lys Asn Ile Glu Asn Leu Asn Arg Phe Glu Lys
405 410 415
Glu Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly
420 425 430
Val Gly Tyr Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Cys
435 440 445
Thr Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg
450 455 460
Cys Val Phe Val Gly
465
<210> 31
<211> 468
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 31
Met Asn Lys Asp Thr Thr Ile Ser Glu Thr Glu Asn Leu Lys Phe Lys
1 5 10 15
Thr Asn Ile Lys Asn Ala Asp Leu Lys Asn Tyr Glu Asn Ser Thr Ser
20 25 30
Tyr Ser Gly Val Phe Glu Asp Val Glu Val Ala Ile Asn Lys Ala Ile
35 40 45
Thr Ala Gln Lys Glu Phe Ser Leu Tyr Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Leu Thr Glu Ile Arg Lys Ala Thr Leu Lys Asn Lys Lys Ile
65 70 75 80
Leu Ala Lys Met Ile Leu Asp Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Ile Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ala Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Ser Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Asp Met Ile Asn Lys Ala Ile Val Ser Cys Gly Gly Pro
180 185 190
Lys Asn Leu Ile Thr Ala Val Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Glu Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Asp Asn Val Ala
275 280 285
Asp Asn Leu Ile Asp Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Lys Asp Lys Ile Thr Lys Leu Leu Asn Leu Ile Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Asn Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asn Glu Ile Asp Val Glu Ala Pro Ser Ser Val Arg
340 345 350
Cys Ile Ile Cys Glu Val Glu Pro Asp His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asn Ile Asp Asp
370 375 380
Ala Ile Gln Tyr Ala Lys Ile Ala Glu Gln Ser Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Asn Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Cys Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 32
<211> 467
<212> PRT
<213> 丝状孢子梭菌（Clostridium taeniosporum）
<400> 32
Met Glu Arg Asn Leu Ser Val Leu Ser Gln Lys Lys Asn Leu Lys Ile
1 5 10 15
Thr Arg Lys Val Glu Gly Asn Lys Ser Ile Asn Lys Glu Ser Tyr Leu
20 25 30
Gly Val Phe Glu Lys Val Asp Asn Ala Ile Thr Lys Ala Ile Tyr Ala
35 40 45
Gln Arg Lys Leu Ser Leu Tyr Tyr Thr Lys Glu Asp Arg Glu Arg Ile
50 55 60
Ile Glu Gly Ile Arg Lys Ala Thr Leu Glu Asn Lys Glu Ile Leu Ala
65 70 75 80
Lys Met Ile Val Asp Glu Thr His Met Gly Arg Tyr Glu Asp Lys Ile
85 90 95
Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu Asp Leu
100 105 110
Ile Thr Thr Ala Trp Ser Gly Asp Gln Gly Leu Thr Leu Val Glu Met
115 120 125
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr
130 135 140
Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ser
145 150 155 160
Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala Phe Ala
165 170 175
Val Asp Met Ile Asn Lys Ala Ile Ile Lys Cys Gly Gly Pro Glu Asn
180 185 190
Leu Val Thr Thr Val Glu Asn Pro Thr Met Asp Ser Leu Asn Val Ile
195 200 205
Met Lys His Pro Tyr Val Lys Leu Leu Cys Gly Thr Gly Gly Pro Gly
210 215 220
Leu Ile Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp Ile Lys His
245 250 255
Ala Ala Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn Leu Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala Asp Asp
275 280 285
Leu Ile Gln Asn Met Leu Lys Asn Asn Ala Val Leu Ile Asn Glu Asn
290 295 300
Glu Val Ser Lys Leu Leu Asp Leu Val Leu Ile Glu Lys Lys Asp Glu
305 310 315 320
Pro Ser Gly Tyr Val Ile Asn Lys Lys Trp Val Gly Lys Asp Ala Lys
325 330 335
Leu Phe Leu Asp Lys Ile Gly Lys Lys Val Ser Asp Asp Val Lys Cys
340 345 350
Ile Ile Cys Glu Val Asp Val Asn His Pro Phe Val Met Thr Glu Leu
355 360 365
Met Met Pro Ile Leu Ala Ile Ala Arg Val Lys Asp Ile Asp Glu Ala
370 375 380
Ile Glu Cys Ala Lys Thr Ala Glu Gln Gly Lys Arg His Ser Ala Tyr
385 390 395 400
Met Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu Ile
405 410 415
Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val Gly
420 425 430
Phe Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys Val
450 455 460
Leu Ala Gly
465
<210> 33
<211> 467
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 33
Met Glu Arg Asn Leu Ser Val Leu Ser Gln Thr Asn Asp Leu Lys Ile
1 5 10 15
Thr Lys Arg Thr Glu Gly Asp Lys Ser Asn Asn Lys Glu Ser Tyr Leu
20 25 30
Gly Val Phe Lys Lys Val Glu Asn Ala Ile Thr Lys Ala Ile Tyr Ala
35 40 45
Gln Lys Lys Leu Ser Leu Tyr Tyr Thr Lys Glu Asp Arg Glu Arg Ile
50 55 60
Ile Lys Ser Ile Arg Lys Ala Thr Leu Glu Asn Lys Glu Ile Leu Ala
65 70 75 80
Lys Met Ile Val Asp Glu Thr His Met Gly Arg Tyr Glu Asp Lys Ile
85 90 95
Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu Asp Leu
100 105 110
Ile Thr Thr Ala Trp Ser Gly Asp Gln Gly Leu Thr Leu Val Glu Met
115 120 125
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr
130 135 140
Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asp Ser
145 150 155 160
Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala Phe Ala
165 170 175
Val Asp Met Ile Asn Lys Ala Val Ile Arg Glu Gly Gly Pro Glu Asn
180 185 190
Leu Val Thr Thr Val Glu Asn Pro Thr Met Glu Ser Leu Asn Val Ile
195 200 205
Met Lys His Pro Tyr Ile Lys Leu Leu Cys Gly Thr Gly Gly Pro Gly
210 215 220
Leu Ile Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp Ile Asp Lys
245 250 255
Ala Ala Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn Leu Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala Asn Asp
275 280 285
Leu Ile Gln Asn Met Ile Lys Asn Asn Ala Val Leu Ile Asn Glu Asn
290 295 300
Gln Val Ser Lys Leu Leu Asp Leu Val Leu Leu Glu Arg Lys Asp Glu
305 310 315 320
Thr Leu Glu Tyr Ala Ile Asn Lys Lys Trp Val Gly Lys Asp Ala Lys
325 330 335
Leu Phe Leu Asp Lys Ile Gly Ile Lys Ala Ser Asp Asn Val Arg Cys
340 345 350
Ile Ile Cys Glu Val Asp Ala Asn His Pro Phe Val Met Thr Glu Leu
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Lys Thr Ala Glu Gln Arg Lys Arg His Ser Ala Tyr
385 390 395 400
Met Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu Ile
405 410 415
Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val Gly
420 425 430
Phe Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys Val
450 455 460
Leu Ala Gly
465
<210> 34
<211> 467
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 34
Met Lys Arg Asn Leu Ser Val Leu Leu Gln Thr Asn Asp Leu Lys Ile
1 5 10 15
Thr Lys Arg Thr Glu Gly Asp Lys Ser Asn Asn Lys Glu Ser Tyr Leu
20 25 30
Gly Val Phe Lys Lys Val Glu Asn Ala Ile Thr Lys Ala Ile Tyr Ala
35 40 45
Gln Lys Lys Leu Ser Leu Tyr Tyr Thr Lys Glu Asp Arg Glu Arg Ile
50 55 60
Ile Lys Gly Ile Arg Lys Ala Thr Leu Glu Asn Lys Glu Ile Leu Ala
65 70 75 80
Lys Met Ile Val Asp Glu Thr His Met Gly Arg Tyr Glu Asp Lys Ile
85 90 95
Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu Asp Leu
100 105 110
Ile Thr Thr Ala Trp Ser Gly Asp Gln Gly Leu Thr Leu Val Glu Met
115 120 125
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr
130 135 140
Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asp Ser
145 150 155 160
Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala Phe Ala
165 170 175
Val Asp Met Ile Asn Lys Ala Val Ile Lys Ala Gly Gly Pro Glu Asn
180 185 190
Leu Val Thr Thr Val Glu Asn Pro Thr Met Glu Ser Leu Asn Val Ile
195 200 205
Met Lys His Pro Tyr Ile Lys Leu Leu Cys Gly Thr Gly Gly Pro Gly
210 215 220
Leu Ile Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp Ile Asn Lys
245 250 255
Ala Ala Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn Ser Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala Asn Asp
275 280 285
Leu Ile Gln Asn Met Ile Lys Asn Asn Ala Val Leu Ile Asn Glu Asn
290 295 300
Gln Val Ser Lys Leu Leu Asp Leu Val Leu Leu Glu Arg Lys Asp Glu
305 310 315 320
Thr Leu Glu Tyr Ala Ile Asn Lys Lys Trp Val Gly Lys Asp Ala Lys
325 330 335
Leu Phe Leu Asp Lys Ile Gly Ile Lys Ser Ser Asp Asn Val Arg Cys
340 345 350
Ile Ile Cys Glu Val Asp Ala Asn His Pro Phe Val Met Thr Glu Leu
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Lys Thr Ala Glu Gln Arg Lys Arg His Ser Ala Tyr
385 390 395 400
Met Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu Ile
405 410 415
Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val Gly
420 425 430
Phe Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys Val
450 455 460
Leu Ala Gly
465
<210> 35
<211> 467
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 35
Met Lys Arg Asn Leu Ser Val Leu Leu Gln Thr Asn Asp Leu Lys Ile
1 5 10 15
Thr Lys Arg Thr Glu Gly Asp Lys Ser Asn Asn Lys Glu Ser Tyr Leu
20 25 30
Gly Val Phe Lys Lys Val Glu Asn Ala Ile Thr Glu Ala Ile Tyr Ala
35 40 45
Gln Lys Lys Leu Ser Leu Tyr Tyr Thr Lys Glu Asp Arg Glu Arg Ile
50 55 60
Ile Lys Gly Ile Arg Lys Ala Thr Leu Glu Asn Lys Glu Ile Leu Ala
65 70 75 80
Lys Met Ile Val Asp Glu Thr His Met Gly Arg Tyr Glu Asp Lys Ile
85 90 95
Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu Asp Leu
100 105 110
Ile Thr Thr Ala Trp Ser Gly Asp Gln Gly Leu Thr Leu Val Glu Met
115 120 125
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr
130 135 140
Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asp Ser
145 150 155 160
Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala Phe Ala
165 170 175
Val Asp Met Ile Asn Lys Ala Val Ile Lys Ala Gly Gly Pro Glu Asn
180 185 190
Leu Val Thr Thr Val Glu Asn Pro Thr Met Glu Ser Leu Asn Val Ile
195 200 205
Met Lys His Pro Tyr Ile Lys Leu Leu Cys Gly Thr Gly Gly Pro Gly
210 215 220
Leu Ile Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp Ile Asn Lys
245 250 255
Ala Ala Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn Ser Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala Asn Asp
275 280 285
Leu Ile Gln Asn Met Ile Lys Asn Asn Ala Val Leu Ile Asn Glu Asn
290 295 300
Gln Val Ser Lys Leu Leu Asp Leu Val Leu Leu Glu Arg Lys Asp Glu
305 310 315 320
Thr Leu Glu Tyr Ala Ile Asn Lys Lys Trp Val Gly Lys Asp Ala Lys
325 330 335
Leu Phe Leu Asp Lys Ile Gly Ile Lys Ser Ser Asp Asn Val Arg Cys
340 345 350
Ile Ile Cys Glu Val Asp Ala Asn His Pro Phe Val Met Thr Glu Leu
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Lys Thr Ala Glu Gln Arg Lys Arg His Ser Ala Tyr
385 390 395 400
Met Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu Ile
405 410 415
Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val Gly
420 425 430
Phe Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys Val
450 455 460
Leu Ala Gly
465
<210> 36
<211> 467
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 36
Met Lys Arg Asn Leu Ser Val Leu Leu Gln Thr Asn Asp Leu Lys Ile
1 5 10 15
Thr Lys Arg Thr Glu Gly Asp Lys Ser Asn Asn Lys Glu Ser Tyr Leu
20 25 30
Gly Val Phe Lys Lys Val Glu Asn Ala Ile Thr Lys Ala Ile Tyr Ser
35 40 45
Gln Lys Lys Leu Ser Leu Tyr Tyr Thr Lys Glu Asp Arg Glu Arg Ile
50 55 60
Ile Lys Gly Ile Arg Lys Ala Thr Leu Glu Asn Lys Glu Ile Leu Ala
65 70 75 80
Lys Met Ile Val Asp Glu Thr His Met Gly Arg Tyr Glu Asp Lys Ile
85 90 95
Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu Asp Leu
100 105 110
Ile Thr Thr Ala Trp Ser Gly Asp Gln Gly Leu Thr Leu Val Glu Met
115 120 125
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr
130 135 140
Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asp Ser
145 150 155 160
Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala Phe Ala
165 170 175
Val Asp Met Ile Asn Lys Ala Val Ile Lys Ala Gly Gly Pro Glu Asn
180 185 190
Leu Val Thr Thr Val Glu Asn Pro Thr Met Glu Ser Leu Asn Val Ile
195 200 205
Met Lys His Pro Tyr Ile Lys Leu Leu Cys Gly Thr Gly Gly Pro Gly
210 215 220
Leu Ile Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp Ile Asn Lys
245 250 255
Ala Ala Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn Ser Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala Asn Asp
275 280 285
Leu Ile Gln Asn Met Ile Lys Asn Asn Ala Val Leu Ile Asn Glu Asn
290 295 300
Gln Val Ser Lys Leu Leu Asp Leu Val Leu Leu Glu Arg Lys Asp Glu
305 310 315 320
Thr Leu Glu Tyr Ala Ile Asn Lys Lys Trp Val Gly Lys Asp Ala Lys
325 330 335
Leu Phe Leu Asp Lys Ile Gly Ile Lys Ser Ser Asp Asn Val Arg Cys
340 345 350
Ile Ile Cys Glu Val Asp Ala Asn His Pro Phe Val Met Thr Glu Leu
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Lys Thr Ala Glu Gln Arg Lys Arg His Ser Ala Tyr
385 390 395 400
Met Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu Ile
405 410 415
Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val Gly
420 425 430
Phe Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys Val
450 455 460
Leu Ala Gly
465
<210> 37
<211> 472
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 37
Met Asp Val Asp Val Val Leu Val Glu Lys Leu Val Arg Gln Ala Ile
1 5 10 15
Glu Glu Val Lys Asn Lys Asn Leu Leu Asn Leu Asp Lys Phe Glu Ser
20 25 30
Val Lys Asn Tyr Gly Ile Phe Gly Thr Met Asp Ala Ala Val Glu Ala
35 40 45
Ser Phe Val Ala Gln Lys Gln Leu Leu Asn Ala Ser Met Thr Asp Lys
50 55 60
Gln Lys Tyr Val Asp Thr Ile Lys Ala Thr Ile Leu Lys Lys Glu Asn
65 70 75 80
Leu Glu Leu Ile Ser Arg Met Ser Val Glu Glu Thr Glu Ile Gly Lys
85 90 95
Tyr Glu His Lys Leu Ile Lys Asn Arg Val Ala Ala Glu Lys Thr Pro
100 105 110
Gly Ile Glu Asp Leu Thr Thr Glu Ala Met Thr Gly Asp Asn Gly Leu
115 120 125
Thr Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro
130 135 140
Thr Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Ser Met Ile
145 150 155 160
Ala Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Asn
165 170 175
Val Ser Ile Lys Leu Val Thr Met Leu Asn Lys Ala Leu Glu Glu Ala
180 185 190
Gly Ala Pro Asp Asn Leu Ile Ala Thr Val Lys Glu Pro Ser Ile Glu
195 200 205
Asn Thr Asn Ile Met Met Glu His Pro Lys Ile Arg Met Leu Val Ala
210 215 220
Thr Gly Gly Pro Ala Ile Val Asn Lys Val Met Ser Thr Gly Lys Lys
225 230 235 240
Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr
245 250 255
Ala Asp Ile Glu Lys Ala Ala Ile Asp Ile Val Asn Gly Cys Ser Phe
260 265 270
Asp Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp
275 280 285
Gln Val Cys Asp Tyr Leu Ile His Tyr Met Lys Leu Asn Gly Ala Tyr
290 295 300
Glu Ile Lys Asp Arg Asp Leu Ile Gln Lys Leu Leu Asp Leu Val Thr
305 310 315 320
Asn Glu Asn Gly Gly Pro Lys Val Ser Phe Val Gly Lys Ser Ala Pro
325 330 335
Tyr Ile Leu Asn Lys Leu Gly Ile Ser Val Asp Glu Asn Ile Lys Val
340 345 350
Ile Ile Met Glu Val Glu Lys Asn His His Phe Val Leu Glu Glu Met
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Thr Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Tyr Val Ala Glu His Gly Asn Arg His Thr Ala Ile
385 390 395 400
Met His Ser Lys Asn Val Asp Lys Leu Thr Lys Met Ala Arg Leu Leu
405 410 415
Glu Thr Thr Ile Phe Val Lys Asn Ser Pro Ser Tyr Ala Gly Ile Gly
420 425 430
Val Gly Gly Glu Gly Thr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Leu Thr Thr Ala Arg Ser Phe Cys Arg Lys Arg Arg Cys Val
450 455 460
Met Val Asp Ala Phe Asn Ile Arg
465 470
<210> 38
<211> 468
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 38
Met Ile Lys Asp Thr Leu Val Ser Ile Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ala Asn Leu Lys Asn Tyr Lys Asp Asp Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Asn Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Asp Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Ser Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Ser Ala Ser His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 39
<211> 468
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 39
Met Ile Lys Asp Thr Leu Val Ser Val Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Cys Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 40
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 40
Met Ile Lys Asp Thr Leu Val Ser Val Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Thr Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Ala Glu Asn Ala Ile Ser Asn Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Asn Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Leu Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Val Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asn
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Glu Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Ile Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 41
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 41
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Asn Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Ile Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Leu Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Ser Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Ile Lys
340 345 350
Cys Ile Val Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Val Lys Tyr Thr Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 42
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 42
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys
1 5 10 15
Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Cys Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 43
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 43
Met Ile Lys Asp Thr Leu Val Ser Val Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
Gln Ala Gln Lys Ile Leu Ser Ile His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 44
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 44
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys
1 5 10 15
Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Ile Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Ile Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Glu Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Arg Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 45
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 45
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys
1 5 10 15
Thr Asn Asp Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Ile His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Ala
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Ser Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 46
<211> 468
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 46
Met Ile Lys Asp Thr Leu Val Ser Ile Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ala Asn Leu Lys Asn Tyr Lys Asp Asp Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Asn Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Asp Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Ser Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Ser Ala Ser His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 47
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 47
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys
1 5 10 15
Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Cys Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 48
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 48
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Leu Lys
1 5 10 15
Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Asn Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Ile Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Leu Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Ser Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Ile Lys
340 345 350
Cys Ile Val Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Val Lys Tyr Thr Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 49
<211> 468
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 49
Met Asn Lys Asp Thr Thr Ile Ser Glu Thr Glu Asn Leu Lys Phe Lys
1 5 10 15
Thr Asn Ile Lys Asn Ala Asp Leu Lys Asn Tyr Glu Asn Ser Thr Ser
20 25 30
Tyr Ser Gly Val Phe Glu Asp Val Glu Val Ala Ile Asn Lys Ala Ile
35 40 45
Thr Ala Gln Lys Glu Phe Ser Leu Tyr Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Leu Thr Glu Ile Arg Lys Ala Thr Leu Lys Asn Lys Lys Ile
65 70 75 80
Leu Ala Lys Met Ile Leu Asp Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Ile Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ala Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Ser Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Asp Met Ile Asn Lys Ala Ile Val Ser Cys Gly Gly Pro
180 185 190
Lys Asn Leu Ile Thr Ala Val Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Glu Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Asp Asn Val Ala
275 280 285
Asp Asn Leu Ile Asp Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Lys Asp Lys Ile Thr Lys Leu Leu Asn Leu Ile Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Asn Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asn Glu Ile Asp Val Glu Ala Pro Ser Ser Val Arg
340 345 350
Cys Ile Ile Cys Glu Val Glu Pro Asp His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asn Ile Asp Asp
370 375 380
Ala Ile Gln Tyr Ala Lys Ile Ala Glu Gln Ser Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Asn Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Cys Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 50
<211> 469
<212> PRT
<213> 糖丁酸梭菌（Clostridium saccharobutylicum）
<400> 50
Met Asn Asn Asn Leu Phe Val Ser Pro Glu Thr Lys Asp Leu Lys Leu
1 5 10 15
Arg Thr Asn Val Glu Asn Leu Lys Phe Lys Gly Cys Glu Gly Gly Ser
20 25 30
Thr Tyr Ile Gly Val Phe Glu Asn Ala Glu Thr Ala Ile Asp Glu Ala
35 40 45
Val Asn Ala Gln Lys Arg Leu Ser Leu Tyr Tyr Thr Lys Glu Gln Arg
50 55 60
Glu Lys Ile Ile Thr Glu Ile Arg Lys Val Thr Leu Lys Asn Lys Glu
65 70 75 80
Ile Leu Ala Gln Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu
85 90 95
Asp Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr
100 105 110
Glu Asp Leu Ala Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val
115 120 125
Val Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr
130 135 140
Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Gly Met Ile Ala Ser
145 150 155 160
Gly Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val
165 170 175
Ala Phe Ala Val Asp Met Ile Asn Arg Ala Ile Ile Ser Cys Gly Gly
180 185 190
Pro Arg Asn Leu Val Thr Ala Ile Lys Asn Pro Thr Met Glu Ser Leu
195 200 205
Asp Ala Ile Ile Lys His Pro Ala Ile Lys Leu Leu Cys Gly Thr Gly
210 215 220
Gly Pro Gly Met Val Lys Thr Leu Leu Ser Ser Gly Lys Lys Ser Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp
245 250 255
Ile Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val
275 280 285
Ala Asp Asp Leu Ile Lys Asn Met Leu Lys Asn Asn Ala Val Ile Ile
290 295 300
Asn Lys Asp Gln Val Ser Arg Leu Val Asn Leu Val Leu Gln Lys Asn
305 310 315 320
Asn Glu Thr Ser Glu Tyr Thr Ile Asn Lys Lys Trp Val Gly Lys Asp
325 330 335
Ala Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Ser Ser Asp Val
340 345 350
Arg Cys Ile Ile Cys Glu Val Asp Ala Asp His Pro Phe Val Met Thr
355 360 365
Glu Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp
370 375 380
Glu Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser
385 390 395 400
Ala Tyr Ile Tyr Ser Lys Asn Ile Glu Asn Leu Asn Arg Phe Glu Lys
405 410 415
Glu Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly
420 425 430
Val Gly Tyr Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Cys
435 440 445
Thr Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg
450 455 460
Cys Val Phe Val Gly
465
<210> 51
<211> 467
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 51
Met Glu Arg Asn Leu Ser Val Leu Ser Gln Thr Asn Asp Leu Lys Ile
1 5 10 15
Thr Lys Arg Thr Glu Gly Asp Lys Ser Asn Asn Lys Glu Ser Tyr Leu
20 25 30
Gly Val Phe Lys Lys Val Glu Asn Ala Ile Thr Lys Ala Ile Tyr Ala
35 40 45
Gln Lys Lys Leu Ser Leu Tyr Tyr Thr Lys Glu Asp Arg Glu Arg Ile
50 55 60
Ile Lys Ser Ile Arg Lys Ala Thr Leu Glu Asn Lys Glu Ile Leu Ala
65 70 75 80
Lys Met Ile Val Asp Glu Thr His Met Gly Arg Tyr Glu Asp Lys Ile
85 90 95
Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu Asp Leu
100 105 110
Ile Thr Thr Ala Trp Ser Gly Asp Gln Gly Leu Thr Leu Val Glu Met
115 120 125
Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr
130 135 140
Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asp Ser
145 150 155 160
Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala Phe Ala
165 170 175
Val Asp Met Ile Asn Lys Ala Val Ile Arg Glu Gly Gly Pro Glu Asn
180 185 190
Leu Val Thr Thr Val Glu Asn Pro Thr Met Glu Ser Leu Asn Val Ile
195 200 205
Met Lys His Pro Tyr Ile Lys Leu Leu Cys Gly Thr Gly Gly Pro Gly
210 215 220
Leu Ile Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp Ile Asp Lys
245 250 255
Ala Ala Lys Asn Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn Leu Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala Asn Asp
275 280 285
Leu Ile Gln Asn Met Ile Lys Asn Asn Ala Val Leu Ile Asn Glu Asn
290 295 300
Gln Val Ser Lys Leu Leu Asp Leu Val Leu Leu Glu Arg Lys Asp Glu
305 310 315 320
Thr Leu Glu Tyr Ala Ile Asn Lys Lys Trp Val Gly Lys Asp Ala Lys
325 330 335
Leu Phe Leu Asp Lys Ile Gly Ile Lys Ala Ser Asp Asn Val Arg Cys
340 345 350
Ile Ile Cys Glu Val Asp Ala Asn His Pro Phe Val Met Thr Glu Leu
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Lys Thr Ala Glu Gln Arg Lys Arg His Ser Ala Tyr
385 390 395 400
Met Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Lys Glu Ile
405 410 415
Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val Gly
420 425 430
Phe Gly Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys Val
450 455 460
Leu Ala Gly
465
<210> 52
<211> 527
<212> PRT
<213> 温泉热碱芽孢杆菌（Caldalkalibacillus thermarum）
<400> 52
Met Asn Met Thr Glu Lys Asp Ile Glu Lys Ile Val Gln Ser Val Leu
1 5 10 15
His Asn Val Glu Ser Ala Leu Gly Lys Ser Ala Ser Ala Ser Pro Ser
20 25 30
Val Ser Ala Val Ser Val Ala Ser Gly Glu Gly Ile Lys Pro Val Gln
35 40 45
Phe Lys Gln Val Pro Val Phe Gln Gln Glu Thr Val Lys Ser Pro Asn
50 55 60
Arg Asn Arg Asn Leu Gly Gly Ala Glu Glu Lys Trp Gly Val Phe Asn
65 70 75 80
His Met Glu Asp Ala Ile Glu Ala Ser Tyr Arg Ala Gln Met Glu Phe
85 90 95
Val Lys His Phe Gln Leu Lys Asp Arg Glu Lys Ile Ile Thr Ala Ile
100 105 110
Arg Glu Ala Val Leu Arg Glu Lys Glu Val Leu Ala Arg Lys Val Tyr
115 120 125
Glu Glu Thr Lys Ile Gly Arg Tyr Glu Asp Lys Val Ala Lys His Glu
130 135 140
Leu Ala Ala Leu Lys Thr Pro Gly Thr Glu Asp Leu Lys Thr Glu Ala
145 150 155 160
Phe Ser Gly Asp Asn Gly Leu Thr Ile Val Glu Arg Ala Pro Tyr Gly
165 170 175
Leu Ile Gly Ala Val Thr Pro Val Thr Asn Pro Thr Glu Thr Ile Ile
180 185 190
Asn Asn Ala Ile Gly Met Leu Ala Ala Gly Asn Ala Val Val Phe Asn
195 200 205
Val His Pro Ser Ser Lys Arg Ser Cys Ala Tyr Ala Val Gln Leu Ile
210 215 220
Asn Lys Ala Ile Thr Glu Ala Gly Gly Pro His His Leu Val Thr Met
225 230 235 240
Val Lys Glu Pro Thr Leu Asp Thr Leu Gln Thr Leu Ile Asp Ser Pro
245 250 255
Lys Val Lys Leu Leu Val Gly Thr Gly Gly Pro Gly Leu Val Gln Thr
260 265 270
Leu Leu Lys Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro
275 280 285
Pro Val Ile Val Asp Asp Thr Ala Asp Leu Glu His Ala Ala Arg Ser
290 295 300
Ile Ile Glu Gly Ala Ala Phe Asp Asn Asn Leu Leu Cys Ile Ala Glu
305 310 315 320
Lys Glu Val Phe Val Leu Glu Ser Val Ala Asp Asp Leu Ile Phe His
325 330 335
Met Leu Asn His Gly Ala Tyr Met Leu Gly Gln His Glu Val Glu Gln
340 345 350
Val Met Ala Phe Ala Leu Glu Glu Gln Gly Asn Glu Gln Asn Arg Gly
355 360 365
Cys Gly Phe Asn Pro Gln Arg His Tyr Gln Val Ser Lys Asp Trp Ile
370 375 380
Gly Gln Asp Ala Arg Leu Phe Leu Glu His Ile Gly Val Gln Pro Pro
385 390 395 400
Thr Glu Val Lys Leu Leu Ile Cys Asp Val Glu Phe Asp His Pro Phe
405 410 415
Val Gln Leu Glu Gln Met Met Pro Val Leu Pro Ile Val Arg Val Lys
420 425 430
Thr Leu Asp Glu Ala Ile Glu Lys Ala Val Met Ala Glu His Gly Asn
435 440 445
Arg His Thr Ala Ile Met His Ser Lys Asn Val Asp His Leu Thr Lys
450 455 460
Phe Ala Arg Ala Ile Gln Thr Thr Leu Phe Val Lys Asn Ala Ser Ser
465 470 475 480
Leu Ala Gly Val Gly Tyr Gly Gly Glu Gly His Thr Thr Met Thr Ile
485 490 495
Ala Gly Pro Thr Gly Glu Gly Val Thr Ser Ala Lys Thr Phe Thr Arg
500 505 510
Glu Arg Arg Cys Val Leu Ala Glu Gly Gly Phe Arg Ile Ile Gly
515 520 525
<210> 53
<211> 480
<212> PRT
<213> 发酵酸酐菌（Pelosinus fermentans）
<400> 53
Met Ser Ile Asp Gln Ala Leu Ile Glu Lys Ile Thr Leu Glu Ile Leu
1 5 10 15
Thr Lys Met Gln Thr Gly Ala Lys Ala Ala Pro Ala Gly Tyr Gly Asp
20 25 30
Gly Ile Phe Glu Thr Val Asp Glu Ala Val Ala Ala Ala Arg Lys Ala
35 40 45
Tyr Gln Glu Leu Lys Thr Leu Ser Leu Glu Lys Arg Glu Val Leu Ile
50 55 60
Lys Ala Met Arg Asp Val Ala Tyr Glu Asn Ala Thr Ile Leu Ala Gln
65 70 75 80
Met Ala Val Asp Glu Ser Gly Met Gly Arg Val Ser Asp Lys Ile Ile
85 90 95
Lys Asn Gln Val Ala Ala Leu Lys Thr Pro Gly Thr Glu Asp Leu Thr
100 105 110
Thr Gln Ala Trp Ser Gly Asp Asn Gly Leu Thr Leu Ile Glu Met Gly
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Val Ile Cys Asn Gly Ile Gly Met Ile Ala Ala Gly Asn Thr Val
145 150 155 160
Phe Phe Ser Pro His Pro Thr Ala Lys Asn Thr Ser Met Lys Ile Ile
165 170 175
Thr Leu Leu Asn Gln Ala Ile Val Lys Ala Gly Gly Pro Asn Asn Leu
180 185 190
Leu Thr Ser Val Ala Asn Pro Ser Ile Lys Ala Ala Asn Glu Met Met
195 200 205
Lys His Pro Gly Ile Asn Met Leu Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Arg Asp Ile Val Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Ile Ala Glu Lys Glu Val Ile Ala Ile Gly Ser Ile Ala Asp Arg Leu
275 280 285
Ile Thr Tyr Met Gln Lys Tyr Gly Ala Tyr Leu Ile Ser Gly Ser Asn
290 295 300
Ile Asp Arg Leu Leu Asp Val Ile Met Thr Val Gln Glu Glu Lys Ile
305 310 315 320
Ala Glu Gly Cys Thr Asp Lys Pro Lys Arg Ser Tyr Gly Ile Asn Lys
325 330 335
Asp Tyr Val Gly Lys Asp Ala Lys Tyr Leu Leu Ser Lys Ile Gly Ile
340 345 350
Asp Val Pro Asp Ser Val Arg Val Val Leu Cys Glu Thr Pro Ala Asp
355 360 365
His Pro Phe Val Ile Glu Glu Leu Met Met Pro Val Leu Pro Val Val
370 375 380
Gln Val Lys Asp Ile Asp Glu Ala Ile Glu Val Ala Val Arg Val Glu
385 390 395 400
His Gly Asn Arg His Thr Ala Ala Met His Ser Lys Asn Val Asp His
405 410 415
Leu Thr Arg Phe Ala Arg Ala Val Glu Thr Thr Ile Phe Val Lys Asn
420 425 430
Ala Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Phe Thr Ser
435 440 445
Phe Thr Leu Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser Pro Arg Ser
450 455 460
Phe Thr Arg Gln Arg Arg Cys Val Leu Val Asp Ala Phe Ser Ile Val
465 470 475 480
<210> 54
<211> 479
<212> PRT
<213> 热解糖热厌氧杆菌（Thermoanaerobacterium thermosaccharolyticum）
<400> 54
Met Glu Ile Asn Asp Asn Met Ile Ser Glu Ile Ile Glu Arg Val Leu
1 5 10 15
Lys Glu Val Gln Lys Lys Ser Ile Asn Asp Arg Tyr Gln Asn Gly Ile
20 25 30
Tyr Asp Arg Met Glu Asp Ala Ile Glu Ala Ala Tyr Glu Ala Gln Lys
35 40 45
Lys Leu Met Lys Met Ser Ile Glu Gln Arg Glu Arg Leu Ile Ser Ala
50 55 60
Met Arg Lys Ala Ile Leu Asp Asn Ala Lys Ser Cys Ala Lys Leu Ser
65 70 75 80
Val Glu Glu Thr Gly Met Gly Arg Val Asp His Lys Tyr Leu Lys Leu
85 90 95
Lys Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Val Leu Thr Thr Lys
100 105 110
Ala Tyr Ser Gly Asp Lys Gly Leu Thr Leu Val Glu Met Ala Pro Phe
115 120 125
Gly Val Ile Gly Ser Ile Thr Pro Ser Thr Asn Pro Ala Glu Thr Val
130 135 140
Cys Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Thr Val Val Phe
145 150 155 160
Ser Pro His Pro Gly Ala Ile Lys Ser Ser Leu Met Ala Val Glu Phe
165 170 175
Leu Asn Lys Ala Ile Ile Glu Ala Gly Gly Pro Glu Asn Leu Ile Thr
180 185 190
Ser Val Arg Lys Pro Ser Ile Glu Phe Thr Asp Val Met Ile Asn His
195 200 205
Pro Lys Ile Asn Leu Leu Val Ala Thr Gly Gly Pro Ala Ile Val Lys
210 215 220
Lys Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Cys Val Val Asp Glu Thr Ala Asp Ile Lys Lys Ala Ala Arg
245 250 255
Asp Ile Ile Leu Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala
260 265 270
Glu Lys Glu Val Ile Ala Val Glu Ser Ile Tyr Glu Glu Leu Ile Glu
275 280 285
Asn Met Lys Lys Asn Gly Ala Tyr Glu Ile Thr Asp Asp Glu Ala Glu
290 295 300
Lys Leu Ala Asp Ile Val Leu Thr Lys Lys Glu Glu Leu Lys Ala Glu
305 310 315 320
Gly Cys Ser Ile Asn Arg Pro Lys Phe Glu Tyr Ser Val Asn Lys Lys
325 330 335
Trp Val Gly Lys Asp Ala Lys Val Leu Leu Glu Gln Ile Gly Ile Asn
340 345 350
Val Gly Asp Asp Ile Val Cys Ile Ile Tyr Arg Cys Asp Lys Gln His
355 360 365
Pro Phe Val Gln Glu Glu Leu Met Met Pro Ile Leu Pro Ile Val Lys
370 375 380
Val Lys Asn Ile Asp Glu Ala Ile Asn Val Ala Val Glu Val Glu His
385 390 395 400
Gly Asn His His Thr Ala Glu Met His Ser Lys Asn Ile Asp Asn Leu
405 410 415
Thr Arg Phe Ala Lys Ala Ile Asn Thr Thr Ile Phe Val Lys Asn Ala
420 425 430
Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly Glu Gly Tyr Thr Thr Phe
435 440 445
Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Cys Ala Ala Thr Phe
450 455 460
Thr Arg Gln Arg Arg Cys Val Met Val Asp Ser Phe Arg Ile Val
465 470 475
<210> 55
<211> 480
<212> PRT
<213> 发酵酸酐菌（Pelosinus fermentans）
<400> 55
Met Ser Ile Asp Gln Ala Leu Ile Glu Lys Ile Thr Leu Glu Ile Leu
1 5 10 15
Thr Lys Met Gln Thr Gly Ala Lys Ala Ala Pro Ala Gly Tyr Gly Asp
20 25 30
Gly Ile Phe Glu Thr Val Asp Glu Ala Val Ala Ala Ala Arg Lys Ala
35 40 45
Tyr Gln Glu Leu Lys Thr Leu Ser Leu Glu Lys Arg Glu Val Leu Ile
50 55 60
Lys Ala Met Arg Asp Val Ala Tyr Glu Asn Ala Thr Ile Leu Ala Gln
65 70 75 80
Met Ala Val Asp Glu Ser Gly Met Gly Arg Val Ser Asp Lys Ile Ile
85 90 95
Lys Asn Gln Val Ala Ala Leu Lys Thr Pro Gly Thr Glu Asp Leu Thr
100 105 110
Thr Gln Ala Trp Ser Gly Asp Asn Gly Leu Thr Leu Ile Glu Met Gly
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Val Ile Cys Asn Gly Ile Gly Met Ile Ala Ala Gly Asn Thr Val
145 150 155 160
Phe Phe Ser Pro His Pro Thr Ala Lys Asn Thr Ser Met Lys Ile Ile
165 170 175
Thr Leu Leu Asn Gln Ala Ile Val Lys Ala Gly Gly Pro Asn Asn Leu
180 185 190
Leu Thr Ser Val Ala Asn Pro Ser Ile Lys Ala Ala Asn Glu Met Met
195 200 205
Lys His Pro Gly Ile Asn Met Leu Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Arg Asp Ile Val Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Ile Ala Glu Lys Glu Val Ile Ala Ile Gly Ser Ile Ala Asp Arg Leu
275 280 285
Ile Thr Tyr Met Gln Lys Tyr Gly Ala Tyr Leu Ile Ser Gly Ser Asn
290 295 300
Ile Asp Arg Leu Leu Asn Val Ile Met Thr Val Gln Glu Glu Lys Ile
305 310 315 320
Ala Glu Gly Cys Thr Asp Lys Pro Lys Arg Ser Tyr Gly Ile Asn Lys
325 330 335
Asp Tyr Val Gly Lys Asp Ala Lys Tyr Leu Leu Ser Lys Ile Gly Ile
340 345 350
Asp Val Pro Asp Ser Val Arg Val Val Leu Cys Glu Thr Pro Ala Asp
355 360 365
His Pro Phe Val Ile Glu Glu Leu Met Met Pro Val Leu Pro Val Val
370 375 380
Gln Val Lys Asp Ile Asp Glu Ala Ile Glu Val Ala Val Arg Val Glu
385 390 395 400
His Gly Asn Arg His Thr Ala Ala Met His Ser Lys Asn Val Asp His
405 410 415
Leu Thr Arg Phe Ala Arg Ala Val Glu Thr Thr Ile Phe Val Lys Asn
420 425 430
Ala Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Phe Thr Ser
435 440 445
Phe Thr Leu Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser Pro Arg Ser
450 455 460
Phe Thr Arg Gln Arg Arg Cys Val Leu Val Asp Ala Phe Ser Ile Val
465 470 475 480
<210> 56
<211> 479
<212> PRT
<213> 脱硫芽孢弯曲菌属（Desulfosporosinus sp.）
<400> 56
Met Ser Val Asp Gln Ala Leu Ile Arg Lys Ile Thr Ser Glu Ile Leu
1 5 10 15
Ala Lys Met Gln Asn Arg Thr Val Ser Ala Cys Gln Asp Cys Asn Gly
20 25 30
Ile Phe Thr Thr Val Asp Glu Ala Val Ala Ala Ala Arg Ile Ala Tyr
35 40 45
Gln Glu Leu Arg Thr Leu Ser Leu Glu Lys Arg Glu Glu Leu Ile Lys
50 55 60
Ala Met Arg Asn Val Ala Leu Glu Asn Ala Thr Met Leu Ala Glu Met
65 70 75 80
Ala Val Lys Glu Ser Gly Met Gly Arg Val Glu Asp Lys Ile Ile Lys
85 90 95
His Lys Leu Val Ala Val Lys Thr Pro Gly Thr Glu Asp Leu Arg Thr
100 105 110
Glu Ala Trp Ser Gly Asp Ser Gly Leu Thr Leu Val Glu Met Gly Pro
115 120 125
Tyr Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Val Ala Thr
130 135 140
Ile Ile Cys Asn Gly Ile Gly Met Ile Ala Ala Gly Asn Ala Val Phe
145 150 155 160
Phe Ser Pro His Pro Thr Ala Lys Asn Thr Ser Ile Lys Thr Ile Thr
165 170 175
Leu Leu Asn Glu Ala Ile Val Lys Ala Gly Gly Pro Met Asn Leu Leu
180 185 190
Thr Ser Val Ala Asp Pro Ser Ile Ser Ala Ala Asn Ala Met Met Lys
195 200 205
His Ala Gly Ile Asn Leu Leu Val Ala Thr Gly Gly Pro Gly Val Val
210 215 220
Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly
225 230 235 240
Asn Pro Pro Val Ile Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala
245 250 255
Arg Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile
260 265 270
Ala Glu Lys Glu Val Ile Ala Val Gly Cys Ile Ala Asp Arg Leu Ile
275 280 285
Ser Asn Met Gln Lys Tyr Gly Ala Tyr Leu Ile Ser Gly Ser Lys Ile
290 295 300
Asp Gln Met Leu Asp Val Val Met Thr Ala Thr Glu Glu Lys Met Ala
305 310 315 320
Glu Gly Cys Thr Ala Lys Pro Ile Lys Arg Tyr Gly Ile Asn Lys Asp
325 330 335
Phe Val Gly Lys Asp Ala Lys Tyr Ile Leu Thr Gln Ile Gly Leu Asp
340 345 350
Val Pro Asp Thr Ile Lys Val Ile Leu Cys Glu Thr Pro Ala Asp His
355 360 365
Pro Phe Val Ile Glu Glu Leu Met Met Pro Ile Leu Pro Val Val Gln
370 375 380
Val Lys Asp Ile Asp Ala Ala Ile Glu Leu Ala Val Lys Val Glu His
385 390 395 400
Gly Asn Arg His Thr Ala Met Met His Ser Lys Asn Val Asp Asn Met
405 410 415
Thr Arg Phe Ala Lys Ala Ile Glu Thr Thr Ile Phe Val Lys Asn Ala
420 425 430
Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Phe Cys Thr Phe
435 440 445
Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Thr Ala Arg Ser Phe
450 455 460
Thr Arg Gln Arg Arg Cys Val Leu Val Asp Ser Phe Ser Ile Ile
465 470 475
<210> 57
<211> 482
<212> PRT
<213> 脱硫芽孢弯曲菌属（Desulfosporosinus sp.）
<400> 57
Met Glu Ile Thr Pro Asn Gln Ile Asp Gln Ile Val Ala Asn Val Met
1 5 10 15
Ala Gln Leu Gly Gly Ser Ala Ala Pro Ala Ala Ser Tyr Asp Ser Thr
20 25 30
Gln Tyr Ser Gly Arg Lys Tyr Ile Gly Ile Tyr Ala Thr Met Thr Glu
35 40 45
Ala Ile Asp Ala Val Ala Asp Ala Tyr Lys Val Leu Arg Ser Met Thr
50 55 60
Val Asp Gln Arg Glu Lys Ile Ile Glu Lys Ile Arg Glu Phe Thr Arg
65 70 75 80
Ala Glu Ala Glu Ile Met Ala Lys Met Gly Val Glu Glu Thr Gly Met
85 90 95
Gly Lys Val Glu His Lys Thr Leu Lys His His Leu Val Ala Asp Lys
100 105 110
Thr Pro Gly Thr Glu Asp Ile Gln Thr Glu Ala Met Ser Gly Asp Gly
115 120 125
Gly Leu Thr Leu Leu Glu Met Ala Pro Phe Gly Ile Ile Gly Ala Ile
130 135 140
Ser Pro Ser Thr Asn Pro Ser Glu Thr Val Leu Cys Asn Ser Met Gly
145 150 155 160
Met Ile Ala Gly Ala Asn Ala Val Val Phe Asn Pro His Pro Ser Ala
165 170 175
Ile Cys Thr Ser Asn Tyr Ala Val Asp Leu Val Asn Arg Ala Ser Leu
180 185 190
Ala Ala Gly Gly Pro Ala Asn Leu Cys Cys Ser Val Val Lys Pro Thr
195 200 205
Met Gln Ser Ala Asp Asp Met Val Lys Asp Pro Arg Val Lys Met Leu
210 215 220
Val Cys Thr Gly Gly Pro Gly Val Val Arg Ala Met Leu Ser Ser Gly
225 230 235 240
Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp
245 250 255
Asp Thr Ala Asp Ile Arg Lys Ala Ala Lys Asp Ile Ile Asp Gly Cys
260 265 270
Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Ala
275 280 285
Phe Ser Asn Ile Ala Asp Glu Leu Met Tyr Tyr Met Gln Gln Asn Gly
290 295 300
Ala Tyr Phe Ile Ser Gly Glu Met Ala Asp Arg Leu Ala Lys Ile Val
305 310 315 320
Leu Val Glu Lys Lys Asn Glu Lys Thr Gly Lys Ile Ser Tyr Ser Val
325 330 335
Ser Arg Asp Trp Val Gly Arg Asp Ala Lys Lys Phe Leu Ala Ala Leu
340 345 350
Asp Ile Glu Val Gly Asp Asp Val Arg Cys Val Ile Cys Glu Thr Asp
355 360 365
Glu Asn His Leu Phe Val Gln Thr Glu Leu Met Met Pro Ile Leu Pro
370 375 380
Ile Val Arg Val Asn Asn Ile Asp Glu Ala Val Arg Met Ala Val Arg
385 390 395 400
Ala Glu His Gly Asn Arg His Thr Ala His Met His Ser Lys Asn Ile
405 410 415
Asp Asn Leu Thr Lys Phe Ala Arg Ala Val Glu Thr Thr Ile Phe Val
420 425 430
Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Phe Gly Ser Glu Gly His
435 440 445
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala
450 455 460
Arg Ser Phe Thr Arg Lys Arg Arg Cys Val Met Ser Asp Ser Phe Asn
465 470 475 480
Ile Val
<210> 58
<211> 467
<212> PRT
<213> 解糖嗜热厌氧杆菌（Thermoanaerobacterium saccharolyticum）
<400> 58
Met Lys Val Lys Glu Glu Asp Ile Glu Ala Ile Val Lys Lys Val Leu
1 5 10 15
Ser Glu Phe Asn Phe Glu Lys Asn Thr Lys Ser Phe Arg Asp Phe Gly
20 25 30
Val Phe Gln Asp Met Asn Asp Ala Ile Arg Ala Ala Lys Asp Ala Gln
35 40 45
Lys Lys Leu Arg Asn Met Ser Met Glu Ser Arg Glu Lys Ile Ile Gln
50 55 60
Asn Ile Arg Lys Lys Ile Met Glu Asn Lys Lys Ile Leu Ala Glu Met
65 70 75 80
Gly Val Ser Glu Thr Gly Met Gly Lys Val Glu His Lys Ile Ile Lys
85 90 95
His Glu Leu Val Ala Leu Lys Thr Pro Gly Thr Glu Asp Ile Val Thr
100 105 110
Thr Ala Trp Ser Gly Asp Lys Gly Leu Thr Leu Val Glu Met Gly Pro
115 120 125
Phe Gly Val Ile Gly Thr Ile Thr Pro Ser Thr Asn Pro Ser Glu Thr
130 135 140
Val Leu Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ser Val Val
145 150 155 160
Phe Asn Pro His Pro Gly Ala Val Asn Val Ser Asn Tyr Ala Val Lys
165 170 175
Leu Val Asn Glu Ala Val Met Glu Ala Gly Gly Pro Glu Asn Leu Val
180 185 190
Ala Ser Val Glu Lys Pro Thr Leu Glu Thr Gly Asn Ile Met Phe Lys
195 200 205
Ser Pro Asp Val Ser Leu Leu Val Ala Thr Gly Gly Pro Gly Val Val
210 215 220
Thr Ser Val Leu Ser Ser Gly Lys Arg Ala Ile Gly Ala Gly Ala Gly
225 230 235 240
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Lys Lys Ala Ala
245 250 255
Lys Asp Ile Val Asp Gly Ala Thr Phe Asp Asn Asn Leu Pro Cys Ile
260 265 270
Ala Glu Lys Glu Val Val Ser Val Asp Lys Ile Thr Asp Glu Leu Ile
275 280 285
Tyr Tyr Met Gln Gln Asn Gly Cys Tyr Lys Ile Glu Gly Arg Glu Ile
290 295 300
Glu Lys Leu Ile Glu Leu Val Leu Asp His Lys Gly Gly Lys Ile Thr
305 310 315 320
Leu Asn Arg Lys Trp Val Gly Lys Asp Ala His Leu Ile Leu Lys Ala
325 330 335
Ile Gly Ile Asp Ala Asp Glu Ser Val Arg Cys Ile Ile Phe Glu Ala
340 345 350
Glu Lys Asp Asn Pro Leu Val Val Glu Glu Leu Met Met Pro Ile Leu
355 360 365
Gly Ile Val Arg Ala Lys Asn Val Asp Glu Ala Ile Met Ile Ala Thr
370 375 380
Glu Leu Glu His Gly Asn Arg His Ser Ala His Met His Ser Lys Asn
385 390 395 400
Val Asp Asn Leu Thr Lys Phe Gly Lys Ile Ile Asp Thr Ala Ile Phe
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ala Ala Leu Gly Tyr Gly Gly Glu Gly
420 425 430
Tyr Cys Thr Phe Thr Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser
435 440 445
Ala Arg Thr Phe Thr Lys Ser Arg Arg Cys Val Leu Ala Asp Gly Leu
450 455 460
Ser Ile Arg
465
<210> 59
<211> 467
<212> PRT
<213> 解木聚糖嗜热厌氧杆菌（Thermoanaerobacterium xylanolyticum）
<400> 59
Met Lys Val Lys Glu Glu Asp Ile Glu Ala Ile Val Lys Lys Val Leu
1 5 10 15
Ser Glu Phe Asn Leu Glu Lys Thr Thr Ser Lys Tyr Gly Asp Val Gly
20 25 30
Ile Phe Gln Asp Met Asn Asp Ala Ile Ser Ala Ala Lys Asp Ala Gln
35 40 45
Lys Lys Leu Arg Asn Met Pro Met Glu Ser Arg Glu Lys Ile Ile Gln
50 55 60
Asn Ile Arg Lys Lys Ile Met Glu Asn Lys Lys Ile Leu Ala Glu Met
65 70 75 80
Gly Val Arg Glu Thr Gly Met Gly Arg Val Glu His Lys Ile Val Lys
85 90 95
His Glu Leu Val Ala Leu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr
100 105 110
Thr Ala Trp Ser Gly Asp Lys Gly Leu Thr Leu Val Glu Met Gly Pro
115 120 125
Phe Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Ser Glu Thr
130 135 140
Val Leu Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ser Val Val
145 150 155 160
Phe Asn Pro His Pro Gly Ala Val Asn Val Ser Asn Tyr Ala Val Lys
165 170 175
Leu Val Asn Glu Ala Ala Met Glu Ala Gly Gly Pro Glu Asn Leu Val
180 185 190
Val Ser Val Glu Lys Pro Thr Leu Glu Thr Gly Asn Val Met Phe Lys
195 200 205
Ser Ser Asp Val Ser Leu Leu Val Ala Thr Gly Gly Pro Gly Val Val
210 215 220
Thr Ala Val Leu Ser Ser Gly Lys Arg Ala Ile Gly Ala Gly Ala Gly
225 230 235 240
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Lys Lys Ala Ala
245 250 255
Lys Asp Ile Ile Asp Gly Ala Thr Phe Asp Asn Asn Leu Pro Cys Ile
260 265 270
Ala Glu Lys Glu Val Val Ser Val Asp Lys Ile Thr Asp Glu Leu Ile
275 280 285
Tyr Tyr Met Gln Lys Asn Gly Cys Tyr Lys Ile Glu Gly Arg Glu Ile
290 295 300
Glu Lys Leu Ile Glu Leu Val Leu Asp His Glu Gly Gly Lys Thr Thr
305 310 315 320
Leu Asn Arg Lys Trp Val Gly Lys Asp Ala His Leu Ile Leu Lys Ala
325 330 335
Ile Gly Ile Asp Ala Asp Glu Ser Val Arg Cys Ile Ile Phe Glu Ala
340 345 350
Glu Lys Asp Asn Pro Leu Val Val Glu Glu Leu Met Met Pro Ile Leu
355 360 365
Gly Ile Val Arg Ala Lys Asn Val Asp Glu Ala Ile Met Ile Ala Thr
370 375 380
Glu Leu Glu His Gly Asn Arg His Ser Ala His Met His Ser Lys Asn
385 390 395 400
Ile Asp Asn Leu Thr Lys Phe Gly Lys Ile Ile Asp Thr Ala Ile Phe
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ala Ala Leu Gly Tyr Gly Gly Glu Gly
420 425 430
Tyr Cys Thr Phe Thr Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser
435 440 445
Ala Arg Thr Phe Thr Lys Ser Arg Arg Cys Val Leu Ala Asp Gly Leu
450 455 460
Ser Ile Arg
465
<210> 60
<211> 477
<212> PRT
<213> 长醋丝菌（Acetonema longum）
<400> 60
Met Val Asp Gln Thr Leu Ile Glu Gln Ile Thr Arg Ala Val Leu Thr
1 5 10 15
Gln Met Lys Ala Gly Lys Asp Ala Ala Val Ser Gly Asp Gly Ile Phe
20 25 30
Ala Thr Val Asp Gln Ala Val Ala Ala Ala Arg Gln Ala Tyr Gln Glu
35 40 45
Leu Arg Leu Leu Thr Leu Glu Lys Arg Glu Thr Leu Ile Arg Ala Ile
50 55 60
Arg Asp Ala Ala Phe Ala Asn Ala Ala Val Ile Ala Gln Met Ala Val
65 70 75 80
Gln Glu Ser Gly Met Gly Arg Val Glu Asp Lys Ile Leu Lys Asn Gln
85 90 95
Leu Ala Ala Arg Lys Thr Pro Gly Thr Glu Asp Leu Thr Ser Arg Ala
100 105 110
Trp Ser Gly Asp His Gly Leu Thr Leu Val Glu Met Ala Pro Tyr Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Ser Glu Thr Val Ile
130 135 140
Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ser Ile Val Phe Ser
145 150 155 160
Pro His Pro Thr Ala Gln Asn Thr Ser Leu Thr Thr Ile Arg Leu Leu
165 170 175
Asn Glu Ala Ile Val Lys Ala Gly Gly Pro Asp Asn Leu Leu Thr Ala
180 185 190
Val Ala Glu Pro Ser Ile Glu Ala Ala Asn Ala Met Met Arg His Pro
195 200 205
Gly Ile Gln Met Leu Val Ala Thr Gly Gly Pro Ala Val Val Lys Ala
210 215 220
Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Val Val Asp Glu Thr Ala Asp Ile Ala Lys Ala Ala Lys Asp
245 250 255
Ile Val Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Ile Ala Val Gly Arg Ile Ala Asp Glu Leu Ile Ser Tyr
275 280 285
Leu Gln Lys Tyr Gly Ala Tyr Leu Ile Ser Gly Arg Asp Ile Glu Arg
290 295 300
Leu Met Glu Val Val Leu Thr Glu Arg Thr Glu Glu Met Ala Pro Gly
305 310 315 320
Cys Val Gly Lys Pro Arg Arg Val Tyr Gly Val Asn Lys Asp Tyr Ile
325 330 335
Gly Lys Asp Ala Lys Phe Ile Leu Ser Lys Ile Asn Ile Gln Ala Pro
340 345 350
Asp His Ile Arg Val Ile Leu Cys Glu Thr Pro Ala Asp His Pro Phe
355 360 365
Val Leu Glu Glu Leu Met Met Pro Val Leu Pro Leu Val Ser Val Arg
370 375 380
Asp Ile Asp Ala Ala Ile Asp Leu Ala Val Lys Val Glu His Gly Asn
385 390 395 400
Arg His Thr Ala Val Met His Ser Lys Asn Val Asp Tyr Met Thr Arg
405 410 415
Leu Ala Lys Ala Ile Glu Thr Thr Ile Phe Val Lys Asn Ala Pro Ser
420 425 430
Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Phe Thr Thr Phe Thr Ile
435 440 445
Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg Ser Phe Thr Arg
450 455 460
Gln Arg Arg Cys Ala Leu Val Asp Ala Phe Ser Ile Val
465 470 475
<210> 61
<211> 465
<212> PRT
<213> 热葡糖苷酶地芽孢杆菌（Geobacillus thermoglucosidans）
<400> 61
Met Ser Val Asp Ala Gln Lys Ile Glu Lys Leu Val Arg Lys Ile Leu
1 5 10 15
Glu Glu Met Glu Glu Lys Lys Lys Pro Ala Glu Thr Glu Cys Glu Trp
20 25 30
Gly Ile Phe Asp His Met Asn Gln Ala Ile Glu Ala Ala Glu Ile Ala
35 40 45
Gln Lys Glu Leu Val Gln Leu Ser Leu Gly Gln Arg Gly Lys Leu Ile
50 55 60
Glu Ala Ile Arg Lys Ala Ala Lys Glu Asn Ala Glu Lys Phe Ala Arg
65 70 75 80
Met Ala Val Asp Glu Thr Gly Met Gly Lys Tyr Glu Asp Lys Ile Val
85 90 95
Lys Asn Leu Leu Ala Ala Glu Lys Thr Pro Gly Ile Glu Asp Leu Arg
100 105 110
Thr Glu Val Phe Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Leu Ser
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Ile Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val
145 150 155 160
Val Phe Ser Pro His Pro Arg Ala Lys Asn Thr Ser Leu Tyr Ala Ile
165 170 175
Lys Ile Phe Asn Gln Ala Ile Val Glu Ala Gly Gly Pro Lys Asn Leu
180 185 190
Ile Thr Thr Val Ala Asn Pro Ser Ile Glu Gln Ala Glu Ile Met Met
195 200 205
Lys His Lys Thr Ile Lys Met Leu Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Val Ala Glu Lys Glu Val Ile Ala Val Glu Ser Ile Ala Asp Arg Leu
275 280 285
Ile Asp Tyr Met Lys Lys His Gly Ala Tyr Glu Ile Thr Asn Lys Glu
290 295 300
Gln Ile Gln Gln Leu Thr Asp Leu Val Val Glu Asn Gly His Ala Asn
305 310 315 320
Lys Glu Phe Val Gly Lys Asp Ala Ala Tyr Ile Leu Lys His Ile Gly
325 330 335
Ile Asn Val Pro Pro Asp Ile Arg Val Ala Ile Met Glu Val Asp Gly
340 345 350
Lys His Pro Leu Val Thr Val Glu Leu Met Met Pro Ile Leu Pro Ile
355 360 365
Val Arg Val Lys Asn Val Asp Gln Ala Ile Glu Leu Ala Val Glu Val
370 375 380
Glu His Gly Phe Arg His Thr Ala Ile Met His Ser Lys Asn Val Asp
385 390 395 400
His Leu Thr Lys Phe Ala Lys Ala Ile Gln Thr Thr Ile Phe Val Lys
405 410 415
Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Tyr Ala
420 425 430
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys
435 440 445
Asp Phe Ala Arg Lys Arg Lys Cys Val Leu Val Asp Ala Leu Ser Ile
450 455 460
Arg
465
<210> 62
<211> 463
<212> PRT
<213> 地芽孢杆菌属（Geobacillus sp.）
<400> 62
Met Asp Ala Gln Lys Ile Glu Lys Leu Val Arg Lys Ile Leu Glu Glu
1 5 10 15
Met Glu Glu Lys Lys Lys Pro Ala Glu Thr Glu Cys Glu Trp Gly Ile
20 25 30
Phe Asp His Met Asn Gln Ala Ile Glu Ala Ala Glu Ile Ala Gln Lys
35 40 45
Glu Leu Val Gln Leu Ser Leu Gly Gln Arg Gly Lys Leu Ile Glu Ala
50 55 60
Ile Arg Lys Ala Ala Lys Glu Asn Ala Glu Lys Phe Ala Arg Met Ala
65 70 75 80
Val Asp Glu Thr Gly Met Gly Lys Tyr Glu Asp Lys Ile Val Lys Asn
85 90 95
Leu Leu Ala Ala Glu Lys Thr Pro Gly Ile Glu Asp Leu Arg Thr Glu
100 105 110
Val Phe Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Leu Ser Pro Tyr
115 120 125
Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu Thr Ile
130 135 140
Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val Val Phe
145 150 155 160
Ser Pro His Pro Arg Ala Lys Asn Thr Ser Leu Tyr Ala Ile Lys Ile
165 170 175
Phe Asn Gln Ala Ile Val Glu Ala Gly Gly Pro Lys Asn Leu Ile Thr
180 185 190
Thr Val Ala Asn Pro Ser Ile Glu Gln Ala Glu Ile Met Met Lys His
195 200 205
Lys Thr Ile Lys Met Leu Val Ala Thr Gly Gly Pro Gly Val Val Lys
210 215 220
Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala Lys
245 250 255
Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Val Ala
260 265 270
Glu Lys Glu Val Ile Ala Val Glu Ser Ile Ala Asp Arg Leu Ile Asp
275 280 285
Tyr Met Lys Lys His Gly Ala Tyr Glu Ile Thr Asn Lys Glu Gln Ile
290 295 300
Gln Gln Leu Thr Asp Leu Val Val Glu Asn Gly His Ala Asn Lys Glu
305 310 315 320
Phe Val Gly Lys Asp Ala Ala Tyr Ile Leu Lys His Ile Gly Ile Asn
325 330 335
Val Pro Pro Asp Thr Arg Val Ala Ile Met Glu Val Asp Gly Lys His
340 345 350
Pro Leu Val Thr Val Glu Leu Met Met Pro Ile Leu Pro Ile Val Arg
355 360 365
Val Lys Asn Val Asp Gln Ala Ile Glu Leu Ala Val Glu Val Glu His
370 375 380
Gly Phe Arg His Thr Ala Ile Met His Ser Lys Asn Val Asp His Leu
385 390 395 400
Thr Lys Phe Ala Lys Ala Ile Gln Thr Thr Ile Phe Val Lys Asn Ala
405 410 415
Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Tyr Ala Thr Phe
420 425 430
Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys Asp Phe
435 440 445
Ala Arg Lys Arg Lys Cys Val Leu Val Asp Ala Leu Ser Ile Arg
450 455 460
<210> 63
<211> 463
<212> PRT
<213> 热葡糖苷酶地芽孢杆菌（Geobacillus thermoglucosidasius）
<400> 63
Met Asp Ala Gln Lys Ile Glu Lys Leu Val Arg Lys Ile Leu Glu Glu
1 5 10 15
Met Glu Glu Lys Lys Lys Pro Ala Glu Thr Glu Cys Glu Trp Gly Ile
20 25 30
Phe Asp His Met Asn Gln Ala Ile Glu Ala Ala Glu Ile Ala Gln Lys
35 40 45
Glu Phe Val Gln Leu Ser Leu Gly Gln Arg Gly Lys Leu Ile Glu Ala
50 55 60
Ile Arg Lys Ala Ala Lys Glu Asn Ala Glu Lys Phe Ala Arg Met Ala
65 70 75 80
Val Asp Glu Thr Gly Met Gly Lys Tyr Glu Asp Lys Ile Val Lys Asn
85 90 95
Leu Leu Ala Ala Glu Lys Thr Pro Gly Ile Glu Asp Leu Arg Thr Glu
100 105 110
Val Phe Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Leu Ser Pro Tyr
115 120 125
Gly Val Ile Gly Ala Ile Thr Pro Thr Thr Asn Pro Thr Glu Thr Ile
130 135 140
Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val Val Phe
145 150 155 160
Ser Pro His Pro Arg Ala Lys Asn Thr Ser Leu Tyr Ala Ile Lys Ile
165 170 175
Phe Asn Gln Ala Ile Val Glu Ala Gly Gly Pro Lys Asn Leu Ile Thr
180 185 190
Thr Val Ala Asn Pro Ser Ile Glu Gln Ala Glu Ile Met Met Lys His
195 200 205
Lys Thr Ile Lys Met Leu Val Ala Thr Gly Gly Pro Gly Val Val Lys
210 215 220
Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala Lys
245 250 255
Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Val Ala
260 265 270
Glu Lys Glu Val Ile Ala Val Glu Ser Ile Ala Asp Arg Leu Ile Asp
275 280 285
Tyr Met Lys Lys His Gly Ala Tyr Glu Ile Thr Asn Lys Glu Gln Ile
290 295 300
Gln Gln Leu Thr Asp Leu Val Val Glu Asn Gly His Ala Asn Lys Glu
305 310 315 320
Phe Val Gly Lys Asp Ala Ala Tyr Ile Leu Lys His Ile Gly Ile Asn
325 330 335
Val Pro Pro Asp Ile Arg Val Ala Ile Met Glu Val Asp Gly Lys His
340 345 350
Pro Leu Val Thr Val Glu Leu Met Met Pro Ile Leu Pro Ile Val Arg
355 360 365
Val Lys Asn Val Asp Gln Ala Ile Glu Leu Ala Val Glu Val Glu His
370 375 380
Gly Phe Arg His Thr Ala Ile Met His Ser Lys Asn Val Asp His Leu
385 390 395 400
Thr Lys Phe Ala Lys Ala Ile Gln Thr Thr Ile Phe Val Lys Asn Ala
405 410 415
Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Tyr Ala Thr Phe
420 425 430
Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys Asp Phe
435 440 445
Ala Arg Lys Arg Lys Cys Val Leu Val Asp Ala Leu Ser Ile Arg
450 455 460
<210> 64
<211> 463
<212> PRT
<213> 产氮芽孢杆菌（Bacillus azotoformans）
<400> 64
Met Ala Val Glu Ala Lys Ala Ile Glu Glu Ile Val Lys Lys Ile Leu
1 5 10 15
Glu Glu Met Met Ile Lys Lys Asp Ala Cys Ile Thr Gly Tyr Gly Ile
20 25 30
Phe Glu Asp Met Asn Glu Ala Ile Glu Ala Ala Thr Ile Ala Gln Lys
35 40 45
Glu Leu Leu Lys Leu Ser Leu Glu Gln Arg Gly Asn Leu Ile Thr Ala
50 55 60
Ile Arg Lys Ala Ala Lys Asp Asn Ala Glu Thr Phe Ala Gln Met Ala
65 70 75 80
Val Asp Glu Thr Gly Met Gly Asn Tyr Gly Asp Lys Val Ile Lys Asn
85 90 95
Leu Ile Ala Ala Glu Lys Thr Pro Gly Ile Glu Asp Leu Thr Thr Glu
100 105 110
Ala Phe Ser Gly Asp His Gly Leu Thr Leu Val Glu Leu Ser Pro Tyr
115 120 125
Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr Glu Thr Val
130 135 140
Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val Val Phe
145 150 155 160
Ser Pro His Pro Thr Ala Lys Asn Thr Ser Leu Lys Ala Ile Glu Val
165 170 175
Ile Asn Lys Ala Ile Ile Lys Ala Gly Gly Pro Pro Asn Leu Ile Thr
180 185 190
Ser Val Ala Asn Pro Thr Ile Asp Gln Ala Asn Ile Met Met Lys His
195 200 205
Lys Lys Ile Lys Leu Leu Val Ala Thr Gly Gly Pro Gly Val Val Lys
210 215 220
Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Ala Val Val Asp Glu Thr Ala Asn Leu Glu Lys Ala Ala Arg
245 250 255
Asp Ile Ile Asp Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Thr Ala
260 265 270
Glu Lys Glu Val Ile Val Val Asp Ser Val Ala Asp Tyr Leu Val Ser
275 280 285
Tyr Met Lys Lys His Gly Ala Phe Leu Ile Thr Asp Lys Glu Gln Ile
290 295 300
Gln Lys Leu Thr Glu Leu Val Val Asp Asn Gly His Ala Asn Lys Glu
305 310 315 320
Leu Val Gly Lys Ser Val Ala His Ile Leu Gln Arg Ile Gly Ile Glu
325 330 335
Val Pro Ser Asp Ala Arg Val Ala Ile Leu Asn Val Glu Arg Asn His
340 345 350
Pro Leu Val Lys Ala Glu Leu Met Met Pro Ile Leu Pro Val Val Arg
355 360 365
Val Glu Asn Val Asp Ala Ala Ile Glu Leu Ala Val Glu Ala Glu Gln
370 375 380
Gly Phe Arg His Thr Ala Ile Met His Ser Thr Asn Ile Asp Asn Leu
385 390 395 400
Thr Lys Phe Ser Lys Glu Ile Gln Thr Thr Ile Phe Val Lys Asn Gly
405 410 415
Pro Ser Tyr Ala Gly Ile Gly Ile Gly Gly Glu Gly Tyr Ala Thr Phe
420 425 430
Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys Asp Phe
435 440 445
Ala Arg Arg Arg Lys Cys Val Leu Val Asp Gly Leu Ser Ile Arg
450 455 460
<210> 65
<211> 503
<212> PRT
<213> 史迪克兰梭菌（Clostridium sticklandii）
<400> 65
Met Lys Ala Gly Asp Ile Val Gln Asp Phe Ile Thr Glu Arg Asp Val
1 5 10 15
Glu Lys Ile Ile Glu Gln Val Leu Ser Lys Leu Glu Pro Val Ile Glu
20 25 30
Gln Val Lys Pro Lys Glu Ile Asn Met Leu Pro Asn Lys Thr Asn Ile
35 40 45
Asp Phe Ser Gln Asn Ala Asn Gly Ile Phe Glu Ser Ile Asp Leu Ala
50 55 60
Val Glu Ser Ala Leu Glu Ala His Ile Ile Leu Thr Ser Tyr Lys Leu
65 70 75 80
Glu Asp Arg Glu Lys Met Ile Gln Ser Ile Arg Lys Glu Val Leu Gly
85 90 95
Asp Ile Glu Asn Ile Ala Arg Leu Val Tyr Glu Glu Thr Lys Leu Gly
100 105 110
Lys Tyr Glu Asp Lys Ile Ala Lys Ile Asn Leu Ala Ala Ser Lys Thr
115 120 125
Pro Gly Thr Glu Asp Ile Lys Thr Ser Ala Ile Ser Gly Asp Tyr Gly
130 135 140
Leu Thr Ile Glu Glu Met Ala Pro Phe Gly Val Ile Gly Ala Val Thr
145 150 155 160
Pro Val Thr Asn Pro Val Glu Thr Leu Ile Asn Asn Ala Ile Ser Met
165 170 175
Ile Ser Gly Gly Asn Ser Val Val Phe Asn Val His Pro Ser Ser Lys
180 185 190
Lys Ser Ser Ala Tyr Thr Val Glu Leu Ile Asn Lys Ala Val Leu Lys
195 200 205
Ala Gly Gly Pro Gln Asn Leu Val Thr Met Val Lys Glu Pro Thr Ile
210 215 220
Glu Thr Val Asn Gln Leu Ser Ser His Pro Arg Ile Ser Met Met Val
225 230 235 240
Gly Thr Gly Gly Pro Gly Leu Val Lys Ser Leu Leu Lys Ser Gly Lys
245 250 255
Lys Thr Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu
260 265 270
Thr Ala Asp Met Asn Leu Ala Ala Lys Gly Ile Ile Glu Gly Ala Ser
275 280 285
Phe Asp Asn Asn Ile Leu Cys Ile Ala Glu Lys Glu Val Phe Val Val
290 295 300
Asn Glu Val Ala Asp Asp Leu Ile Tyr Asn Met Leu Ser Ser Gly Ala
305 310 315 320
Tyr Met Leu Asn Gln Glu Glu Leu Glu Lys Val Met Lys Leu Thr Leu
325 330 335
Val Glu Asp Glu Asp Leu Gly Ala Lys Ser Cys Thr Leu Ser Pro Lys
340 345 350
Lys Lys Tyr His Val His Lys Asn Trp Val Gly Lys Asp Ala Ser Lys
355 360 365
Ile Leu Ser Glu Ile Gly Ile Thr Lys Gln Asp Val Lys Leu Leu Ile
370 375 380
Cys Glu Val Asp Ser Asp His Pro Tyr Val Thr Leu Glu Gln Met Met
385 390 395 400
Pro Ile Leu Pro Leu Val Arg Cys Ser Asp Val Asp Glu Ala Ile Lys
405 410 415
Leu Ala Val Lys Ala Glu Gly Thr Asn Lys His Thr Ala Ser Ile Phe
420 425 430
Ser Arg Asn Val Asp Asn Met Thr Lys Phe Ala Arg Ala Ile Asn Thr
435 440 445
Thr Ile Phe Val Lys Asn Ala Pro Thr Leu Ala Gly Val Gly Tyr Lys
450 455 460
Gly Glu Gly Asn Ala Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
465 470 475 480
Ile Thr Ser Ala Lys Thr Phe Thr Arg Val Arg Arg Cys Val Leu Ala
485 490 495
Glu Gly Gly Phe Arg Ile Val
500
<210> 66
<211> 482
<212> PRT
<213> 潜能栖热泉菌（Thermincola potens）
<400> 66
Met Ala Ile Glu Ala Tyr Gln Ile Glu Lys Ile Val Glu Glu Val Met
1 5 10 15
Arg Lys Met Val Ser Gly Gly Ser Gly Asp Ser Phe Ala Gly Lys Ala
20 25 30
Lys Gly Ile Phe Glu Ser Val Asp Glu Ala Val Lys Ala Ala Lys Ala
35 40 45
Ala Gln Lys Glu Leu Val Ala Met Arg Ile Glu Lys Arg Glu Met Leu
50 55 60
Leu Lys Ala Met Arg Glu Ala Ala Ile Ala His Ala Glu Glu Leu Ala
65 70 75 80
Arg Leu Ala Val Glu Glu Thr Gly Met Gly Arg Val Thr Asp Lys Ile
85 90 95
Ile Lys Asn Arg Val Ala Ala Glu Lys Thr Pro Gly Thr Glu Asn Leu
100 105 110
Gln Pro Ser Ala Val Thr Gly Asp Arg Gly Leu Thr Leu Ile Glu Arg
115 120 125
Ala Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Cys
130 135 140
Ala Thr Val Ile Asn Asn Ser Ile Ser Met Val Ala Ala Gly Asn Ser
145 150 155 160
Val Val Phe Ser Val His Pro Gly Ala Lys Lys Ala Ser Leu Leu Thr
165 170 175
Val Glu Ile Leu Asn Glu Ala Ile Glu Lys Ala Gly Gly Pro Ala Asn
180 185 190
Val Leu Thr Ala Val Ala Ser Pro Ser Leu Glu Asn Thr Asn Ala Leu
195 200 205
Met Lys His Pro Asp Ile Lys Leu Leu Val Ala Thr Gly Gly Pro Gly
210 215 220
Leu Val Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Ala Leu Val Asp Glu Thr Ala Asp Leu Glu Arg
245 250 255
Ala Ala Lys Ser Ile Val Ala Gly Ala Ser Phe Asp Asn Asn Leu Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Ile Val Val Asp Tyr Val Ala Asn Gln
275 280 285
Leu Ile Ser Tyr Met Lys Gln Asn Gly Ala Tyr Leu Ala Asn Asp Arg
290 295 300
Glu Ile Lys Ala Leu Met Asp Leu Val Leu Thr Lys Asn Glu Asn Leu
305 310 315 320
Lys Ala Glu Gly Cys Thr Val Lys Pro Glu Lys Leu Tyr Gly Gly Ile
325 330 335
Asn Lys Glu Tyr Val Gly Lys Asp Ala Ala Tyr Ile Met Lys Lys Ile
340 345 350
Gly Val Asp Ile Pro Glu Asp Thr Lys Leu Ile Ile Cys Glu Val Asp
355 360 365
Glu Asp His Pro Phe Val Leu Glu Glu Leu Met Met Pro Ile Leu Pro
370 375 380
Ile Val Arg Val Pro Asn Val Gln Lys Ala Ile Glu Val Gly Val Arg
385 390 395 400
Val Glu His Gly Asn Arg His Thr Ala Val Met His Ser Gln Asn Ile
405 410 415
Asp Asn Leu Ser Ala Phe Ala Arg Ala Val Gln Thr Thr Ile Phe Val
420 425 430
Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly Gly Glu Gly Tyr
435 440 445
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ala Ala
450 455 460
Ser Ser Phe Thr Arg Gln Arg Arg Cys Val Leu Val Asp Gly Phe Ser
465 470 475 480
Ile Val
<210> 67
<211> 462
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 67
Met Ser Val Asn Glu Gln Met Ile Gln Asp Ile Val Ser Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Ser Glu Val Ser Asp Asn His Gly Ile Phe
20 25 30
Ala Asp Met Asn Glu Ala Ile Glu Ala Ala Lys Lys Ala Gln Lys Ile
35 40 45
Val Gly Arg Met Ser Met Asp Gln Arg Glu Lys Ile Ile Ser Asn Ile
50 55 60
Arg Lys Lys Thr Val Glu Asn Ala Glu Ile Leu Ala Arg Met Gly Val
65 70 75 80
Gln Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Gly Ala Ile Lys Thr Ser Ile Phe Ala Ile Asn Met Ile
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Ser Ser Asn Ile Met Met Lys His Lys
195 200 205
Ala Ile His Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Val Ala Asp Glu Leu Met His Tyr
275 280 285
Met Val Ser Glu Gln Gly Cys Tyr Leu Ala Ser Lys Glu Glu Gln Glu
290 295 300
Ala Leu Thr Ala Val Val Leu Lys Asp Gly Arg Leu Asn Arg Asn Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Val Ser Val
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Ala Thr Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ser Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 68
<211> 470
<212> PRT
<213> 梭杆菌属（Fusobacterium sp.）
<400> 68
Met Arg Gly Glu Leu Met Glu Phe Glu Val Asn Asn Ile Glu Lys Ile
1 5 10 15
Val Glu Leu Ile Met Lys Lys Met Ala Glu Ser Asn Ile Ser Thr Ala
20 25 30
Gly Asn Ser Lys Asn Gly Val Phe Asp Asn Val Asp Glu Ala Ile Glu
35 40 45
Glu Ala Lys Lys Ala Gln Ala Ile Leu Phe Ser Ser Lys Leu Glu Leu
50 55 60
Arg Glu Lys Ile Ile Ala Ser Ile Arg Asp Thr Leu Lys Ser His Val
65 70 75 80
Thr Glu Leu Ala Glu Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val
85 90 95
Ser Asp Lys Glu Leu Lys Asn Lys Ile Ala Ile Glu Lys Thr Pro Gly
100 105 110
Leu Glu Asp Leu Lys Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr
115 120 125
Val Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser
130 135 140
Thr Asn Pro Ser Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala
145 150 155 160
Ala Gly Asn Ala Val Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr
165 170 175
Ser Ile Arg Thr Val Glu Leu Ile Asn Glu Ala Ile Arg Lys Val Gly
180 185 190
Gly Pro Asp Asn Leu Val Val Thr Ile Arg Glu Pro Ser Ile Glu Asn
195 200 205
Thr Glu Lys Ile Ile Ala Asn Pro Asn Ile Lys Met Leu Val Ala Thr
210 215 220
Gly Gly Pro Gly Val Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp
260 265 270
Asn Asn Leu Pro Cys Thr Ala Glu Lys Glu Val Val Ala Val Asp Ser
275 280 285
Ile Val Asn Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu
290 295 300
Leu Lys Asp Lys Lys Leu Ile Glu Lys Leu Leu Ser Leu Val Leu Lys
305 310 315 320
Asn Asn Ser Pro Asp Arg Lys Tyr Val Gly Arg Asp Ala Lys Tyr Leu
325 330 335
Leu Lys Gln Ile Gly Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile
340 345 350
Val Glu Thr Asp Lys Asn His Pro Phe Ala Val Glu Glu Leu Leu Met
355 360 365
Pro Ile Leu Pro Ile Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys
370 375 380
Val Ala Lys Glu Leu Glu Arg Gly Leu Arg His Thr Ala Val Ile His
385 390 395 400
Ser Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Glu Met Glu Thr
405 410 415
Thr Ile Leu Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly
420 425 430
Gly Glu Gly His Val Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
435 440 445
Leu Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val
450 455 460
Gly Gly Phe Ser Ile Lys
465 470
<210> 69
<211> 470
<212> PRT
<213> 梭杆菌属（Fusobacterium sp.）
<400> 69
Met Arg Gly Glu Leu Met Glu Phe Glu Val Asn Asn Ile Glu Glu Ile
1 5 10 15
Val Glu Leu Ile Met Lys Lys Met Ala Glu Ser Asn Ile Ser Thr Ala
20 25 30
Gly Asn Ser Lys Asn Gly Val Phe Asp Asn Val Asp Glu Ala Ile Glu
35 40 45
Glu Ala Lys Lys Ala Gln Ala Ile Leu Phe Ser Ser Lys Leu Glu Leu
50 55 60
Arg Glu Lys Ile Ile Ala Ser Ile Arg Asp Thr Leu Lys Asn His Val
65 70 75 80
Ser Glu Leu Ala Glu Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val
85 90 95
Ala Asp Lys Glu Leu Lys Asn Lys Ile Ala Ile Glu Lys Thr Pro Gly
100 105 110
Leu Glu Asp Leu Lys Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr
115 120 125
Val Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser
130 135 140
Thr Asn Pro Ser Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala
145 150 155 160
Ala Gly Asn Ala Val Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr
165 170 175
Ser Ile Arg Thr Val Glu Leu Ile Asn Glu Ala Ile Arg Lys Val Gly
180 185 190
Gly Pro Asp Asn Leu Val Val Thr Ile Arg Glu Pro Ser Ile Glu Asn
195 200 205
Thr Glu Lys Ile Ile Ala Asn Pro Asn Ile Lys Met Leu Val Ala Thr
210 215 220
Gly Gly Pro Gly Val Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp
260 265 270
Asn Asn Leu Pro Cys Thr Ala Glu Lys Glu Val Val Ala Val Asp Ser
275 280 285
Ile Val Asn Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu
290 295 300
Leu Lys Asp Lys Glu Leu Ile Glu Lys Leu Leu Ser Leu Val Leu Lys
305 310 315 320
Asn Asn Ser Pro Asp Arg Lys Tyr Val Gly Arg Asp Ala Lys Tyr Leu
325 330 335
Leu Lys Gln Ile Gly Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile
340 345 350
Val Glu Thr Asp Lys Asn His Pro Phe Ala Val Glu Glu Leu Leu Met
355 360 365
Pro Ile Leu Pro Ile Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys
370 375 380
Val Ala Lys Glu Leu Glu Arg Gly Leu Arg His Thr Ala Val Ile His
385 390 395 400
Ser Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Glu Met Glu Thr
405 410 415
Thr Ile Leu Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly
420 425 430
Gly Glu Gly His Val Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
435 440 445
Leu Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val
450 455 460
Gly Gly Phe Ser Ile Lys
465 470
<210> 70
<211> 462
<212> PRT
<213> 瘤胃球菌属（Ruminococcus sp.）
<400> 70
Met Pro Ile Ser Glu Asn Met Val Gln Glu Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Asp Ala Pro Ala Gly Lys His Gly Val Phe
20 25 30
Lys Asp Met Asn Asp Ala Ile Glu Ala Ala Lys Lys Ala Gln Leu Val
35 40 45
Val Lys Thr Met Ser Met Asp Gln Arg Glu Lys Ile Ile Thr Cys Ile
50 55 60
Arg Lys Lys Ile Lys Glu Asn Ala Glu Val Leu Ala Arg Met Gly Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His His
85 90 95
Leu Val Ala Asp Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Met Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Val Lys Thr Ser Ile Tyr Ala Ile Asn Leu Leu
165 170 175
Asn Glu Ala Ser Leu Glu Ser Gly Gly Pro Asp Asn Ile Ala Val Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Thr Ser Asn Ile Met Met Lys His Lys
195 200 205
Asp Ile His Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Asp Thr Ala Asp Ile Arg Lys Ala Ala Gln Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Val Val Ala Val Ser Ser Val Val Asp Glu Leu Met His Tyr
275 280 285
Met Ile Thr Glu Asn Asp Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Lys Leu Val Glu Thr Val Leu Ala Gly Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Ser Met Ile Gly Val Gln Ala
325 330 335
Pro Ala Asn Thr Arg Cys Ile Ile Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Ile Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Ile Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ser Ala Leu Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Thr Asp Ser Leu Cys Ile Arg
450 455 460
<210> 71
<211> 465
<212> PRT
<213> 具核梭杆菌（Fusobacterium nucleatum）
<400> 71
Met Glu Phe Glu Val Asn Asn Ile Glu Glu Ile Val Glu Leu Ile Met
1 5 10 15
Lys Lys Met Ala Glu Ser Asn Ile Ser Thr Ala Gly Asn Ser Lys Asn
20 25 30
Gly Val Phe Asp Asn Val Asp Glu Ala Ile Glu Glu Ala Lys Lys Ala
35 40 45
Gln Ala Ile Leu Phe Ser Ser Lys Leu Glu Leu Arg Glu Lys Ile Ile
50 55 60
Ala Ser Ile Arg Asp Thr Leu Lys Asn His Val Thr Glu Leu Ala Glu
65 70 75 80
Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val Ala Asp Lys Glu Leu
85 90 95
Lys Asn Lys Ile Ala Ile Glu Lys Thr Pro Gly Leu Glu Asp Leu Lys
100 105 110
Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val Met Glu Leu Ser
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Ser Glu
130 135 140
Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val
145 150 155 160
Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser Ile Arg Thr Val
165 170 175
Glu Leu Ile Asn Glu Ala Ile Arg Lys Val Gly Gly Pro Asp Asn Leu
180 185 190
Val Val Thr Ile Arg Glu Pro Ser Ile Glu Asn Thr Glu Lys Ile Ile
195 200 205
Ala Asn Pro Asn Ile Lys Met Leu Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Thr Ala Glu Lys Glu Val Val Ala Val Asp Ser Ile Val Asn Tyr Leu
275 280 285
Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Leu Lys Asp Lys Glu
290 295 300
Leu Ile Glu Lys Leu Leu Ser Leu Val Leu Lys Asn Asn Ser Pro Asp
305 310 315 320
Arg Lys Tyr Val Gly Arg Asp Ala Lys Tyr Leu Leu Lys Gln Ile Gly
325 330 335
Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile Val Glu Thr Asp Lys
340 345 350
Asn His Pro Phe Ala Val Glu Glu Leu Leu Met Pro Ile Leu Pro Ile
355 360 365
Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys Val Ala Lys Glu Leu
370 375 380
Glu Arg Gly Leu Arg His Thr Ala Val Ile His Ser Lys Asn Ile Asp
385 390 395 400
Ile Leu Thr Lys Tyr Ala Arg Glu Met Glu Thr Thr Ile Leu Val Lys
405 410 415
Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly Gly Glu Gly His Val
420 425 430
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys
435 440 445
Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly Gly Phe Ser Ile
450 455 460
Lys
465
<210> 72
<211> 465
<212> PRT
<213> 具核梭杆菌（Fusobacterium nucleatum）
<400> 72
Met Glu Phe Glu Val Asn Asn Ile Glu Glu Ile Val Glu Leu Ile Met
1 5 10 15
Lys Lys Met Thr Glu Gly Gly Val Ser Thr Ser Asn Asn Ser Thr Asn
20 25 30
Gly Val Phe Lys Asn Val Asp Glu Ala Ile Ala Glu Ala Lys Lys Ala
35 40 45
Gln Thr Val Leu Phe Ser Ser Lys Leu Glu Leu Arg Glu Arg Ile Ile
50 55 60
Ala Ser Ile Arg Asp Thr Leu Lys Ser His Ile Thr Glu Leu Ser Glu
65 70 75 80
Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val Ala Asp Lys Glu Leu
85 90 95
Lys Asn Arg Ile Ala Ile Glu Lys Thr Pro Gly Leu Glu Asp Leu Lys
100 105 110
Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val Met Glu Leu Ser
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Ser Glu
130 135 140
Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val
145 150 155 160
Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser Ile Arg Ala Val
165 170 175
Glu Leu Ile Asn Glu Ala Ile Lys Lys Ala Gly Gly Pro Asp Asn Leu
180 185 190
Val Val Thr Ile Ala Glu Pro Ser Ile Glu Asn Thr Glu Lys Ile Ile
195 200 205
Ala Asn Pro Asn Ile Lys Met Val Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Thr Ala Glu Lys Glu Val Ile Ala Val Asp Ser Ile Val Asn Tyr Leu
275 280 285
Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Leu Lys Asp Lys Glu
290 295 300
Leu Ile Glu Lys Leu Leu Ser Ile Val Leu Lys Asn Asn Ser Pro Asp
305 310 315 320
Arg Lys Tyr Val Gly Lys Asp Ala Lys Tyr Leu Leu Lys Gln Ile Gly
325 330 335
Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile Val Glu Thr Asp Lys
340 345 350
Asn His Pro Phe Ala Val Glu Glu Leu Leu Met Pro Ile Leu Pro Ile
355 360 365
Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys Val Ala Lys Glu Leu
370 375 380
Glu Lys Gly Leu Arg His Thr Ala Val Ile His Ser Lys Asn Ile Asp
385 390 395 400
Ile Leu Ser Lys Tyr Ala Arg Glu Met Glu Thr Thr Ile Leu Val Lys
405 410 415
Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly Gly Glu Gly His Val
420 425 430
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg
435 440 445
Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly Gly Phe Ser Ile
450 455 460
Lys
465
<210> 73
<211> 470
<212> PRT
<213> 梭杆菌属（Fusobacterium sp.）
<400> 73
Met Arg Gly Glu Leu Met Glu Phe Glu Val Asn Asn Ile Glu Glu Ile
1 5 10 15
Val Glu Leu Ile Met Lys Lys Met Ala Glu Ser Asn Ile Ser Thr Ala
20 25 30
Gly Asn Ser Lys Asn Gly Val Phe Asp Asn Val Asp Gly Ala Ile Glu
35 40 45
Glu Ala Lys Lys Ala Gln Ala Ile Leu Phe Ser Ser Lys Leu Glu Leu
50 55 60
Arg Glu Lys Ile Ile Ala Ser Ile Arg Asp Thr Leu Lys Asn His Val
65 70 75 80
Thr Glu Leu Ala Glu Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val
85 90 95
Ala Asp Lys Glu Leu Lys Asn Lys Ile Ala Ile Glu Lys Thr Pro Gly
100 105 110
Leu Glu Asp Leu Lys Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr
115 120 125
Val Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser
130 135 140
Thr Asn Pro Ser Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala
145 150 155 160
Ala Gly Asn Ala Val Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr
165 170 175
Ser Ile Arg Thr Val Glu Leu Ile Asn Glu Ala Ile Arg Lys Val Gly
180 185 190
Gly Pro Asp Asn Leu Ile Val Thr Ile Arg Glu Pro Ser Ile Glu Asn
195 200 205
Thr Glu Lys Ile Ile Ala Asn Pro Asn Ile Lys Met Leu Val Ala Thr
210 215 220
Gly Gly Pro Gly Val Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp
260 265 270
Asn Asn Leu Pro Cys Thr Ala Glu Lys Glu Val Val Ala Val Asp Ser
275 280 285
Ile Val Asn Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu
290 295 300
Leu Lys Asp Lys Glu Leu Ile Glu Lys Leu Leu Ser Leu Val Leu Lys
305 310 315 320
Asn Asn Ser Pro Asp Arg Lys Tyr Val Gly Arg Asp Ala Lys Tyr Leu
325 330 335
Leu Lys Gln Ile Gly Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile
340 345 350
Val Glu Thr Asp Lys Asn His Pro Phe Ala Val Glu Glu Leu Leu Met
355 360 365
Pro Ile Leu Pro Ile Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys
370 375 380
Val Ala Lys Glu Leu Glu Arg Gly Leu Arg His Thr Ala Val Ile His
385 390 395 400
Ser Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Glu Met Glu Thr
405 410 415
Thr Ile Leu Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly
420 425 430
Gly Glu Gly His Val Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
435 440 445
Leu Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val
450 455 460
Gly Gly Phe Ser Ile Lys
465 470
<210> 74
<211> 465
<212> PRT
<213> 具核梭杆菌（Fusobacterium nucleatum）
<400> 74
Met Glu Phe Glu Val Asn Asn Leu Glu Glu Ile Val Glu Leu Ile Met
1 5 10 15
Lys Lys Met Ser Glu Ser Ser Ile Ser Thr Ser Ser Asn Ser Lys Asn
20 25 30
Gly Val Phe Glu Asn Val Asp Glu Ala Ile Ala Glu Ala Lys Lys Ala
35 40 45
Gln Thr Ile Leu Phe Ser Ser Lys Leu Glu Leu Arg Glu Arg Ile Ile
50 55 60
Ala Ser Ile Arg Asp Thr Leu Lys Pro Tyr Ile Thr Glu Leu Ser Glu
65 70 75 80
Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val Ser Asp Lys Glu Ile
85 90 95
Lys Asn Arg Ile Ala Ile Glu Lys Thr Pro Gly Leu Glu Asp Leu Lys
100 105 110
Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val Met Glu Leu Ser
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Ser Glu
130 135 140
Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val
145 150 155 160
Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser Ile Arg Ala Val
165 170 175
Glu Leu Ile Asn Glu Ala Ile Lys Lys Val Gly Gly Pro Asp Asn Leu
180 185 190
Ile Val Thr Ile Thr Glu Pro Ser Ile Glu Asn Thr Glu Lys Ile Ile
195 200 205
Ala Asn Pro Asn Ile Lys Met Val Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Thr Ala Glu Lys Glu Val Ile Ala Val Asp Ser Ile Val Asn Tyr Leu
275 280 285
Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Leu Lys Asp Lys Asp
290 295 300
Leu Ile Glu Lys Leu Leu Ser Ile Val Leu Lys Asn Asn Ser Pro Asp
305 310 315 320
Arg Lys Tyr Val Gly Lys Asp Ala Lys Tyr Leu Leu Lys Gln Ile Gly
325 330 335
Ile Glu Val Gly Asp Glu Ile Arg Val Ile Ile Val Glu Thr Ser Lys
340 345 350
Asp His Pro Phe Ala Val Glu Glu Leu Leu Met Pro Ile Leu Pro Ile
355 360 365
Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys Val Ala Lys Glu Leu
370 375 380
Glu Lys Gly Leu Arg His Thr Ala Ile Ile His Ser Lys Asn Ile Asp
385 390 395 400
Ile Leu Ser Lys Tyr Ala Arg Glu Met Glu Thr Thr Ile Leu Val Lys
405 410 415
Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly Gly Glu Gly His Val
420 425 430
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg
435 440 445
Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly Gly Phe Ser Ile
450 455 460
Lys
465
<210> 75
<211> 465
<212> PRT
<213> 具核梭杆菌（Fusobacterium nucleatum）
<400> 75
Met Glu Phe Glu Val Asn Asn Ile Glu Glu Ile Val Glu Leu Ile Met
1 5 10 15
Lys Lys Met Ser Glu Ser Gly Val Ser Thr Ser Asn Asn Ser Thr Asn
20 25 30
Gly Val Phe Glu Asn Val Asp Glu Ala Ile Ala Glu Ala Lys Lys Ala
35 40 45
Gln Thr Val Leu Phe Ser Ser Lys Leu Glu Leu Arg Glu Arg Ile Ile
50 55 60
Ala Ser Ile Arg Asp Thr Leu Lys Thr His Ile Thr Glu Leu Ser Glu
65 70 75 80
Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val Ala Asp Lys Glu Leu
85 90 95
Lys Asn Arg Ile Ala Ile Glu Lys Thr Pro Gly Leu Glu Asp Leu Lys
100 105 110
Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val Met Glu Leu Ser
115 120 125
Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Ser Glu
130 135 140
Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ala Val
145 150 155 160
Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser Ile Arg Ala Val
165 170 175
Glu Leu Ile Asn Glu Ala Ile Lys Lys Ala Gly Gly Pro Asp Asn Leu
180 185 190
Val Val Thr Ile Ala Glu Pro Ser Ile Glu Asn Thr Glu Lys Ile Ile
195 200 205
Ala Asn Pro Asn Ile Lys Met Val Val Ala Thr Gly Gly Pro Gly Val
210 215 220
Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Thr Ala Glu Lys Glu Val Ile Ala Val Asp Ser Ile Val Asn Tyr Leu
275 280 285
Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Leu Lys Asp Lys Glu
290 295 300
Leu Ile Glu Lys Leu Leu Ser Ile Val Leu Lys Asn Asn Ser Pro Asp
305 310 315 320
Arg Lys Tyr Val Gly Lys Asp Ala Lys Tyr Leu Leu Lys Gln Ile Gly
325 330 335
Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile Val Glu Thr Asp Lys
340 345 350
Asn His Pro Phe Ala Val Glu Glu Leu Leu Met Pro Ile Leu Pro Ile
355 360 365
Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys Val Ala Lys Glu Leu
370 375 380
Glu Lys Gly Leu Arg His Thr Ala Val Ile His Ser Lys Asn Ile Asp
385 390 395 400
Ile Leu Ser Lys Tyr Ala Arg Glu Met Glu Thr Thr Ile Leu Val Lys
405 410 415
Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly Gly Glu Gly His Val
420 425 430
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg
435 440 445
Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly Gly Phe Ser Ile
450 455 460
Lys
465
<210> 76
<211> 470
<212> PRT
<213> 梭杆菌属（Fusobacterium sp.）
<400> 76
Met Arg Gly Glu Leu Met Glu Phe Glu Val Asn Asn Ile Glu Glu Ile
1 5 10 15
Val Glu Leu Ile Met Lys Lys Met Ala Glu Ser Asn Ile Ser Thr Ala
20 25 30
Gly Asn Ser Lys Asn Gly Val Phe Asp Asn Val Asp Glu Ala Ile Glu
35 40 45
Glu Ala Lys Lys Ala Gln Ala Ile Leu Phe Ser Ser Lys Leu Glu Leu
50 55 60
Arg Glu Lys Ile Ile Ala Ser Ile Arg Asp Thr Leu Lys Asn His Val
65 70 75 80
Thr Glu Leu Ala Glu Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val
85 90 95
Ala Asp Lys Glu Leu Lys Asn Lys Ile Ala Ile Glu Lys Thr Pro Gly
100 105 110
Leu Glu Asp Leu Lys Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr
115 120 125
Val Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser
130 135 140
Thr Asn Pro Ser Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala
145 150 155 160
Ala Gly Asn Ala Val Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr
165 170 175
Ser Ile Arg Thr Val Glu Leu Ile Asn Glu Ala Ile Arg Lys Val Gly
180 185 190
Gly Pro Asp Asn Leu Ile Val Thr Ile Arg Glu Pro Ser Ile Glu Asn
195 200 205
Thr Glu Lys Ile Ile Ala Asn Pro Asn Ile Lys Met Leu Val Ala Thr
210 215 220
Gly Gly Pro Gly Val Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Lys Asp Ile Ile Ala Gly Cys Ser Phe Asp
260 265 270
Asn Asn Leu Pro Cys Thr Ala Glu Lys Glu Val Val Ala Val Asp Ser
275 280 285
Ile Val Asn Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu
290 295 300
Leu Lys Asp Lys Glu Leu Ile Glu Lys Leu Leu Ser Leu Val Leu Lys
305 310 315 320
Asn Asn Ser Pro Asp Arg Lys Tyr Val Gly Arg Asp Ala Lys Tyr Leu
325 330 335
Leu Lys Gln Ile Gly Ile Glu Val Gly Asp Glu Ile Lys Val Ile Ile
340 345 350
Val Glu Thr Asp Lys Asn His Pro Phe Ala Val Glu Glu Leu Leu Met
355 360 365
Pro Ile Leu Pro Ile Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys
370 375 380
Val Ala Lys Glu Leu Glu Arg Gly Leu Arg His Thr Ala Val Ile His
385 390 395 400
Ser Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Glu Met Glu Thr
405 410 415
Thr Ile Leu Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly
420 425 430
Gly Glu Gly His Val Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
435 440 445
Leu Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val
450 455 460
Gly Gly Phe Ser Ile Lys
465 470
<210> 77
<211> 481
<212> PRT
<213> 天门冬形梭菌（Clostridium asparagiforme）
<400> 77
Met Glu Ile Glu Thr Arg Asp Ile Glu Arg Ile Val Arg Gln Val Met
1 5 10 15
Ala Ala Met Glu Gln Gln Gly Thr Ile Ala Gly Gly Ala Tyr Pro Pro
20 25 30
Ala Pro Gly Ile Thr Ala Pro Arg Gly Asp Asn Gly Val Phe Glu Arg
35 40 45
Val Glu Asp Ala Ile Asp Ala Ala Trp Ala Ala Gly Arg Val Trp Ala
50 55 60
Phe His Tyr Lys Val Glu Asp Arg Arg Arg Val Ile Glu Ala Ile Arg
65 70 75 80
Val Met Ala Arg Glu Asn Ala Arg Thr Leu Ala Gln Met Val Arg Asp
85 90 95
Glu Thr Gly Met Gly Arg Val Glu Asp Lys Val Glu Lys His Leu Ala
100 105 110
Val Ala Asp Lys Thr Pro Gly Val Glu Cys Leu Thr Thr Asp Ala Ile
115 120 125
Ser Gly Asp Gly Gly Leu Met Ile Glu Glu Tyr Ala Pro Phe Gly Val
130 135 140
Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Thr Glu Thr Val Ile His
145 150 155 160
Asn Thr Ile Ser Met Ile Ala Gly Gly Asn Ser Val Val Phe Asn Val
165 170 175
His Pro Gly Ala Lys Lys Cys Cys Ala Phe Cys Leu Gln Leu Leu Asn
180 185 190
Lys Thr Ile Val Glu Asn Gly Gly Pro Ala Asn Leu Ile Thr Met Gln
195 200 205
Arg Asp Pro Thr Met Asp Ala Val Asn Lys Met Thr Ser Ser Pro Lys
210 215 220
Ile Arg Leu Met Val Gly Thr Gly Gly Met Gly Met Val Asn Ala Leu
225 230 235 240
Leu Arg Ser Gly Lys Lys Thr Ile Gly Ala Gly Ala Gly Asn Pro Pro
245 250 255
Val Ile Val Asp Asp Thr Ala Asp Val Lys Leu Ala Ala Arg Glu Leu
260 265 270
Tyr Trp Gly Ala Ser Phe Asp Asn Asn Leu Phe Cys Phe Ala Glu Lys
275 280 285
Glu Val Phe Val Met Glu Ala Ser Ala Asp Gly Leu Ile Arg Gly Leu
290 295 300
Val Glu Gln Gly Ala Tyr Leu Leu Thr Pro Ala Glu Thr Glu Ala Ile
305 310 315 320
Val Lys Leu Ala Leu Ile Gln Lys Asp Gly Lys Tyr Glu Val Asn Lys
325 330 335
Lys Trp Val Gly Lys Asp Ala Gly Leu Phe Leu Gln Ala Ile Gly Val
340 345 350
Ser Gly His Glu Asn Thr Arg Leu Leu Ile Cys Asp Val Pro Lys Cys
355 360 365
His Pro Tyr Val Met Val Glu Gln Leu Met Pro Val Leu Pro Ile Val
370 375 380
Arg Cys Arg Thr Phe Asp Glu Cys Ile Gln Cys Ser Val Glu Ala Glu
385 390 395 400
Gln Gly Asn Arg His Thr Ser Ser Ile Phe Ser Thr Asn Val Tyr Asn
405 410 415
Met Thr Lys Phe Gly Lys Glu Ile Glu Thr Thr Ile Tyr Val Lys Asn
420 425 430
Gly Ala Thr Leu Arg Gly Leu Gly Ile Gly Gly Glu Gly His Thr Thr
435 440 445
Met Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Cys Ala Arg Ser
450 455 460
Phe Thr Arg Arg Arg Arg Cys Met Leu Ala Glu Gly Gly Leu Arg Ile
465 470 475 480
Ile
<210> 78
<211> 462
<212> PRT
<213> 植物发酵梭菌（Clostridium phytofermentans）
<400> 78
Met Thr Val Asn Glu Gln Leu Val Gln Asp Ile Ile Lys Asn Val Val
1 5 10 15
Ala Ser Met Gln Leu Thr Gln Thr Asn Lys Thr Glu Leu Gly Val Phe
20 25 30
Asp Asp Met Asn Gln Ala Ile Glu Ala Ala Lys Glu Ala Gln Leu Val
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Ser Ala Ile
50 55 60
Arg Lys Lys Thr Ile Glu His Ala Glu Thr Leu Ala Arg Met Ala Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Val Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Ile
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Asn Phe Ala Val Gln Leu Ile
165 170 175
Asn Glu Ala Ser Leu Ser Ala Gly Gly Pro Val Asn Ile Ala Cys Ser
180 185 190
Val Arg Lys Pro Thr Leu Asp Ser Ser Lys Ile Met Met Ser His Gln
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Gln Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Val Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Ile Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Val Val Ala Ile Asp Ala Ile Ala Asn Glu Leu Met Asn Tyr
275 280 285
Met Val Lys Glu Gln Gly Cys Tyr Ala Ile Thr Lys Glu Gln Gln Glu
290 295 300
Lys Leu Thr Asn Leu Val Ile Thr Pro Lys Gly Leu Asn Arg Asn Cys
305 310 315 320
Val Gly Lys Asp Ala Arg Thr Leu Leu Gly Met Ile Gly Ile Asp Val
325 330 335
Pro Ser Asn Ile Arg Cys Ile Ile Phe Glu Gly Glu Lys Glu His Pro
340 345 350
Leu Ile Ser Glu Glu Leu Met Met Pro Ile Leu Gly Ile Val Arg Ala
355 360 365
Lys Ser Phe Asp Asp Ala Val Glu Lys Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Arg Ile Thr
385 390 395 400
Thr Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Ala Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 79
<211> 470
<212> PRT
<213> 梭杆菌属（Fusobacterium sp.）
<400> 79
Met Arg Gly Glu Leu Met Glu Leu Glu Val Lys Asn Ile Glu Glu Ile
1 5 10 15
Val Asp Leu Ile Met Lys Lys Met Thr Glu Ser Asn Val Ala Val Ser
20 25 30
Tyr Asp Ser Lys Asn Gly Val Phe Asp Asp Val Asp Val Ala Ile Ala
35 40 45
Glu Ala Lys Lys Ala Gln Thr Val Leu Phe Ser Ser Lys Leu Glu Leu
50 55 60
Arg Glu Arg Ile Ile Ala Ser Ile Arg Glu Thr Met Arg Ala His Ile
65 70 75 80
Thr Glu Leu Ser Glu Leu Ala Val Lys Glu Thr Gly Met Gly Arg Val
85 90 95
Lys Asp Lys Glu Gln Lys Asn Arg Val Ala Ile Asp Arg Thr Pro Gly
100 105 110
Leu Glu Asp Leu Lys Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr
115 120 125
Val Met Glu Phe Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser
130 135 140
Thr Asn Pro Ser Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala
145 150 155 160
Ala Gly Asn Ala Val Ile Phe Ala Pro His Pro Gly Ala Lys Arg Thr
165 170 175
Ser Ile Arg Ala Val Glu Leu Ile Asn Glu Ala Ile Lys Lys Val Gly
180 185 190
Gly Pro Glu Asn Leu Val Val Thr Ile Ser Glu Pro Ser Ile Glu Asn
195 200 205
Thr Glu Lys Ile Ile Ala Asn Pro Asn Ile Lys Met Leu Val Ala Thr
210 215 220
Gly Gly Pro Gly Val Val Lys Thr Val Met Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Lys Asp Ile Ile Asp Gly Cys Ser Phe Asp
260 265 270
Asn Asn Leu Pro Cys Thr Ala Glu Lys Glu Val Ile Ala Val Asp Ser
275 280 285
Ile Val Asn Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu
290 295 300
Leu Lys Asp Lys Glu Leu Ile Glu Lys Leu Val Ser Leu Val Leu Lys
305 310 315 320
Asn Asn Ser Pro Asp Arg Lys Tyr Val Gly Lys Asp Ala Lys Tyr Ile
325 330 335
Leu Lys Gln Leu Gly Ile Glu Val Gly Asp Glu Ile Arg Val Ile Ile
340 345 350
Val Glu Thr Asp Lys Asn His Pro Phe Ala Val Glu Glu Leu Leu Met
355 360 365
Pro Val Leu Pro Ile Val Lys Val Lys Asp Ala Leu Glu Gly Ile Lys
370 375 380
Val Ala Lys Glu Leu Glu Arg Gly Leu Arg His Thr Ala Ile Ile His
385 390 395 400
Ser Lys Asn Ile Asp Ile Leu Ser Lys Tyr Ala Arg Glu Met Glu Thr
405 410 415
Thr Ile Leu Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Ile Gly
420 425 430
Gly Glu Gly His Val Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly
435 440 445
Leu Thr Ser Ala Arg Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val
450 455 460
Gly Gly Phe Ser Ile Lys
465 470
<210> 80
<211> 462
<212> PRT
<213> 毛螺科菌（Lachnospiraceae bacterium）
<400> 80
Met Ser Val Asn Glu Lys Met Val Gln Asp Ile Val Gln Glu Val Val
1 5 10 15
Ala Lys Met Gln Ile Ser Ser Asp Val Ser Gly Lys Lys Gly Val Phe
20 25 30
Ser Asp Met Asn Glu Ala Ile Glu Ala Ser Lys Lys Ala Gln Lys Ile
35 40 45
Val Ala Lys Met Ser Met Asp Gln Arg Glu Ala Ile Ile Ser Lys Ile
50 55 60
Arg Glu Lys Ile Lys Glu Asn Ala Glu Ile Leu Ala Arg Met Gly Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Tyr Ala Val Asn Leu Leu
165 170 175
Asn Glu Ala Ser Val Glu Val Gly Gly Pro Glu Asn Ile Ala Val Thr
180 185 190
Val Glu His Pro Thr Met Glu Thr Ser Asp Val Met Met Lys His Lys
195 200 205
Asp Ile His Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Leu His Tyr
275 280 285
Met Val Asn Glu Gln Gly Cys Tyr Met Ile Ser Lys Glu Glu Gln Asp
290 295 300
Ala Leu Thr Glu Val Val Leu Lys Gly Gly Arg Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Ile Thr Val
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Thr Asp Ser Leu Cys Ile Arg
450 455 460
<210> 81
<211> 462
<212> PRT
<213> 活泼瘤胃球菌（Ruminococcus gnavus）
<400> 81
Met Ser Val Asn Glu Lys Met Val Gln Asp Ile Val Gln Glu Val Val
1 5 10 15
Ala Lys Met Gln Ile Ser Ser Asp Val Ser Gly Lys Lys Gly Val Phe
20 25 30
Ser Asp Met Asn Glu Ala Ile Glu Ala Ser Lys Lys Ala Gln Lys Ile
35 40 45
Val Ala Lys Met Ser Met Asp Gln Arg Glu Ala Ile Ile Ser Lys Ile
50 55 60
Arg Glu Lys Ile Lys Glu Asn Ala Glu Ile Leu Ala Arg Met Gly Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Tyr Ala Val Asn Leu Leu
165 170 175
Asn Glu Ala Ser Val Glu Val Gly Gly Pro Glu Asn Ile Ala Val Thr
180 185 190
Val Glu His Pro Thr Met Glu Thr Ser Asp Ile Met Met Lys His Lys
195 200 205
Asp Ile His Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Leu His Tyr
275 280 285
Met Val Ser Glu Gln Gly Cys Tyr Met Ile Ser Lys Glu Glu Gln Asp
290 295 300
Ala Leu Thr Glu Val Val Leu Lys Gly Gly Arg Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Ile Thr Val
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Thr Asp Ser Leu Cys Ile Arg
450 455 460
<210> 82
<211> 462
<212> PRT
<213> 卵形瘤胃球菌（Ruminococcus obeum）
<400> 82
Met Pro Ile Ser Glu Ser Met Val Gln Asp Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Asp Ala Pro Thr Gly Lys His Gly Val Phe
20 25 30
Lys Glu Met Asn Asp Ala Ile Glu Ala Ala Lys Lys Ala Glu Leu Ile
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Thr Cys Ile
50 55 60
Arg Lys Lys Ile Lys Glu Asn Ala Glu Val Met Ala Arg Met Gly Val
65 70 75 80
Asp Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His His
85 90 95
Leu Val Ala Asp Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Phe Ala Ile Asn Leu Val
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Val Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Thr Ser Asn Ile Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Val Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Gln Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Val Val Ala Val Ser Ser Val Val Asp Glu Leu Met His Tyr
275 280 285
Met Leu Thr Glu Asn Asp Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Lys Leu Thr Glu Val Val Leu Ala Gly Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Ser Met Ile Gly Val Asn Ala
325 330 335
Pro Ala Asn Thr Arg Cys Ile Ile Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Arg Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Ile Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ser Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 83
<211> 473
<212> PRT
<213> 解糖梭菌（Clostridium saccharolyticum）
<400> 83
Met Glu Ile Gly Ala Lys Glu Ile Glu Leu Ile Val Arg Glu Val Leu
1 5 10 15
Ala Gly Ile Glu Ser Arg Gly Pro Lys Leu Ser Tyr Ile Pro Ala Gln
20 25 30
Ser Asp Asn Gly Val Phe Glu Arg Val Glu Asp Ala Ile Gly Ala Ala
35 40 45
His Thr Ala Gln Arg Glu Trp Val Glu His Tyr Arg Val Glu Asp Arg
50 55 60
Arg Arg Ile Ile Glu Ala Ile Arg Met Thr Ala Lys Ser His Ala Lys
65 70 75 80
Thr Leu Ala Lys Leu Val Trp Glu Glu Thr Gly Met Gly Arg Phe Glu
85 90 95
Asp Lys Ile Gln Lys His Met Ala Val Ile Glu Lys Thr Pro Gly Val
100 105 110
Glu Cys Leu Thr Thr Asp Ala Ile Ser Gly Asp Glu Gly Leu Met Ile
115 120 125
Glu Glu Tyr Ala Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Ser Thr
130 135 140
Asn Pro Thr Glu Thr Ile Ile Asn Asn Thr Ile Ser Met Ile Ala Gly
145 150 155 160
Gly Asn Ala Val Val Phe Asn Val His Pro Gly Ala Lys Lys Cys Cys
165 170 175
Ala His Cys Leu Lys Leu Leu His Gln Ala Ile Val Glu Asn Gly Gly
180 185 190
Pro Ala Asn Leu Ile Thr Met Gln Lys Glu Pro Thr Met Glu Ala Val
195 200 205
Thr Lys Met Thr Ser Asp Pro Arg Ile Arg Leu Met Val Gly Thr Gly
210 215 220
Gly Met Pro Met Val Asn Ala Leu Leu Arg Ser Gly Lys Lys Thr Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Ser Ala Asp
245 250 255
Val Ser Leu Ala Ala Arg Glu Ile Tyr Arg Gly Ala Ser Phe Asp Asn
260 265 270
Asn Ile Leu Cys Leu Ala Glu Lys Glu Val Phe Val Met Glu Lys Ala
275 280 285
Ala Asp Glu Leu Val Asn Asn Leu Val Lys Glu Gly Ala Tyr Leu Leu
290 295 300
Asn Pro Met Glu Leu Asn Glu Ile Leu Lys Phe Ala Met Ile Glu Lys
305 310 315 320
Asn Gly Ser Cys Glu Val Asn Lys Lys Trp Val Gly Lys Asp Ala Gly
325 330 335
Leu Phe Leu Glu Ala Ile Gly Val Ser Gly His Lys Asp Val Arg Leu
340 345 350
Leu Ile Cys Glu Thr Asp Arg Asn His Pro Phe Val Met Val Glu Gln
355 360 365
Leu Met Pro Ile Leu Pro Ile Val Arg Leu Arg Thr Phe Glu Glu Cys
370 375 380
Val Glu Ser Ala Val Ala Ala Glu Ser Gly Asn Arg His Thr Ala Ser
385 390 395 400
Met Phe Ser Arg Asn Val Glu Asn Met Thr Arg Phe Gly Lys Val Ile
405 410 415
Glu Thr Thr Ile Phe Thr Lys Asn Gly Ser Thr Leu Lys Gly Val Gly
420 425 430
Ile Gly Gly Glu Gly His Thr Thr Met Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Leu Thr Cys Ala Arg Ser Phe Thr Arg Arg Arg Arg Cys Met
450 455 460
Leu Ala Glu Gly Gly Leu Arg Ile Ile
465 470
<210> 84
<211> 471
<212> PRT
<213> 普氏梭杆菌（Flavonifractor plautii）
<400> 84
Met Asn Ile Asp Glu Asn Val Val Glu Ser Ile Val Lys Arg Val Val
1 5 10 15
Ser Gln Leu Ser Thr Glu Thr Ala Ser Ala Gln Thr Cys Pro Ser Gly
20 25 30
Gly Asp Trp Gly Val Phe Glu Ser Met Asn Asp Ala Val Asp Ala Ala
35 40 45
Val Glu Ala Gln Arg Glu Tyr Leu Asn Arg Ser Met His Asp Arg Ala
50 55 60
Cys Tyr Val Gln Ala Ile Arg Asp Val Val Leu Asp Gln Glu Asn Leu
65 70 75 80
Glu Tyr Ile Ser Arg Leu Ala Val Glu Glu Thr Gly Met Gly Gly Tyr
85 90 95
Glu Tyr Lys Leu Ile Lys Asn Arg Leu Ala Ala Val Lys Thr Pro Gly
100 105 110
Ile Glu Asp Leu Thr Thr Asp Ala Met Ser Gly Asp Asp Gly Leu Thr
115 120 125
Leu Val Glu Tyr Ser Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr
130 135 140
Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Gly Met Leu Ala
145 150 155 160
Ala Gly Asn Ala Val Val Phe Ser Pro His Pro Arg Ala Lys Lys Val
165 170 175
Ser Leu His Leu Ile Gln Leu Ile Asn Lys Ala Leu Cys Lys Ala Gly
180 185 190
Ala Pro Ala Asn Leu Val Val Thr Val Ser Ala Pro Ser Ile Glu Asn
195 200 205
Thr Asn Ala Met Met Ser His Pro Lys Ile Arg Met Leu Val Ala Thr
210 215 220
Gly Gly Pro Ala Ile Val Lys Thr Val Leu Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Lys Asp Ile Val Asp Gly Cys Ser Phe Asp
260 265 270
Asn Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Ile Ala Val Asp Ser
275 280 285
Val Ala Asp Tyr Leu Ile Phe Asn Met Lys Lys Asn Gly Ala Tyr Glu
290 295 300
Val Lys Asp Pro Ala Val Ile Ser Gln Leu Val Glu Leu Val Thr Lys
305 310 315 320
Glu Gly Lys Ser Pro Lys Thr Glu Phe Val Gly Lys Ser Ala Lys Tyr
325 330 335
Ile Leu Asp Lys Ile Gly Ile Thr Val Gly Asp Asp Val Lys Val Ile
340 345 350
Leu Met Glu Ala Lys Glu Asp His Pro Phe Val Gln Val Glu Leu Met
355 360 365
Met Pro Ile Leu Pro Leu Val Arg Val Pro Asp Val Asp Gln Ala Ile
370 375 380
Glu Met Ala Val Arg Val Glu His Gly Asn Arg His Thr Ala Met Met
385 390 395 400
His Ser Arg Asn Val Glu Lys Leu Thr Lys Met Ala Lys Leu Ile Gln
405 410 415
Thr Thr Ile Phe Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Val
420 425 430
Gly Gly Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu
435 440 445
Gly Leu Thr Ser Ala Lys Ser Phe Ala Arg Arg Arg Arg Cys Val Leu
450 455 460
Val Gly Gly Met Asp Val Arg
465 470
<210> 85
<211> 462
<212> PRT
<213> 卵形瘤胃球菌（Ruminococcus obeum）
<400> 85
Met Pro Ile Ser Glu Ser Met Val Gln Asp Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Asp Ala Pro Thr Gly Lys His Gly Ile Phe
20 25 30
Lys Asp Met Asn Asp Ala Ile Glu Ala Ala Lys Lys Ser Glu Leu Ile
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Thr Cys Ile
50 55 60
Arg Lys Lys Ile Lys Glu Asn Ala Glu Val Met Ala Arg Met Gly Val
65 70 75 80
Asp Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His His
85 90 95
Leu Val Ala Asp Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Leu
130 135 140
Cys Asn Thr Met Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Phe Ala Ile Asn Leu Val
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Val Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Thr Ser Asn Ile Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Val Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Gln Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Val Val Ala Val Ser Ser Val Val Asp Glu Leu Met His Tyr
275 280 285
Met Leu Thr Glu Asn Asp Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Lys Leu Thr Glu Val Val Leu Ala Gly Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Arg Thr Leu Leu Ser Met Ile Gly Val Asn Ala
325 330 335
Pro Ala Asn Ile Arg Cys Ile Ile Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Ile Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ser Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 86
<211> 469
<212> PRT
<213> 食一氧化碳梭菌（Clostridium carboxidivorans）
<400> 86
Met Glu Leu Gln Ser Asn Glu Leu Ser Leu Ile Ile Glu Lys Val Leu
1 5 10 15
Lys Glu Met Asn Lys Lys Glu Leu Lys Glu Glu Val Ser Asp Gly Val
20 25 30
Phe Asp Thr Met Glu Glu Ala Ile Glu Ala Ala Tyr Glu Ala Gln Lys
35 40 45
Lys Phe Ser Ser Tyr Thr Ile Glu Gln Arg Glu Lys Leu Ile Ala Ala
50 55 60
Met Arg Lys Ala Ile Ile Asp Asn Ala Met Glu Ile Ala Asn Leu Cys
65 70 75 80
Val Asn Glu Ser Gly Met Gly Arg Val Asp His Lys Tyr Leu Lys Leu
85 90 95
Lys Leu Thr Ala Glu Lys Thr Pro Gly Thr Glu Val Leu Gln Thr Thr
100 105 110
Ala Phe Thr Gly Asp Lys Gly Leu Thr Leu Val Glu Asn Gly Ala Phe
115 120 125
Gly Val Ile Gly Ser Ile Thr Pro Ser Thr Asn Pro Ala Ala Thr Val
130 135 140
Ala Cys Asn Gly Ile Gly Met Leu Ala Gly Gly Asn Thr Ala Val Phe
145 150 155 160
Ser Pro His Pro Gly Ala Phe Arg Ser Ser Leu Ala Met Leu Arg Ala
165 170 175
Leu Asn Lys Ala Ile Lys Glu Ala Gly Gly Pro Asp Asn Leu Leu Thr
180 185 190
Ser Val Lys Lys Pro Ser Ile Glu Ser Thr Asn Ser Met Met Lys Asn
195 200 205
Asp Lys Ile Arg Met Val Val Ala Thr Gly Gly Pro Gly Ile Val Lys
210 215 220
Met Val Leu Ser Ser Gly Arg Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Lys Lys Ala Ala Arg
245 250 255
Asp Ile Ile Ala Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala
260 265 270
Glu Lys Glu Ala Leu Val Val Glu Ala Val Tyr Glu Glu Leu Ile Lys
275 280 285
Glu Met Lys Asn Asn Arg Ala Val Tyr Glu Leu Asn Asp Glu Glu Ala
290 295 300
Ala Lys Val Ala Glu Leu Val Leu Val His Asn Lys Glu Lys Asn Thr
305 310 315 320
Tyr Ser Ile Asn Lys Ala Phe Val Gly Lys Asp Ala Lys Tyr Ile Leu
325 330 335
Gln Asn Ile Gly Lys Asn Asp Ala Glu Gly Val Glu Cys Leu Ile Tyr
340 345 350
Arg Ala Glu Asn Ser His Pro Phe Val Gln Glu Glu Leu Met Met Pro
355 360 365
Ile Leu Pro Ile Val Lys Thr Lys Asp Phe Glu Glu Ala Leu Lys Leu
370 375 380
Ala Val Gln Asp Glu His Gly Asn Arg His Thr Ala Ile Met His Ser
385 390 395 400
Lys Asn Val Asp Asn Leu Thr Lys Met Ala Arg Ala Ile Asp Thr Thr
405 410 415
Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly
420 425 430
Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu
435 440 445
Thr Asn Ala Val Ser Phe Thr Arg Lys Arg Arg Cys Thr Met Ala Glu
450 455 460
Ser Phe Arg Ile Val
465
<210> 87
<211> 469
<212> PRT
<213> 溃疡梭杆菌（Fusobacterium ulcerans）
<400> 87
Met Asn Leu Glu Ala Asn Asn Met Asp Glu Ile Val Ala Leu Ile Met
1 5 10 15
Lys Glu Leu Lys Lys Thr Asp Ile Lys Ala Gly Cys Gln Ser Cys Glu
20 25 30
Ser Pro Lys Asn Gly Val Phe Ser Ser Met Asp Glu Ala Ile Ala Ala
35 40 45
Ala Lys Lys Ala Gln Glu Ile Leu Phe Ser Ser Arg Leu Glu Met Arg
50 55 60
Glu Lys Ile Val Ala Ser Ile Arg Glu Val Met Lys Asp Tyr Val Val
65 70 75 80
Glu Leu Ala Glu Leu Gly Val Lys Glu Thr Gly Met Gly Arg Ala Ala
85 90 95
Asp Lys Ala Leu Lys His Gln Val Thr Ile Glu Lys Thr Pro Gly Val
100 105 110
Glu Asp Leu Arg Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val
115 120 125
Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr
130 135 140
Asn Pro Ser Glu Thr Ile Ile Cys Asn Ser Ile Gly Met Ile Ser Ala
145 150 155 160
Gly Asn Ser Val Val Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser
165 170 175
Ile Lys Thr Val Glu Ile Ile Asn Glu Ala Val Arg Lys Ala Gly Gly
180 185 190
Pro Glu Asn Leu Val Val Thr Ile Ala Glu Pro Ser Ile Glu Asn Thr
195 200 205
Asn Arg Met Met Glu Asn Pro Asp Ile Lys Met Leu Val Ala Thr Gly
210 215 220
Gly Pro Gly Val Val Lys Ser Val Met Ser Ser Gly Lys Lys Ala Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp
245 250 255
Ile Glu Lys Ala Ala Arg Asp Ile Val Ala Gly Cys Ser Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Val Ala Val Asp Ser Ile
275 280 285
Thr Asp Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Ile
290 295 300
Lys Asp Lys Ser Val Ile Asp Arg Leu Val Ala Met Val Leu Lys Asn
305 310 315 320
Gly Ser Pro Asn Arg Ala Tyr Val Gly Lys Asp Ala Ser Tyr Ile Leu
325 330 335
Lys Asp Leu Gly Ile Asn Val Gly Gly Glu Ile Arg Val Ile Ile Thr
340 345 350
Glu Ala Asp Lys Asp His Pro Phe Ala Val Glu Glu Leu Leu Met Pro
355 360 365
Ile Leu Pro Ile Ile Arg Val Lys Asn Ala Leu Glu Gly Ile Glu Val
370 375 380
Ser Lys Lys Leu Glu His Gly Leu Arg His Thr Ala Met Ile His Ser
385 390 395 400
Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Asp Met Glu Thr Thr
405 410 415
Ile Leu Val Lys Asn Gly Pro Ser Phe Ala Gly Ile Gly Val Gly Gly
420 425 430
Glu Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu
435 440 445
Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly
450 455 460
Gly Leu Ser Ile Lys
465
<210> 88
<211> 469
<212> PRT
<213> 梭杆菌属（Fusobacterium sp.）
<400> 88
Met Asn Leu Glu Ala Asn Asn Met Asp Glu Ile Val Ala Leu Ile Met
1 5 10 15
Lys Glu Leu Lys Lys Thr Asp Ile Lys Ala Gly Cys Gln Ser Cys Glu
20 25 30
Ser Leu Lys Asn Gly Val Phe Ser Ser Met Asp Glu Ala Ile Ala Ala
35 40 45
Ala Lys Lys Ala Gln Glu Ile Leu Phe Ser Ser Arg Leu Glu Met Arg
50 55 60
Glu Lys Ile Val Ala Ser Ile Arg Glu Val Met Lys Asp Tyr Val Val
65 70 75 80
Glu Leu Ala Glu Leu Gly Val Lys Glu Thr Gly Met Gly Arg Ala Ala
85 90 95
Asp Lys Ala Leu Lys His Gln Val Thr Ile Glu Lys Thr Pro Gly Val
100 105 110
Glu Asp Leu Arg Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val
115 120 125
Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr
130 135 140
Asn Pro Ser Glu Thr Ile Ile Cys Asn Ser Ile Gly Met Ile Ser Ala
145 150 155 160
Gly Asn Ser Val Val Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser
165 170 175
Ile Lys Thr Val Glu Ile Ile Asn Glu Ala Val Arg Arg Ala Gly Gly
180 185 190
Pro Glu Asn Leu Val Val Thr Ile Ala Glu Pro Ser Ile Glu Asn Thr
195 200 205
Asn Arg Met Met Glu Asn Pro Asp Ile Lys Met Leu Val Ala Thr Gly
210 215 220
Gly Pro Gly Val Val Lys Ser Val Met Ser Ser Gly Lys Lys Ala Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp
245 250 255
Ile Glu Lys Ala Ala Arg Asp Ile Val Ala Gly Cys Ser Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Val Ala Val Asp Ser Ile
275 280 285
Thr Asp Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Ile
290 295 300
Lys Asp Lys Ser Val Ile Asp Arg Leu Val Ala Met Val Leu Lys Asn
305 310 315 320
Gly Ser Pro Asn Arg Ala Tyr Val Gly Lys Asp Ala Ser Tyr Ile Leu
325 330 335
Lys Asp Leu Gly Ile Asn Val Gly Asp Glu Ile Arg Val Ile Ile Thr
340 345 350
Glu Thr Asp Lys Asp His Pro Phe Ala Val Glu Glu Leu Leu Met Pro
355 360 365
Ile Leu Pro Ile Ile Arg Val Lys Asn Ala Leu Glu Gly Ile Glu Val
370 375 380
Ser Lys Lys Leu Glu His Gly Leu Arg His Thr Ala Met Ile His Ser
385 390 395 400
Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Asp Met Glu Thr Thr
405 410 415
Ile Leu Val Lys Asn Gly Pro Ser Phe Ala Gly Ile Gly Val Gly Gly
420 425 430
Glu Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu
435 440 445
Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly
450 455 460
Gly Leu Ser Ile Lys
465
<210> 89
<211> 469
<212> PRT
<213> 食一氧化碳梭菌（Clostridium carboxidivorans）
<400> 89
Met Glu Leu Glu Ser Asn Glu Leu Ser Val Ile Ile Glu Lys Val Leu
1 5 10 15
Lys Glu Met Asn Lys Lys Glu Phe Gly Lys Lys Glu Ser Asp Gly Ile
20 25 30
Phe Asp Thr Met Asp Glu Ala Val Glu Ala Ser Tyr Glu Ala Gln Lys
35 40 45
Lys Tyr Ser Ser Tyr Ser Leu Glu Gln Arg Glu Lys Leu Ile Gln Ala
50 55 60
Met Arg Lys Ala Ile Met Asp Asn Ala Met Glu Val Ala Asn Leu Cys
65 70 75 80
Val Lys Glu Ser Gly Met Gly Arg Val Asp His Lys Tyr Leu Lys Leu
85 90 95
Lys Leu Ile Val Glu Lys Thr Gln Gly Thr Glu Ile Leu Arg Pro Glu
100 105 110
Val Tyr Thr Gly Asp Asn Gly Leu Thr Leu Ile Glu His Gly Ala Phe
115 120 125
Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn Pro Ala Ala Thr Val
130 135 140
Ala Cys Asn Ser Ile Cys Met Leu Ala Gly Gly Asn Thr Val Val Phe
145 150 155 160
Ser Pro His Pro Gly Ala Leu Asn Ser Cys Leu Thr Met Ile Arg Ile
165 170 175
Leu Asn Lys Ala Ile Lys Glu Ala Gly Gly Pro Glu Asn Leu Ile Thr
180 185 190
Ser Val Lys Ala Pro Ser Ile Glu Asn Thr Asn Ile Met Ile Asn His
195 200 205
Lys Arg Ile Arg Leu Val Val Ala Thr Gly Gly Pro Gly Ile Val Lys
210 215 220
Leu Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Pro Lys Ala Ala Arg
245 250 255
Asp Ile Ile Ala Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile Ala
260 265 270
Glu Lys Glu Ala Ile Val Val Glu Ser Val Tyr Glu Glu Leu Ile Lys
275 280 285
Glu Phe Lys Lys Asn Arg Val Val Tyr Glu Leu Thr Asp Glu Glu Ala
290 295 300
Glu Lys Leu Val Gly Lys Val Leu Asn Tyr Asp Glu Lys Asn Lys Lys
305 310 315 320
Tyr Ser Ile Asn Lys Lys Phe Val Gly Lys Asp Ala Lys Tyr Leu Leu
325 330 335
Glu Ser Ile Gly Lys Asp Ala Gly Thr Gly Val Glu Cys Leu Ile Tyr
340 345 350
Arg Ala Glu Asn Ser His Pro Phe Val Gln Glu Glu Leu Met Met Pro
355 360 365
Ile Leu Pro Ile Val Lys Val Lys Asn Val Asp Glu Ala Ile Glu Thr
370 375 380
Ala Val Glu Asp Glu His Gly Asn Arg His Thr Ala Met Met His Ser
385 390 395 400
Lys Asn Val Val Asn Leu Thr Lys Met Ala Arg Ala Ile Asp Thr Thr
405 410 415
Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly
420 425 430
Glu Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile
435 440 445
Thr Asn Ala Val Thr Phe Thr Arg Gln Arg Arg Cys Thr Met Val Asp
450 455 460
Ser Phe Arg Ile Val
465
<210> 90
<211> 471
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 90
Met Glu Met Asp Met Lys Val Ile Glu Gln Leu Val Ala Gln Ala Leu
1 5 10 15
Lys Glu Met Lys Ala Glu Glu Pro Ala Ala Phe Ala Glu Lys Lys Glu
20 25 30
Glu Asn Tyr Gly Val Phe Ser Thr Met Asp Glu Ala Ile Glu Ala Ser
35 40 45
Glu Lys Ala Gln Lys Ala Leu Leu Phe Ser Lys Ile Gln Asp Arg Gln
50 55 60
Lys Tyr Val Asp Ile Ile Arg Ala Ala Ile Leu Lys Arg Glu Asn Leu
65 70 75 80
Glu Leu Ile Ser Arg Met Ala Val Glu Glu Thr Glu Ile Gly Lys Tyr
85 90 95
Glu His Lys Leu Ile Lys Asn Arg Leu Ala Ala Glu Lys Thr Pro Gly
100 105 110
Thr Glu Asp Leu Thr Thr Glu Ala Gln Thr Gly Asp His Gly Leu Thr
115 120 125
Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Thr
130 135 140
Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Ser Met Ile Ala
145 150 155 160
Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Lys Val
165 170 175
Ser Gln Leu Leu Val Lys Met Leu Asn Lys Ala Leu Met Glu Gly Gly
180 185 190
Ala Pro Ala Asn Leu Ile Thr Met Val Glu Glu Pro Ser Ile Glu Asn
195 200 205
Thr Asn Lys Met Ile Glu His Pro Gly Val Arg Leu Leu Val Ala Thr
210 215 220
Gly Gly Pro Ala Ile Val Lys Lys Val Leu Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala
245 250 255
Asp Ile Glu Lys Ala Ala Arg Asp Ile Val Asp Gly Cys Ser Phe Asp
260 265 270
Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser
275 280 285
Ile Cys Asp Tyr Leu Ile Gln Asn Met Lys Leu Asn Gly Ala Tyr Glu
290 295 300
Ile Arg Asp Ala Glu Thr Ile Glu Arg Leu Asp Ala Leu Val Thr Asn
305 310 315 320
Glu Lys Gly Gly Pro Lys Thr Ser Phe Val Gly Lys Ser Ala Lys Tyr
325 330 335
Ile Leu Asp Lys Met Gly Ile Pro Ala Asp Asp Ser Val Lys Val Ile
340 345 350
Ile Met Glu Val Arg Arg Asp His His Leu Val Thr Glu Glu Met Met
355 360 365
Met Pro Ile Leu Pro Ile Val Arg Val Ser Asp Val Asp Thr Ala Ile
370 375 380
Glu Tyr Ala His Asp Ala Glu His Gly Asn Arg His Thr Ala Met Met
385 390 395 400
His Ser Lys Asn Val Glu Lys Leu Ser Lys Met Ala Lys Leu Leu Glu
405 410 415
Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Ala
420 425 430
Gly Gly Glu Gly His Ala Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu
435 440 445
Gly Leu Thr Ser Ala Arg Ser Phe Cys Arg Lys Arg Arg Cys Val Met
450 455 460
Ser Asp Ala Phe Ser Ile Arg
465 470
<210> 91
<211> 469
<212> PRT
<213> 可变梭杆菌（Fusobacterium varium）
<400> 91
Met Asn Leu Glu Ala Asn Asn Met Asp Glu Ile Val Ala Leu Ile Met
1 5 10 15
Lys Glu Leu Lys Lys Thr Asp Ile Lys Thr Val Cys Gln Ser Cys Glu
20 25 30
Asn Pro Lys Asn Gly Val Phe Ser Ser Met Asp Glu Ala Ile Thr Ala
35 40 45
Ala Lys Lys Ala Gln Glu Ile Leu Phe Ser Ser Arg Leu Glu Met Arg
50 55 60
Glu Lys Ile Val Ala Ser Ile Arg Glu Val Met Lys Asp Tyr Val Leu
65 70 75 80
Glu Leu Ala Glu Leu Gly Val Lys Glu Thr Gly Met Gly Arg Val Ala
85 90 95
Asp Lys Ala Leu Lys His Gln Val Thr Ile Glu Lys Thr Pro Gly Val
100 105 110
Glu Asp Leu Lys Ala Phe Ala Phe Ser Gly Asp Asp Gly Leu Thr Val
115 120 125
Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr
130 135 140
Asn Pro Ser Glu Thr Ile Ile Cys Asn Ser Ile Gly Met Ile Ser Ala
145 150 155 160
Gly Asn Ser Ile Val Phe Ala Pro His Pro Gly Ala Lys Arg Thr Ser
165 170 175
Ile Lys Thr Val Glu Ile Ile Asn Glu Ala Val Arg Lys Val Gly Gly
180 185 190
Pro Glu Asn Leu Val Val Thr Ile Ala Glu Pro Ser Ile Glu Asn Thr
195 200 205
Asn Lys Met Met Ala Asn Pro Asp Ile Lys Met Leu Val Ala Thr Gly
210 215 220
Gly Pro Gly Val Val Lys Ser Val Met Ser Ser Gly Lys Lys Ala Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu Thr Ala Asp
245 250 255
Ile Glu Lys Ala Ala Lys Asp Ile Val Ala Gly Cys Ser Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Val Ala Val Asp Ser Ile
275 280 285
Thr Asp Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala Tyr Leu Ile
290 295 300
Lys Asp Lys Ala Val Ile Glu Arg Leu Ala Gly Met Val Leu Lys Asn
305 310 315 320
Gly Ser Pro Asn Arg Ala Tyr Val Gly Lys Asp Ala Ser Tyr Ile Leu
325 330 335
Lys Asp Leu Gly Ile Asn Val Gly Asp Glu Ile Arg Val Ile Ile Ala
340 345 350
Glu Thr Asp Lys Glu His Pro Phe Ala Val Glu Glu Leu Leu Met Pro
355 360 365
Ile Leu Pro Ile Ile Arg Val Lys Asn Ala Leu Glu Gly Ile Glu Val
370 375 380
Ser Lys Lys Leu Glu His Gly Leu Arg His Thr Ala Met Ile His Ser
385 390 395 400
Lys Asn Ile Asp Val Leu Thr Lys Tyr Ala Arg Asp Met Glu Thr Thr
405 410 415
Ile Leu Val Lys Asn Gly Pro Ser Phe Ala Gly Ile Gly Val Gly Gly
420 425 430
Glu Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu
435 440 445
Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Val Leu Val Gly
450 455 460
Gly Leu Ser Ile Lys
465
<210> 92
<211> 482
<212> PRT
<213> 隐藏梭菌（Clostridium celatum）
<400> 92
Met Asp Asp Asn Thr Lys Leu Ile Gln Asp Ile Val Ala Lys Val Ile
1 5 10 15
Ser Glu Ile Gly Thr Lys Glu Ile Glu Glu Glu Ala Cys Cys Gly Asn
20 25 30
Gly Ser Cys Gly Gly Ser Cys Gly Cys Asn Lys Glu Lys Tyr Val Phe
35 40 45
Glu Asp Val Asp Ser Ala Val Ala Ala Ala Lys Lys Ala Tyr Lys Glu
50 55 60
Leu Lys Gln Leu Thr Ile Lys Asp Arg Glu Asn Ile Ile Thr Lys Ile
65 70 75 80
Arg Glu Lys Cys Leu Thr Tyr Ser Glu Arg Leu Ser Ile Met Ala Val
85 90 95
Asp Glu Thr Gly Met Gly Lys Val Glu Asp Lys Ile Thr Lys His Val
100 105 110
Leu Val Ala Arg Lys Thr Pro Gly Thr Glu Asp Leu Thr Thr Thr Ala
115 120 125
Trp Ser Gly Asp Gly Gly Leu Thr Leu Val Glu Arg Gly Ala Phe Gly
130 135 140
Val Ile Ala Ala Ile Thr Pro Ser Thr Asn Pro Thr Ala Thr Ile Phe
145 150 155 160
Cys Asn Ser Ile Gly Met Ile Ala Ala Gly Asn Ser Val Val Phe Ala
165 170 175
Pro His Pro Ala Ala Lys Ser Cys Ser Lys Phe Ala Val Lys Leu Ile
180 185 190
Asn Glu Ala Ser Ile Glu Val Gly Gly Pro Glu Asn Ile Val Val Thr
195 200 205
Phe Glu Asn Pro Ser Ile Glu Ile Thr Ser Ala Leu Met Lys His Lys
210 215 220
Asp Ile Pro Phe Ile Ser Ala Thr Gly Gly Pro Gly Val Val Thr Gln
225 230 235 240
Ala Cys Ser Ser Gly Lys Arg Ala Ile Gly Ala Gly Ala Gly Asn Pro
245 250 255
Pro Val Leu Val Asp Glu Thr Ala Asp Ile Lys His Ala Ala Lys Ser
260 265 270
Ile Ile Ala Gly Ala Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
275 280 285
Lys Glu Val Val Ala Leu Asp Ser Ile Cys Asp Glu Leu Ile Glu Asp
290 295 300
Met Gln Lys Glu Gly Ala Tyr Phe Leu Asn Ser Thr Glu Leu Ile Asn
305 310 315 320
Arg Leu Ile Asp Thr Val Leu Ile Arg Lys Asp Gly Lys Val Thr Leu
325 330 335
Asn Arg Asn Phe Val Gly Arg Asp Ala Lys Ile Ile Leu Asp Ala Ile
340 345 350
Gly Val Tyr Ala Asp Asp Ser Val Lys Cys Ile Ile Phe Glu Gly Cys
355 360 365
Lys Ser Asn Leu Leu Ile Val Glu Glu Leu Met Met Pro Ile Leu Gly
370 375 380
Ile Val Arg Val Lys Asp Phe Asn Thr Ala Val Asp Val Ala Val Glu
385 390 395 400
Leu Glu His Gly Asn Arg His Ser Ala His Ile His Ser Lys Arg Ile
405 410 415
Asp Arg Leu Thr Tyr Phe Ala Arg Glu Ile Asp Thr Ala Ile Phe Val
420 425 430
Lys Asn Ala Pro Ser Tyr Ser Ala Leu Gly Val Glu Ala Glu Gly Tyr
435 440 445
Pro Thr Phe Thr Ile Ala Ser Arg Thr Gly Glu Gly Leu Ser Ser Ala
450 455 460
Lys Thr Phe Ser Lys Ser Arg Arg Cys Ile Met Lys Asp Ala Leu Ser
465 470 475 480
Ile Lys
<210> 93
<211> 462
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 93
Met Ser Val Asn Glu Arg Met Val Gln Asp Ile Val Gln Glu Val Val
1 5 10 15
Ala Lys Met Gln Ile Ala Ser Asp Val Thr Gly Asn His Gly Val Phe
20 25 30
Gln Asp Met Asn Ala Ala Ile Glu Ala Ala Lys Lys Thr Gln Lys Val
35 40 45
Val Ala Arg Met Ser Met Asp Gln Arg Glu Lys Ile Ile Ser Asn Ile
50 55 60
Arg Ala Lys Ile Lys Glu His Ala Glu Ile Phe Ala Arg Met Gly Val
65 70 75 80
Gln Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Gln Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Tyr Ala Val Asn Leu Ile
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr
180 185 190
Val Glu Asn Pro Thr Leu Glu Ser Ser Asn Ile Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Met His Tyr
275 280 285
Met Ile Ser Glu Gln Gly Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Ala Leu Thr Glu Val Val Leu Lys Gly Gly Arg Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Val Thr Val
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Ala Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Cys Asp Ser Leu Cys Ile Arg
450 455 460
<210> 94
<211> 462
<212> PRT
<213> 毛螺科菌（Lachnospiraceae bacterium）
<400> 94
Met Ser Val Asn Glu Gln Met Val Gln Asp Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Thr Ser Asp Val Ser Gly Ser His Gly Val Phe
20 25 30
Lys Asp Met Asn Glu Ala Ile Ala Ala Ala Lys Lys Thr Gln Lys Ile
35 40 45
Val Gly Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Ser Asn Ile
50 55 60
Arg Thr Lys Ile Lys Glu Asn Ala Glu Ile Met Ala Arg Met Gly Val
65 70 75 80
Gln Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Val
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu
130 135 140
Cys Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Phe Ala Ile Asn Leu Leu
165 170 175
Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr
180 185 190
Val Glu Lys Pro Thr Leu Ala Ser Ser Asp Ile Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Met Tyr Tyr
275 280 285
Met Val Ser Glu Gln Gly Cys Tyr Lys Ile Thr Lys Glu Glu Gln Asp
290 295 300
Ala Leu Thr Ala Val Val Leu Lys Asp Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Val Thr Val
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Lys Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile Thr
385 390 395 400
Thr Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Thr Asp Ser Leu Cys Ile Arg
450 455 460
<210> 95
<211> 463
<212> PRT
<213> 毛螺科菌（Lachnospiraceae bacterium）
<400> 95
Met Pro Val Ser Glu Ser Met Val Gln Asp Ile Val Lys Glu Val Val
1 5 10 15
Ala Arg Met Gln Leu Ser Gly Ser Ala Gly Thr Ala Gln His Gly Val
20 25 30
Phe Thr Asp Met Asn Gln Ala Ile Glu Ala Ala Lys Glu Ala Glu Ala
35 40 45
Lys Val Arg Cys Met Thr Met Asp Gln Arg Glu Gln Ile Val Ser Asn
50 55 60
Ile Arg Arg Lys Thr His Glu Asn Ala Glu Leu Leu Ala Arg Met Gly
65 70 75 80
Val Glu Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His
85 90 95
His Leu Leu Ala Asp Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Thr
100 105 110
Ala Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe
115 120 125
Gly Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val
130 135 140
Leu Cys Asn Ser Met Gly Met Ile Ala Ala Gly Asn Thr Val Val Phe
145 150 155 160
Asn Pro His Pro Gln Ala Ile Lys Thr Ser Ile Phe Ala Ile Asn Met
165 170 175
Val Asn Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Val Ala Cys
180 185 190
Thr Val Ser Lys Pro Thr Leu Glu Thr Ser Asn Ile Met Met Lys His
195 200 205
Lys Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr
210 215 220
Ala Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn
225 230 235 240
Pro Pro Ala Leu Val Asp Glu Thr Ala Asp Val Arg Lys Ala Ala Ala
245 250 255
Asp Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala
260 265 270
Glu Lys Glu Ile Val Ala Val Asp Ser Val Ala Asp Glu Leu Met Asn
275 280 285
Tyr Met Ile Ser Glu Gln Gly Cys Tyr Leu Ile Ser Lys Glu Glu Gln
290 295 300
Asp Lys Leu Thr Ala Thr Val Ile Thr Pro Lys Gly Leu Asn Arg Lys
305 310 315 320
Cys Val Gly Arg Asp Ala Arg Thr Leu Leu Ser Met Ile Gly Ile Gln
325 330 335
Ala Pro Glu Asn Ile Arg Cys Ile Val Phe Glu Gly Glu Lys Glu His
340 345 350
Pro Leu Ile Ala Glu Glu Leu Met Met Pro Ile Leu Gly Leu Val Arg
355 360 365
Ala Lys Asp Phe Asp Asp Ala Val Glu Lys Ala Val Trp Leu Glu His
370 375 380
Gly Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp Asn Ile
385 390 395 400
Thr Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Ala
405 410 415
Pro Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe
420 425 430
Thr Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Thr Phe
435 440 445
Thr Lys Arg Arg Arg Cys Val Met Ser Asp Ser Leu Cys Ile Arg
450 455 460
<210> 96
<211> 462
<212> PRT
<213> 瘤胃球菌属（Ruminococcus sp.）
<400> 96
Met Pro Ile Asn Glu Asn Met Val Gln Glu Ile Val Gln Glu Val Met
1 5 10 15
Ala Lys Met Gln Ile Ala Asp Ala Pro Thr Gly Lys His Gly Ile Phe
20 25 30
Lys Glu Met Asn Asp Ala Ile Glu Ala Ala Lys Lys Ser Gln Leu Ile
35 40 45
Val Lys Lys Met Ser Met Asp Gln Arg Glu Lys Ile Ile Thr Cys Ile
50 55 60
Arg Lys Lys Ile Lys Glu Asn Ala Glu Val Met Ala Arg Met Gly Val
65 70 75 80
Glu Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His His
85 90 95
Leu Val Ala Asp Lys Thr Pro Gly Thr Glu Val Ile Thr Thr Thr Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Leu
130 135 140
Cys Asn Thr Met Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Tyr Ala Ile Asn Leu Leu
165 170 175
Asn Glu Ala Ser Leu Glu Ser Gly Gly Pro Asp Asn Ile Ala Val Thr
180 185 190
Val Glu Lys Pro Thr Leu Glu Thr Ser Asn Val Met Met Lys His Lys
195 200 205
Asp Ile Pro Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Thr Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Ser Ser Ile Val Asp Glu Leu Met His Tyr
275 280 285
Leu Val Thr Glu Asn Asp Cys Tyr Leu Ala Ser Lys Glu Glu Gln Asp
290 295 300
Lys Leu Thr Glu Val Val Leu Ala Gly Gly Lys Leu Asn Arg Lys Cys
305 310 315 320
Val Gly Arg Asp Ala Arg Thr Leu Leu Ser Met Ile Gly Val Asn Ala
325 330 335
Pro Ala Asn Ile Arg Cys Ile Val Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Arg Asp Phe Asp Asp Ala Val Glu Gln Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Ile Asp Asn Ile Thr
385 390 395 400
Lys Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Ala Pro
405 410 415
Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Cys Ala Ser Thr Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Ala Asp Ser Leu Cys Ile Arg
450 455 460
<210> 97
<211> 469
<212> PRT
<213> 伍迪乙酸梭菌（Acetobacterium woodii）
<400> 97
Met Asn Ile Asp Thr Thr Gly Ile Glu Tyr Ile Val Lys Lys Val Met
1 5 10 15
Ala Glu Ile Asp Cys Ala Glu Glu Gly Gly Lys Pro Leu Lys Asp Gly
20 25 30
Glu Leu Gly Ile Phe Asn Asp Met Glu Asn Ala Ile Asp Ala Ala Phe
35 40 45
Ile Ala Gln Lys Ser Phe Met Arg Ala Ser Met Ala Phe Arg Ser Lys
50 55 60
Ile Ile Ala Ala Met Arg Ala Glu Met Leu Lys Lys Glu Asn Met Glu
65 70 75 80
Met Ile Cys Gln Met Ala Val Glu Glu Thr Gly Met Gly Asn Tyr Glu
85 90 95
His Lys Leu Leu Lys His Glu Leu Ala Ala Thr Lys Thr Pro Gly Val
100 105 110
Glu Asp Leu Val Ala Asp Ala Phe Thr Gly Asp Asp Gly Leu Thr Leu
115 120 125
Ile Glu Gln Ser Pro Phe Gly Val Ile Gly Ala Val Ser Pro Ser Thr
130 135 140
Asn Pro Ser Glu Thr Ile Ile Cys Asn Gly Ile Gly Met Leu Ala Gly
145 150 155 160
Gly Asn Thr Val Val Phe Ala Pro His Pro Ser Ala Lys Lys Thr Ser
165 170 175
Ala Leu Val Val Lys Leu Leu Asn Lys Ala Ile Leu Glu Ala Gly Gly
180 185 190
Pro Glu Asn Leu Ile Val Thr Thr Val Lys Pro Thr Ile Asp Ser Ala
195 200 205
Asn Thr Met Phe Ala Ser Pro Lys Ile Thr Met Leu Cys Ala Thr Gly
210 215 220
Gly Pro Gly Val Val Lys Ser Val Leu Gln Ser Gly Lys Lys Ala Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Ala Leu Val Asp Glu Thr Ala Asp
245 250 255
Ile Glu Lys Ala Gly Lys Asp Ile Ile Asp Gly Cys Cys Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Val Val Val Glu Gln Val
275 280 285
Ala Asp Tyr Leu Ile Phe Asn Met Lys Lys Asn Gly Ala Tyr Glu Leu
290 295 300
Lys Asp Ala Gln Lys Ile Lys Glu Leu Glu Glu Leu Val Ile Pro Gly
305 310 315 320
Gly Arg Leu Ser Arg Asp Tyr Val Gly Arg Ser Ala Lys Val Ile Leu
325 330 335
Lys Gly Ile Gly Ile Glu Val Asp Asp Ser Val Arg Val Val Ile Ile
340 345 350
Glu Thr Ser Lys Asp His Ile Phe Ala Val Glu Glu Leu Met Met Pro
355 360 365
Ile Leu Ala Ile Val Arg Val Lys Asp Val Ala Glu Gly Ile Asp Leu
370 375 380
Ala Val Ser Leu Glu His Gly Asn Arg His Thr Ala Ile Met His Ser
385 390 395 400
Thr Asn Ile Asn Asn Leu Thr Glu Met Ala Lys Arg Val Gln Thr Thr
405 410 415
Ile Phe Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly
420 425 430
Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu
435 440 445
Thr Ser Ala Lys Thr Phe Thr Arg Lys Arg Arg Cys Val Leu Val Gly
450 455 460
Gly Phe Thr Ile Lys
465
<210> 98
<211> 497
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 98
Met Asn Asp Phe Asn Met Ile Asp Ile Glu Ser Ile Val Lys Asn Ile
1 5 10 15
Val Lys Glu Leu Thr Gly Asn Glu Lys Gly Gln Gly Ala Ile Thr Thr
20 25 30
Ala Thr Ala Pro Lys Glu Ala Asn Pro Leu Val Asp Ile Glu Lys Lys
35 40 45
Ile Met Gly Phe Met Asn Thr Pro Thr Met Pro Val Gly Glu Tyr Gly
50 55 60
Val Phe Glu Asp Ile Asn Asp Ala Ile Glu Gln Ala Trp Leu Ala Glu
65 70 75 80
Gln Glu Tyr Arg Lys Val Gly Leu Asp Lys Arg Thr Glu Ile Ile Glu
85 90 95
Ala Phe Lys Ala Glu Val Arg Lys Asn Val Glu Glu Ile Ser Arg Arg
100 105 110
Thr Phe Glu Glu Thr Gly Met Gly Arg Tyr Glu Asp Lys Ile Leu Lys
115 120 125
Asn Asn Leu Ala Leu Asp Lys Thr Pro Gly Val Glu Asp Leu Glu Ala
130 135 140
Gly Val Lys Thr Gly Asp Gly Gly Leu Thr Leu Tyr Glu Met Ser Pro
145 150 155 160
Phe Gly Val Ile Gly Ala Ile Ala Pro Ser Thr Asn Pro Thr Glu Thr
165 170 175
Ile Ile Asn Asn Gly Ile Ser Met Leu Ala Gly Gly Asn Thr Val Val
180 185 190
Phe Ser Pro His Pro Gly Ala Lys Asp Val Ser Val Phe Ile Val Gln
195 200 205
Leu Ile Asn Lys Ala Ile Glu Arg Ile Asn Gly Pro Lys Asn Leu Ile
210 215 220
Val Thr Val Lys Asn Pro Asn Ile Glu Ser Thr Asn Ile Met Leu Ala
225 230 235 240
His Pro Lys Val Asn Met Ile Cys Ala Thr Gly Gly Pro Gly Ile Val
245 250 255
Lys Val Ala Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly
260 265 270
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala
275 280 285
Val Asp Ile Ile Asp Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile
290 295 300
Cys Glu Lys Glu Val Ile Val Val Asp Lys Val Ala Asp Tyr Leu Lys
305 310 315 320
Thr Cys Met Ser Lys Tyr Cys Ala Leu Glu Ile Thr Asp Lys Asn Met
325 330 335
Leu Ala Gln Leu Glu Lys Leu Val Leu Thr Glu Asn Gly Thr Ile Asn
340 345 350
Lys Gln Phe Val Gly Lys Asn Ala Asp Tyr Ile Met Ser Lys Leu Gly
355 360 365
Val Asn Ile Asp Pro Ser Ile Arg Val Ile Phe Ala Glu Val Glu Ala
370 375 380
Asn His Pro Phe Ala Val Glu Glu Leu Met Met Pro Ile Leu Pro Val
385 390 395 400
Ile Arg Val Arg Asn Val Asp Glu Ala Ile Asp Leu Gly Val Glu Leu
405 410 415
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys His Ile Asp
420 425 430
Asn Leu Ser Lys Phe Ala Lys Ala Val Gln Thr Thr Ile Phe Val Lys
435 440 445
Asn Ala Pro Ser Tyr Ala Gly Ile Gly Tyr Gly Ala Glu Gly His Gly
450 455 460
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg
465 470 475 480
Thr Phe Thr Arg Lys Arg Arg Cys Val Met Val Asp Asn Phe Ser Ile
485 490 495
Lys
<210> 99
<211> 497
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 99
Met Asn Asp Phe Asn Met Ile Asp Ile Glu Ser Ile Val Lys Asn Ile
1 5 10 15
Val Lys Glu Leu Thr Gly Asn Glu Lys Glu Gln Gly Ala Ile Ile Thr
20 25 30
Ala Thr Ala Pro Lys Glu Val Asn Pro Leu Val Asp Ile Glu Lys Lys
35 40 45
Ile Met Gly Phe Met Asn Thr Pro Thr Met Gln Ala Gly Glu Tyr Gly
50 55 60
Val Phe Glu Asp Ile Asn Asp Ala Ile Glu Gln Ala Trp Leu Ala Glu
65 70 75 80
Gln Glu Tyr Arg Lys Val Gly Leu Asp Lys Arg Thr Glu Ile Ile Glu
85 90 95
Val Phe Lys Ala Glu Val Arg Lys Asn Val Glu Glu Ile Ser Arg Arg
100 105 110
Thr Phe Glu Glu Thr Gly Met Gly Arg Tyr Glu Asp Lys Ile Leu Lys
115 120 125
Asn Asn Leu Ala Leu Asp Lys Thr Pro Gly Val Glu Asp Leu Glu Ala
130 135 140
Gly Val Lys Thr Gly Asp Gly Gly Leu Thr Leu Tyr Glu Met Ser Pro
145 150 155 160
Phe Gly Val Ile Gly Ala Ile Ala Pro Ser Thr Asn Pro Thr Glu Thr
165 170 175
Ile Ile Asn Asn Gly Ile Ser Met Leu Ala Gly Gly Asn Thr Val Val
180 185 190
Phe Ser Pro His Pro Gly Ala Lys Asp Val Ser Val Phe Ile Ile Gln
195 200 205
Leu Ile Asn Lys Ala Ile Glu Arg Val Asn Gly Pro Lys Asn Leu Ile
210 215 220
Val Thr Val Arg Asn Pro Asn Ile Glu Ser Thr Asn Ile Met Leu Ser
225 230 235 240
His Pro Lys Val Asn Met Ile Cys Ala Thr Gly Gly Pro Gly Ile Val
245 250 255
Lys Val Ala Leu Ser Ser Gly Lys Lys Ala Val Gly Ala Gly Ala Gly
260 265 270
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala
275 280 285
Val Asp Ile Ile Asp Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile
290 295 300
Cys Glu Lys Glu Val Ile Val Val Asp Lys Val Thr Asp Tyr Leu Lys
305 310 315 320
Thr Cys Met Ser Lys Tyr Cys Ala Leu Glu Ile Thr Asp Lys Asn Met
325 330 335
Leu Ala Gln Leu Glu Lys Leu Val Leu Thr Glu Asn Gly Thr Ile Asn
340 345 350
Lys Lys Phe Val Gly Lys Asn Ala Asp Tyr Ile Met Ser Lys Leu Gly
355 360 365
Ile Asn Ile Asp Pro Ser Ile Arg Val Ile Phe Ala Glu Val Gly Ala
370 375 380
Asn His Pro Phe Ala Val Glu Glu Leu Met Met Pro Ile Leu Pro Ile
385 390 395 400
Ile Arg Val Arg Asn Val Asp Glu Ala Ile Glu Leu Gly Val Glu Leu
405 410 415
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys His Ile Asp
420 425 430
Asn Leu Ser Lys Phe Ala Lys Ala Val Gln Thr Thr Ile Phe Val Lys
435 440 445
Asn Ala Pro Ser Tyr Ala Gly Ile Gly Tyr Gly Ala Glu Gly His Gly
450 455 460
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg
465 470 475 480
Thr Phe Thr Arg Lys Arg Arg Cys Val Met Val Asp Asn Phe Ser Ile
485 490 495
Lys
<210> 100
<211> 497
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 100
Met Asn Asp Phe Asn Met Ile Asp Ile Glu Ser Ile Val Lys Asn Ile
1 5 10 15
Val Lys Glu Leu Thr Gly Asn Glu Lys Glu Gln Gly Thr Ile Thr Thr
20 25 30
Ala Ala Val Pro Lys Glu Val Asn Pro Leu Val Asp Ile Glu Lys Lys
35 40 45
Ile Met Gly Phe Val Asn Thr Pro Thr Met Pro Ile Gly Glu His Gly
50 55 60
Val Phe Glu Asp Ile Asn Asp Ala Ile Glu Gln Ala Trp Ile Ala Glu
65 70 75 80
Gln Glu Tyr Arg Lys Val Gly Leu Asp Lys Arg Thr Glu Ile Ile Glu
85 90 95
Ala Phe Lys Ala Glu Val Arg Lys Asn Val Glu Glu Ile Ser Arg Arg
100 105 110
Thr Phe Glu Glu Thr Gly Met Gly Arg Tyr Glu Asp Lys Ile Leu Lys
115 120 125
Asn Asn Leu Ala Leu Asp Lys Thr Pro Gly Val Glu Asp Leu Glu Ala
130 135 140
Gly Val Lys Thr Gly Asp Gly Gly Leu Thr Leu Tyr Glu Met Ser Pro
145 150 155 160
Phe Gly Val Ile Gly Ala Ile Ala Pro Ser Thr Asn Pro Thr Glu Thr
165 170 175
Ile Ile Asn Asn Gly Ile Ser Met Leu Ala Gly Gly Asn Thr Val Val
180 185 190
Phe Ser Pro His Pro Gly Ala Lys Asp Val Ser Val Phe Ile Ile Gln
195 200 205
Leu Ile Asn Lys Ala Ile Glu Arg Val Asn Gly Pro Lys Asn Leu Ile
210 215 220
Val Thr Val Arg Asn Pro Asn Ile Glu Ser Thr Asn Ile Met Leu Ala
225 230 235 240
His Pro Lys Val Asn Met Ile Cys Ala Thr Gly Gly Pro Gly Ile Val
245 250 255
Lys Val Ala Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly
260 265 270
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala
275 280 285
Val Asp Ile Ile Asp Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile
290 295 300
Cys Glu Lys Glu Val Ile Val Val Asp Lys Val Ala Asp Tyr Leu Lys
305 310 315 320
Thr Cys Met Ser Lys Tyr Cys Ala Leu Glu Ile Thr Asp Lys Asn Met
325 330 335
Leu Ala Gln Leu Glu Lys Leu Val Leu Thr Glu Asn Gly Thr Ile Asn
340 345 350
Lys Lys Phe Val Gly Lys Asn Ala Asp Tyr Ile Met Ser Lys Leu Gly
355 360 365
Val Asn Ile Asp Pro Ser Ile Arg Val Ile Phe Ala Glu Val Glu Ala
370 375 380
Asn His Pro Phe Ala Val Glu Glu Leu Met Met Pro Ile Leu Pro Val
385 390 395 400
Ile Arg Val Arg Asn Val Asp Glu Ala Ile Asp Leu Gly Val Glu Leu
405 410 415
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys His Ile Asp
420 425 430
Asn Leu Ser Lys Phe Ala Lys Ala Val Gln Thr Thr Ile Phe Val Lys
435 440 445
Asn Ala Pro Ser Tyr Ala Gly Ile Gly Tyr Gly Ala Glu Gly His Gly
450 455 460
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Arg
465 470 475 480
Thr Phe Thr Arg Lys Arg Arg Cys Val Met Val Asp Asn Phe Ser Ile
485 490 495
Lys
<210> 101
<211> 462
<212> PRT
<213> 绳尾真杆菌（Eubacterium plexicaudatum）
<400> 101
Met Ser Val Asn Asp Gln Met Val Gln Asp Ile Val Arg Gln Val Leu
1 5 10 15
Ala Asn Met Arg Ile Ser Ser Asp Ala Ser Gly Ser Arg Gly Val Phe
20 25 30
Ser Asp Met Asn Glu Ala Val Glu Ala Ala Lys Lys Ala Gln Ala Val
35 40 45
Ile Gly Lys Met Pro Met Asp His Arg Glu Lys Ile Ile Ser Ser Ile
50 55 60
Arg Ala Lys Ile Met Glu Asn Ala Glu Ile Leu Ala Arg Met Gly Val
65 70 75 80
Lys Glu Thr Gly Met Gly Asn Val Gly His Lys Ile Leu Lys His Gln
85 90 95
Leu Val Ala Glu Lys Thr Pro Gly Thr Glu Asp Ile Thr Thr Lys Ala
100 105 110
Trp Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly
115 120 125
Val Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Ile Leu
130 135 140
Cys Asn Thr Ile Gly Met Val Ala Gly Gly Asn Thr Val Val Phe Asn
145 150 155 160
Pro His Pro Ala Ala Ile Lys Thr Ser Ile Phe Ala Val Asn Leu Val
165 170 175
Asn Glu Ala Ser Val Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr
180 185 190
Val Glu His Pro Thr Leu Asp Thr Ser Ala Ile Met Met Lys His Lys
195 200 205
Asp Ile His Leu Ile Ala Ala Thr Gly Gly Pro Gly Val Val Thr Ala
210 215 220
Val Leu Ser Ser Gly Lys Arg Gly Ile Gly Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp
245 250 255
Ile Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu
260 265 270
Lys Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Met His Tyr
275 280 285
Met Ile Ser Glu Gln Gly Cys Tyr Leu Ala Ser Ala Lys Glu Gln Glu
290 295 300
Ala Leu Ile Ser Val Val Leu Lys Gly Gly Gln Leu Asn Arg Asp Cys
305 310 315 320
Val Gly Arg Asp Ala Lys Thr Leu Leu Gly Met Ile Gly Val Gln Ala
325 330 335
Pro Asp Asn Ile Arg Cys Ile Thr Phe Glu Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Glu Glu Leu Met Met Pro Ile Leu Gly Val Val Arg Ala
355 360 365
Asp Ser Phe Glu Asp Ala Val Glu Lys Ala Val Trp Leu Glu His Gly
370 375 380
Asn Arg His Ser Ala His Ile His Ser Lys Asn Val Asp His Ile Thr
385 390 395 400
Thr Tyr Ala Lys Ala Ile Asp Thr Ala Ile Leu Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Ser Ala Phe Thr
435 440 445
Lys Arg Arg Arg Cys Val Met Cys Asp Ser Leu Cys Ile Arg
450 455 460
<210> 102
<211> 467
<212> PRT
<213> 海洋热沉积杆菌（Thermosediminibacter oceani）
<400> 102
Met Val Asp Glu Lys Val Val Glu Ala Ile Ala Lys Arg Ile Ile Glu
1 5 10 15
Glu Leu Asn Leu Cys Glu Ser Gly Ser Ser Gly Gly Glu Ser Arg Glu
20 25 30
Glu Leu Gly Ile Phe Asp Asn Leu Asp Asp Ala Val Glu Ala Ala Ser
35 40 45
Gln Ala Gln Lys Arg Phe Ala Ala Leu Asp Leu Glu Lys Arg Glu Glu
50 55 60
Ile Ile Gln Ala Ile Arg Glu Ala Cys Leu Asn Asn Ala Arg Tyr Leu
65 70 75 80
Ala Glu Leu Thr Val Asn Glu Thr Gly Ile Gly Arg Val Glu Asp Lys
85 90 95
Ile Val Lys Asn Ile Leu Ala Ala Lys Lys Thr Pro Gly Thr Glu Asp
100 105 110
Leu Arg Pro Ser Cys Trp Thr Gly Asp His Gly Leu Thr Leu Val Glu
115 120 125
Met Ala Pro Val Gly Val Ile Gly Ser Ile Thr Pro Val Thr Asn Pro
130 135 140
Val Ala Thr Val Ile Asn Asn Ser Ile Ser Met Leu Ala Ala Gly Asn
145 150 155 160
Ala Val Val Phe Asn Pro His Pro Ser Ala Lys Arg Ser Ser Asn Lys
165 170 175
Ala Val Glu Ile Ile Asn Glu Ala Ile Met Lys Val Gly Gly Pro Arg
180 185 190
His Leu Val Asn Ser Val Ala Glu Pro Thr Ile Glu Thr Ala Lys Ala
195 200 205
Leu Met Ala His Pro Lys Val Asn Leu Val Ser Val Thr Gly Gly Lys
210 215 220
Ala Val Val Ser Glu Ala Leu Arg Ser Gly Lys Lys Val Ile Gly Ala
225 230 235 240
Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Val
245 250 255
Lys Ala Ala His Asp Ile Tyr Cys Gly Ala Ser Phe Asp Asn Asn Leu
260 265 270
Pro Cys Ile Ala Glu Lys Glu Leu Ile Ala Val Glu Ala Val Ala Asp
275 280 285
Met Leu Leu Glu Arg Leu Ala Arg Glu Gly Ala Tyr Ile Leu Arg Gly
290 295 300
Lys Asp Val Glu Lys Ile Thr Glu Val Val Phe Asp Glu Asn His Arg
305 310 315 320
Ile Asn Lys Lys Leu Val Gly Lys Asp Ala Ser Phe Ile Leu Glu Gln
325 330 335
Ile Gly Ile Gln Val Gly Lys Asp Val Arg Leu Val Val Val Pro Val
340 345 350
Asn Pro Glu His Pro Leu Val His His Glu Gln Leu Met Pro Val Leu
355 360 365
Pro Phe Val Arg Val Pro Asn Ile Gln Glu Ala Val Glu Leu Ala Val
370 375 380
Arg Ala Glu Gly Gly Asn Arg His Thr Ala Val Met His Ser Lys Asn
385 390 395 400
Val Asp Asn Met Thr Asn Phe Ala Arg Ala Ile Gln Thr Thr Ile Phe
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly Glu Gly
420 425 430
Tyr Ala Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser
435 440 445
Ala Arg Thr Phe Thr Arg Gln Arg Arg Cys Val Leu Val Asp Ala Phe
450 455 460
Arg Ile Ile
465
<210> 103
<211> 479
<212> PRT
<213> 梭状梭菌（Clostridium clostridioforme）
<400> 103
Met Glu Ile Ser Glu Lys Glu Val Glu Ala Ile Val Arg Ser Val Leu
1 5 10 15
Ser Gly Leu Gly Gln Lys Ser Phe Gln Ala Glu Ala Leu His Val Lys
20 25 30
Asp Lys Met Cys Ser Asp Gly Glu Asp Gly Ile Phe Glu Leu Val Glu
35 40 45
Asp Ala Ile Glu Ala Ala Ser Lys Ala Gln Lys Glu Trp Val His Arg
50 55 60
Tyr Lys Leu Lys Asp Arg Lys Arg Ile Ile Glu Ala Ile Arg Val Thr
65 70 75 80
Ser Arg Ala His Ala Glu Ser Leu Ala Arg Met Val His Glu Glu Thr
85 90 95
Gly Met Gly Arg Tyr Glu Asp Lys Ile Thr Lys His Met Ala Val Ile
100 105 110
Asp Lys Thr Pro Gly Val Glu Cys Leu Val Thr Asp Ala Ile Ser Gly
115 120 125
Asp Glu Gly Leu Met Ile Glu Glu Pro Ala Pro Phe Gly Val Ile Gly
130 135 140
Ala Ile Thr Pro Ser Thr Asn Pro Thr Glu Thr Met Ile Asn Asn Thr
145 150 155 160
Ile Ser Met Ile Ala Gly Gly Asn Ala Val Val Phe Asn Val His Pro
165 170 175
Gly Ala Lys Lys Cys Cys Ala Tyr Cys Leu Gln Ile Leu His Arg Ala
180 185 190
Ile Val Glu Asn Gly Gly Pro Lys Asn Leu Ile Thr Met Gln Arg Glu
195 200 205
Pro Asp Met Asp Ala Val His Lys Leu Thr Ser Ser Pro His Ile Arg
210 215 220
Leu Met Val Gly Thr Gly Gly Met Gly Met Val His Ala Leu Leu Cys
225 230 235 240
Ser Gly Lys Arg Thr Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val
245 250 255
Val Asp Asp Thr Ala Asp Leu Ser Leu Ala Ala Arg Glu Leu Tyr Arg
260 265 270
Gly Ala Ser Phe Asp Asn Asn Leu Leu Cys Leu Ala Glu Lys Glu Val
275 280 285
Phe Val Met Asp Asn Val Ala Glu Glu Leu Val Asp Arg Leu Val Gly
290 295 300
Glu Gly Ala Tyr Leu Leu Asp Asp Leu Gln Leu Lys Lys Ile Thr Glu
305 310 315 320
Leu Ala Met Val Asn Lys Asp Gly Lys Tyr Glu Val Asn Lys Lys Trp
325 330 335
Val Gly Lys Asp Ala Gly Lys Phe Leu Glu Ala Ile Gly Ile Gln Glu
340 345 350
His Arg Glu Pro Arg Leu Leu Ile Cys Val Thr Asp Arg Ser His Pro
355 360 365
Phe Val Lys Val Glu Gln Leu Met Pro Val Leu Pro Ile Val Arg Cys
370 375 380
Gly Ser Phe Glu Lys Cys Val Glu Trp Ala Val Asp Thr Glu Ala Gly
385 390 395 400
Asn Arg His Thr Ala Ser Ile Phe Ser Lys Asn Val Glu His Met Thr
405 410 415
Leu Phe Gly Lys Glu Ile Glu Thr Thr Ile Tyr Thr Lys Asn Gly Ala
420 425 430
Thr Leu Lys Gly Ile Gly Ile Gly Gly Glu Gly His Thr Thr Met Thr
435 440 445
Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Cys Ala Arg Ser Phe Thr
450 455 460
Arg Arg Arg Arg Cys Met Leu Ala Glu Gly Gly Leu Arg Ile Ile
465 470 475
<210> 104
<211> 471
<212> PRT
<213> 梭状梭菌（Clostridium clostridioforme）
<400> 104
Met Asp Met Asp Ile Lys Val Ile Glu Gln Met Val Glu Gln Ala Leu
1 5 10 15
Lys Glu Ile Lys Ala Glu Gln Pro Gln Lys Phe Thr Met Pro Lys Ala
20 25 30
Glu Leu Tyr Gly Val Phe Lys Thr Met Asp Glu Ala Ile Ala Ala Ser
35 40 45
Glu Glu Ala Gln Lys Lys Leu Leu Phe Ser Lys Ile Ser Asp Arg Gln
50 55 60
Lys Tyr Val Asp Val Ile Arg Arg Thr Ile Leu Lys Arg Glu Asn Leu
65 70 75 80
Glu Met Ile Ser Arg Leu Ser Val Glu Glu Thr Glu Ile Gly Asp Tyr
85 90 95
Glu His Lys Leu Ile Lys Asn Arg Leu Ala Ala Glu Lys Thr Pro Gly
100 105 110
Thr Glu Asp Leu Leu Thr Glu Ala Met Thr Gly Asp Asn Gly Leu Thr
115 120 125
Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Thr
130 135 140
Thr Asn Pro Thr Glu Thr Ile Ile Asn Asn Ser Ile Ser Met Ile Ala
145 150 155 160
Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Lys Val
165 170 175
Ser Gln Met Thr Val Lys Leu Leu Asn Lys Ala Leu Thr Glu Ser Gly
180 185 190
Ala Pro Glu Asn Leu Ile Thr Met Val Glu Glu Pro Ser Ile Glu Asn
195 200 205
Thr Asn Lys Met Ile Glu Asn Pro Ser Val Arg Leu Leu Val Ala Thr
210 215 220
Gly Gly Pro Ser Ile Val Lys Lys Val Leu Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala
245 250 255
Asp Ile Val Lys Ala Ala Lys Asp Ile Val Asp Gly Cys Ser Phe Asp
260 265 270
Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser
275 280 285
Ile Cys Asp Tyr Leu Ile His Asn Met Lys Glu Asn Gly Ala Tyr Gln
290 295 300
Ile Thr Asp Pro Ala Leu Leu Glu Lys Leu Val Thr Leu Val Thr Asn
305 310 315 320
Glu Lys Gly Gly Pro Lys Thr Ser Phe Val Gly Lys Ser Ala Arg Tyr
325 330 335
Ile Leu Asp Lys Leu Gly Ile Thr Ala Asp Ala Ser Val Arg Val Ile
340 345 350
Ile Met Glu Val Pro Lys Glu His Leu Leu Val Gln Glu Glu Met Met
355 360 365
Met Pro Ile Leu Pro Val Val Arg Val Cys Asp Val Asp Thr Ala Ile
370 375 380
Glu Tyr Ala Arg Gln Ala Glu His Gly Asn Arg His Thr Ala Met Met
385 390 395 400
His Ser Arg Asn Val Glu Lys Leu Ser Lys Met Ala Lys Ile Met Glu
405 410 415
Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val
420 425 430
Gly Gly Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu
435 440 445
Gly Leu Thr Ser Pro Arg Ala Phe Cys Arg Lys Arg Lys Cys Val Met
450 455 460
Thr Asp Ala Phe Ser Ile Arg
465 470
<210> 105
<211> 472
<212> PRT
<213> 多养型泥杆菌（Ilyobacter polytropus）
<400> 105
Met Asn Leu Asp Ala Asn Asn Leu Asn Asn Ile Val Ser Leu Ile Met
1 5 10 15
Lys Glu Leu Asp Lys Asn Asn Asn Ile Asp Asp Thr Gly Gln Gly Cys
20 25 30
Gly Gly Glu Glu Gly Lys Asn Gly Ile Phe Ser Ser Met Asp Thr Ala
35 40 45
Val Ser Lys Ala Lys Glu Ala Gln Val Thr Leu Phe Ala Ser Lys Leu
50 55 60
Glu Leu Arg Glu Arg Ile Ile Lys Ala Ile Arg Glu Asp Val Arg Glu
65 70 75 80
Ala Ala Ala Glu Leu Ala Glu Ile Ala Val Glu Glu Thr Gly Met Gly
85 90 95
Arg Val Asp Asp Lys Thr Leu Lys His Tyr Val Thr Val Asp Lys Thr
100 105 110
Pro Gly Val Glu Asp Leu Arg Ala Phe Ala Tyr Ser Gly Asp Asn Gly
115 120 125
Leu Thr Val Met Glu Leu Ser Pro Tyr Gly Val Ile Gly Ser Ile Thr
130 135 140
Pro Ser Thr Asn Pro Ser Glu Thr Ile Val Cys Asn Ala Ile Gly Met
145 150 155 160
Ile Ala Ala Gly Asn Ser Val Val Phe Ala Pro His Pro Gly Ala Lys
165 170 175
Lys Thr Ser Leu Arg Ala Val Glu Ile Leu Asn Lys Ala Val Ala Arg
180 185 190
Ala Gly Gly Pro Asn Asn Leu Val Val Thr Ile Phe Glu Pro Ser Ile
195 200 205
Glu Asn Thr Asn Lys Met Val Lys Asn Pro Asp Ile Lys Met Val Val
210 215 220
Ala Thr Gly Gly Pro Gly Val Val Lys Ser Val Met Ser Ser Gly Lys
225 230 235 240
Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Glu
245 250 255
Thr Ala Asp Ile Glu Lys Ala Ala Lys Asp Ile Val Asn Gly Cys Ser
260 265 270
Phe Asp Asn Asn Leu Pro Cys Ile Thr Glu Lys Glu Val Val Ala Val
275 280 285
Asp Ser Ile Thr Asp Tyr Leu Ile Phe Glu Met Gln Lys Asn Gly Ala
290 295 300
Tyr Leu Val Gln Asp Ser Lys Thr Ile Lys Lys Leu Cys Glu Met Val
305 310 315 320
Ile Asn Asp Gly Ser Pro Asn Arg Ala Tyr Val Gly Lys Asn Ala Ser
325 330 335
Tyr Ile Leu Lys Asp Leu Gly Ile Asp Val Gly Asp Glu Ile Lys Val
340 345 350
Ile Ile Val Glu Thr Asp Ala Gly His Pro Leu Ala Val Leu Glu Met
355 360 365
Leu Met Pro Val Leu Pro Ile Val Arg Val Lys Asp Ala Leu Glu Gly
370 375 380
Ile Lys Val Cys Lys Lys Leu Glu Asp Gly Leu Arg His Thr Ala Met
385 390 395 400
Ile His Ser Lys Asn Ile Asp Ile Leu Thr Lys Tyr Ala Arg Asp Met
405 410 415
Glu Thr Thr Ile Leu Val Lys Asn Gly Pro Ser Tyr Ser Gly Ile Gly
420 425 430
Val Gly Gly Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Leu Thr Ser Ala Lys Ser Phe Ala Arg Asn Arg Arg Cys Ala
450 455 460
Leu Val Gly Gly Leu Ser Ile Lys
465 470
<210> 106
<211> 462
<212> PRT
<213> 沙棘（Shuttleworthia satelles）
<400> 106
Met Ala Asp Glu Gln Leu Val Gln Asn Val Val Arg Glu Val Val Ala
1 5 10 15
Arg Met Gln Ile Ser Ala Pro Ala Arg Gly Met His Gly Val Phe Ser
20 25 30
Asp Met Glu Glu Ala Ile Glu Ala Ala Arg Thr Ala Gln Gln Thr Val
35 40 45
Arg Leu Leu Pro Met Asp Gln Arg Glu Lys Ile Ile Gly Ala Ile Arg
50 55 60
Arg Lys Thr Arg Glu Asn Ala Glu Ile Leu Ala Arg Met Ala Val Asn
65 70 75 80
Glu Thr Gly Met Gly Asn Val Gly Asp Lys Ile Leu Lys His Leu Leu
85 90 95
Val Ala Asp Lys Val Pro Gly Thr Glu Asp Ile Ser Thr Arg Ala Phe
100 105 110
Ser Gly Asp Arg Gly Leu Thr Leu Ile Glu Met Gly Pro Phe Gly Val
115 120 125
Ile Gly Ala Ile Thr Pro Cys Thr Asn Pro Ser Glu Thr Val Leu Cys
130 135 140
Asn Thr Ile Gly Met Leu Ala Gly Gly Asn Thr Val Val Phe Asn Pro
145 150 155 160
His Pro Gln Ala Ile Lys Thr Thr Leu Phe Thr Ile Gln Met Val Asn
165 170 175
Glu Ala Ser Leu Glu Ala Gly Gly Pro Asp Asn Ile Ala Cys Thr Val
180 185 190
Asp Ala Pro Thr Leu Ala Thr Ser Glu Ile Met Met Lys Ser Pro His
195 200 205
Ile Lys Leu Leu Val Ala Thr Gly Gly Pro Gly Val Val Thr Ala Val
210 215 220
Leu Ser Ser Gly Lys Arg Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro
225 230 235 240
Ala Leu Val Asp Glu Thr Ala Asp Ile Arg Lys Ala Ala Glu Asp Ile
245 250 255
Val Asn Gly Cys Thr Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu Lys
260 265 270
Glu Ile Val Ala Val Asp Ser Ile Ala Asp Glu Leu Leu His Tyr Met
275 280 285
Leu Thr Glu Gln Gly Cys Tyr Gln Ala Ser Glu Glu Glu Leu Asp Arg
290 295 300
Leu Thr Lys Ala Val Met Asp Glu Lys Gly Arg Leu Asn Arg Lys Ala
305 310 315 320
Val Gly Arg Ser Ala Arg Lys Leu Leu Ser Met Ile Gly Val Glu Val
325 330 335
Asp Ala Asn Ile Arg Cys Ile Thr Phe Phe Gly Pro Lys Glu His Pro
340 345 350
Leu Ile Thr Thr Glu Leu Met Met Pro Ile Leu Gly Ile Val Arg Val
355 360 365
Lys Asp Phe Ala Glu Gly Leu Glu Thr Ala Ala Trp Leu Glu His Gly
370 375 380
Asn Lys His Ser Ala His Ile His Ser Lys Asn Val Asp Arg Ile Thr
385 390 395 400
Glu Tyr Ala Arg Arg Leu Asp Thr Thr Ile Thr Val Lys Asn Gly Pro
405 410 415
Ser Tyr Ala Ala Leu Gly Phe Gly Gly Glu Ser Tyr Cys Thr Phe Thr
420 425 430
Ile Ala Ser Arg Thr Gly Glu Gly Leu Thr Ser Ala Arg Ser Phe Ile
435 440 445
Lys Ser Arg His Cys Val Met Thr Asp Ser Leu Cys Val Arg
450 455 460
<210> 107
<211> 472
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 107
Met Asp Val Asp Val Val Leu Val Glu Lys Leu Val Arg Gln Ala Ile
1 5 10 15
Glu Glu Val Lys Asn Lys Asn Leu Leu Asn Leu Asp Lys Phe Glu Ser
20 25 30
Val Lys Asn Tyr Gly Ile Phe Gly Thr Met Asp Ala Ala Val Glu Ala
35 40 45
Ser Phe Val Ala Gln Lys Gln Leu Leu Asn Ala Ser Met Thr Asp Lys
50 55 60
Gln Lys Tyr Val Asp Thr Ile Lys Ala Thr Ile Leu Lys Lys Glu Asn
65 70 75 80
Leu Glu Leu Ile Ser Arg Met Ser Val Glu Glu Thr Glu Ile Gly Lys
85 90 95
Tyr Glu His Lys Leu Ile Lys Asn Arg Val Ala Ala Glu Lys Thr Pro
100 105 110
Gly Ile Glu Asp Leu Thr Thr Glu Ala Met Thr Gly Asp Asn Gly Leu
115 120 125
Thr Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro
130 135 140
Thr Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Ser Met Ile
145 150 155 160
Ala Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Asn
165 170 175
Val Ser Ile Lys Leu Val Thr Met Leu Asn Lys Ala Leu Glu Glu Ala
180 185 190
Gly Ala Pro Asp Asn Leu Ile Ala Thr Val Lys Glu Pro Ser Ile Glu
195 200 205
Asn Thr Asn Ile Met Met Glu His Pro Lys Ile Arg Met Leu Val Ala
210 215 220
Thr Gly Gly Pro Ala Ile Val Asn Lys Val Met Ser Thr Gly Lys Lys
225 230 235 240
Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr
245 250 255
Ala Asp Ile Glu Lys Ala Ala Ile Asp Ile Val Asn Gly Cys Ser Phe
260 265 270
Asp Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp
275 280 285
Gln Ile Cys Asp Tyr Leu Ile His Tyr Met Lys Leu Asn Gly Ala Tyr
290 295 300
Glu Ile Lys Asp Arg Asp Leu Ile Gln Lys Leu Leu Asp Leu Val Thr
305 310 315 320
Asn Glu Asn Gly Gly Pro Lys Val Ser Phe Val Gly Lys Ser Ala Pro
325 330 335
Tyr Ile Leu Asn Lys Leu Gly Ile Ser Val Asp Glu Asn Ile Lys Val
340 345 350
Ile Ile Met Glu Val Glu Lys Asn His His Phe Val Leu Glu Glu Met
355 360 365
Met Met Pro Ile Leu Pro Ile Val Arg Thr Lys Asp Val Asp Glu Ala
370 375 380
Ile Glu Cys Ala Tyr Val Ala Glu His Gly Asn Arg His Thr Ala Ile
385 390 395 400
Met His Ser Lys Asn Val Asp Lys Leu Thr Lys Met Ala Arg Leu Leu
405 410 415
Glu Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly
420 425 430
Val Gly Gly Glu Gly Thr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly
435 440 445
Glu Gly Leu Thr Thr Ala Arg Ser Phe Cys Arg Lys Arg Arg Cys Val
450 455 460
Met Val Asp Ala Phe Asn Ile Arg
465 470
<210> 108
<211> 471
<212> PRT
<213> 梭状梭菌（Clostridium clostridioforme）
<400> 108
Met Asp Met Asp Ile Lys Val Ile Glu Gln Met Val Glu Gln Ala Leu
1 5 10 15
Lys Glu Ile Lys Ala Glu Gln Pro Gln Lys Phe Thr Met Pro Lys Ala
20 25 30
Glu Leu Tyr Gly Val Phe Lys Thr Met Asp Glu Ala Ile Ala Ala Ser
35 40 45
Glu Glu Ala Gln Lys Lys Leu Leu Phe Ser Lys Ile Ser Asp Arg Gln
50 55 60
Lys Tyr Val Asp Val Ile Arg Arg Thr Ile Leu Lys Arg Glu Asn Leu
65 70 75 80
Glu Met Ile Ser Arg Leu Ser Val Glu Glu Thr Glu Ile Gly Asp Tyr
85 90 95
Glu His Lys Leu Ile Lys Asn Arg Leu Ala Ala Glu Lys Thr Pro Gly
100 105 110
Thr Glu Asp Leu Leu Thr Glu Ala Met Thr Gly Asp Asn Gly Leu Thr
115 120 125
Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Thr
130 135 140
Thr Asn Pro Thr Glu Thr Ile Ile Asn Asn Ser Ile Ser Met Ile Ala
145 150 155 160
Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Lys Val
165 170 175
Ser Gln Met Thr Val Lys Leu Leu Asn Lys Ala Leu Thr Glu Ser Gly
180 185 190
Ala Pro Glu Asn Leu Ile Thr Met Val Glu Glu Pro Ser Ile Glu Asn
195 200 205
Thr Asn Lys Met Ile Glu Asn Pro Ser Val Arg Leu Leu Val Ala Thr
210 215 220
Gly Gly Pro Ser Ile Val Lys Lys Val Leu Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala
245 250 255
Asp Ile Val Lys Ala Ala Lys Asp Ile Val Asp Gly Cys Ser Phe Asp
260 265 270
Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser
275 280 285
Ile Cys Asp Tyr Leu Ile His Asn Met Lys Glu Asn Gly Ala Tyr Gln
290 295 300
Ile Thr Asp Pro Ala Leu Leu Glu Lys Leu Val Thr Leu Val Thr Asn
305 310 315 320
Glu Lys Gly Gly Pro Lys Thr Ser Phe Val Gly Lys Ser Ala Arg Tyr
325 330 335
Ile Leu Asp Lys Leu Gly Ile Thr Ala Asp Ala Ser Val Arg Val Ile
340 345 350
Ile Met Glu Val Pro Lys Glu His Leu Leu Val Gln Glu Glu Met Met
355 360 365
Met Pro Ile Leu Pro Val Val Arg Val Cys Asp Val Asp Thr Ala Ile
370 375 380
Glu Tyr Ala Arg Gln Ala Glu His Gly Asn Arg His Thr Ala Met Met
385 390 395 400
His Ser Arg Asn Val Glu Lys Leu Ser Lys Met Ala Lys Ile Met Glu
405 410 415
Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val
420 425 430
Gly Gly Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu
435 440 445
Gly Leu Thr Ser Pro Lys Ala Phe Cys Arg Lys Arg Lys Cys Val Met
450 455 460
Thr Asp Ala Phe Ser Ile Arg
465 470
<210> 109
<211> 473
<212> PRT
<213> 梭菌属（Clostridium sp.）
<400> 109
Met Lys Leu Asp Asp Lys Leu Ile Glu Gln Val Ala Arg Leu Val Met
1 5 10 15
Glu Glu Met Lys Ser Gly Ser Ala Ala Ala Cys Glu Glu Asn Gly Thr
20 25 30
Cys Gly Asp Ser Tyr Gly Ile Phe Asp Ser Met Asp Asp Ala Val Gln
35 40 45
Ala Ser Glu Ala Ala Gln Arg Lys Tyr Leu Phe Ser Thr Met Glu Asp
50 55 60
Arg Gln Lys Tyr Val Asp Val Ile Arg Gln Thr Val Leu Glu Pro Glu
65 70 75 80
Met Leu Gln Lys Ile Ser Arg Met Ala Val Glu Glu Thr Gly Met Gly
85 90 95
Asn Tyr Glu His Lys Leu Ile Lys Asn Arg Leu Ala Ala Glu Lys Ser
100 105 110
Pro Gly Thr Glu Asp Leu Val Thr Glu Ala Met Thr Gly Asp Arg Gly
115 120 125
Leu Thr Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Val Thr
130 135 140
Pro Ala Thr Asn Pro Thr Glu Thr Ile Ile Cys Asn Ser Ile Ala Met
145 150 155 160
Leu Ala Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys
165 170 175
Asn Val Thr His Val Leu Val Thr Ala Leu Asn Gln Ala Leu Glu Lys
180 185 190
Val Gly Ala Pro Thr Asn Leu Ile Val Thr Val Arg Glu Pro Ser Val
195 200 205
Glu Asn Thr Asn Leu Met Ile Lys His Pro Lys Ile Arg Val Leu Val
210 215 220
Ala Thr Gly Gly Pro Gly Ile Val Lys Met Val Met Ser Thr Gly Lys
225 230 235 240
Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu
245 250 255
Thr Ala Asp Ile Glu Lys Ala Ala Lys Asp Ile Val Asp Gly Cys Ser
260 265 270
Phe Asp Asn Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Ile Ala Val
275 280 285
Asp Thr Ile Ala Asp Cys Leu Ile Trp His Met Lys Arg Val Gly Ala
290 295 300
Phe Glu Leu Lys Glu Glu Ser Ala Ile Ser Arg Leu Leu Gln Leu Val
305 310 315 320
Thr Asn Glu Lys Gly Gly Pro Lys Val Glu Phe Val Gly Lys Ser Ala
325 330 335
Pro Tyr Ile Leu Asn Lys Leu Gly Ile Ser Gly Gly Glu Asn Ala Arg
340 345 350
Val Ile Leu Met Glu Thr Gln Lys Asp His Pro Phe Val Met Glu Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Ala Ala Asp Val Asp Glu
370 375 380
Ala Ile Glu Ile Ala Leu Val Ala Glu Arg Gly Asn Arg His Thr Ala
385 390 395 400
Met Met His Ser Lys Asn Val Asp Lys Leu Thr Lys Met Ala Lys Leu
405 410 415
Leu Gln Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile
420 425 430
Gly Val Gly Gly Glu Gly Cys Thr Thr Phe Thr Ile Ala Gly Pro Thr
435 440 445
Gly Glu Gly Leu Thr Thr Ala Arg Ser Phe Cys Arg Lys Arg Arg Cys
450 455 460
Val Met Ser Asp Ala Leu His Ile Arg
465 470
<210> 110
<211> 471
<212> PRT
<213> 鲍氏梭菌（Clostridium bolteae）
<400> 110
Met Asp Met Asp Ile Lys Val Ile Glu Gln Leu Val Glu Gln Ala Leu
1 5 10 15
Lys Glu Ile Lys Ala Glu Gln Pro Leu Lys Phe Thr Ala Pro Lys Leu
20 25 30
Glu Arg Tyr Gly Val Phe Lys Thr Met Asp Glu Ala Ile Ala Ala Ser
35 40 45
Glu Glu Ala Gln Lys Lys Leu Leu Phe Ser Lys Ile Ser Asp Arg Gln
50 55 60
Lys Tyr Val Asp Val Ile Arg Ser Thr Ile Ile Lys Arg Glu Asn Leu
65 70 75 80
Glu Leu Ile Ser Arg Leu Ser Val Glu Glu Thr Glu Ile Gly Asp Tyr
85 90 95
Glu His Lys Leu Ile Lys Asn Arg Leu Ala Ala Glu Lys Thr Pro Gly
100 105 110
Thr Glu Asp Leu Leu Thr Glu Ala Ile Thr Gly Asp Asn Gly Leu Thr
115 120 125
Leu Val Glu Tyr Cys Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Thr
130 135 140
Thr Asn Pro Thr Glu Thr Ile Ile Asn Asn Ser Ile Ser Met Ile Ala
145 150 155 160
Gly Gly Asn Thr Val Val Phe Ser Pro His Pro Arg Ala Lys Lys Val
165 170 175
Ser Gln Met Thr Val Lys Met Leu Asn Lys Ala Leu Ile Asp Asn Gly
180 185 190
Ala Pro Pro Asn Leu Ile Thr Met Val Glu Glu Pro Ser Ile Glu Asn
195 200 205
Thr Asn Lys Met Ile Asp Asn Pro Ser Val Arg Leu Leu Val Ala Thr
210 215 220
Gly Gly Pro Ser Ile Val Lys Lys Val Leu Ser Ser Gly Lys Lys Ala
225 230 235 240
Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala
245 250 255
Asp Ile Asp Lys Ala Ala Lys Asp Ile Val Asp Gly Cys Ser Phe Asp
260 265 270
Asn Asn Val Pro Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser
275 280 285
Ile Cys Asp Tyr Leu Ile His His Met Lys Glu Asn Gly Ala Tyr Gln
290 295 300
Ile Thr Asp Pro Met Leu Leu Glu Gln Leu Val Ala Leu Val Thr Thr
305 310 315 320
Glu Lys Gly Gly Pro Lys Thr Ser Phe Val Gly Lys Ser Ala Arg Tyr
325 330 335
Ile Leu Asp Lys Leu Gly Ile Thr Val Asp Ala Ser Val Arg Val Ile
340 345 350
Ile Met Glu Val Pro Lys Asp His Leu Leu Val Gln Glu Glu Met Met
355 360 365
Met Pro Ile Leu Pro Val Val Arg Val Ser Asp Val Asp Thr Ala Ile
370 375 380
Glu Tyr Ala His Gln Ala Glu His Gly Asn Arg His Thr Ala Met Met
385 390 395 400
His Ser Lys Asn Val Glu Lys Leu Ser Lys Met Ala Lys Ile Met Glu
405 410 415
Thr Thr Ile Phe Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val
420 425 430
Gly Gly Glu Gly Tyr Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu
435 440 445
Gly Leu Thr Ser Pro Arg Thr Phe Cys Arg Lys Arg Lys Cys Val Met
450 455 460
Thr Asp Ala Phe Ser Ile Arg
465 470
<210> 111
<211> 468
<212> PRT
<213> 霍氏真杆菌（Eubacterium hallii）
<400> 111
Met Asn Ile Asp Val Glu Leu Ile Glu Lys Val Val Lys Lys Val Leu
1 5 10 15
Asn Asp Val Glu Thr Gly Ser Ser Glu Ser Glu Tyr Gly Tyr Gly Ile
20 25 30
Phe Asp Thr Met Asp Glu Ala Ile Glu Ala Ser Ala Lys Ala Gln Lys
35 40 45
Glu Tyr Met Asn His Ser Met Ala Asp Arg Gln Arg Tyr Val Glu Gly
50 55 60
Ile Arg Glu Val Val Cys Thr Lys Glu Asn Leu Glu Tyr Met Ser Lys
65 70 75 80
Leu Ala Val Glu Glu Ser Gly Met Gly Ala Tyr Glu Tyr Lys Val Ile
85 90 95
Lys Asn Arg Leu Ala Ala Val Lys Ser Pro Gly Val Glu Asp Leu Thr
100 105 110
Thr Glu Ala Leu Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Tyr Cys
115 120 125
Pro Phe Gly Val Ile Gly Ala Ile Ala Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Val Ile Cys Asn Ser Ile Ala Met Leu Ala Gly Gly Asn Thr Val
145 150 155 160
Val Phe Ser Pro His Pro Arg Ser Lys Gly Val Ser Ile Trp Leu Ile
165 170 175
Lys Lys Leu Asn Ala Lys Leu Glu Glu Leu Gly Ala Pro Arg Asn Leu
180 185 190
Ile Val Thr Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Ile Met Met
195 200 205
Asn His Pro Lys Val Arg Met Leu Val Ala Thr Gly Gly Pro Gly Ile
210 215 220
Val Lys Ala Val Met Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Val Asn Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys
260 265 270
Ile Ala Glu Lys Glu Val Ile Ala Val Asp Gln Ile Ala Asp Tyr Leu
275 280 285
Ile Phe Asn Met Lys Asn Asn Gly Ala Tyr Glu Val Lys Asp Pro Glu
290 295 300
Ile Ile Glu Lys Met Val Asp Leu Val Thr Lys Asp Arg Lys Lys Pro
305 310 315 320
Ala Val Asn Phe Val Gly Lys Ser Ala Gln Tyr Ile Leu Asp Lys Val
325 330 335
Gly Ile Lys Val Gly Pro Glu Val Lys Cys Ile Ile Met Glu Ala Pro
340 345 350
Lys Asp His Pro Phe Val Gln Ile Glu Leu Met Met Pro Ile Leu Pro
355 360 365
Ile Val Arg Val Pro Asn Val Asp Glu Ala Ile Asp Phe Ala Val Glu
370 375 380
Val Glu His Gly Asn Arg His Thr Ala Met Met His Ser Lys Asn Val
385 390 395 400
Asp Lys Leu Thr Lys Met Ala Lys Glu Ile Glu Thr Thr Ile Phe Val
405 410 415
Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Met Gly Tyr
420 425 430
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala
435 440 445
Lys Ser Phe Cys Arg Lys Arg Arg Cys Val Leu Gln Asp Gly Leu His
450 455 460
Ile Arg Met Lys
465
<210> 112
<211> 467
<212> PRT
<213> 解糖盐厌氧菌（Halanaerobium saccharolyticum）
<400> 112
Met Lys Ile Lys Glu Asn Glu Leu Asp Lys Ile Val Asn Gln Val Ile
1 5 10 15
Ser Ser Leu Asn Asn Lys Gln Asn Ser Asn Asp Phe Asn Thr Lys Ile
20 25 30
Asn Tyr Gly Ile Phe Ser Thr Met Asp Glu Ala Ile Ala Glu Ala Val
35 40 45
Lys Ala Gln Ala Cys Leu Gln Leu Asn Tyr Ser Thr Glu Ala Arg Glu
50 55 60
Lys Ile Ile Lys Ser Ile Arg Lys Asn Val Ser Lys His Val Glu Lys
65 70 75 80
Ile Ser Glu Met Ala Val Glu Glu Thr Asp Met Gly Arg Ile Glu Asp
85 90 95
Lys Ile Ile Lys Asn Asn Leu Ala Ile Asn Lys Thr Pro Gly Thr Glu
100 105 110
Asp Leu Arg Thr Glu Ala Phe Ser Gly Lys Lys Gly Leu Thr Ile Val
115 120 125
Glu Glu Ala Pro Phe Gly Val Ile Cys Ser Ile Ala Pro Val Thr Asn
130 135 140
Pro Thr Glu Thr Ile Ile Ser Asn Ala Ile Ser Met Ile Ala Ser Cys
145 150 155 160
Asn Gly Val Val Phe Asn Ser His Pro Gly Ala Lys Lys Val Ser Lys
165 170 175
Tyr Ile Ile Glu Val Leu Asn Lys Val Ile Met Glu Ala Gly Gly Pro
180 185 190
Glu Asn Leu Leu Thr Ala Val Asn Glu Pro Thr Leu Gln Thr Val Glu
195 200 205
Ser Cys Met Arg Asp Asp Arg Ile Ala Met Ile Val Ala Thr Gly Gly
210 215 220
Pro Gly Val Val Asn Ala Ala Leu Ser Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Leu Val Asp Asp Thr Val Asp Leu
245 250 255
Lys Arg Val Ala Lys Asp Ile Ile Asn Gly Ala Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Thr Ser Glu Lys Ala Ile Val Ala Leu Glu Ser Ile Ala
275 280 285
Asp Ser Leu Leu Asn Glu Met Thr Asn Gln Asn Ala Gln Leu Val His
290 295 300
Asp Ile Lys Ala Leu Glu Arg Val Ile Leu Asn Asp Asp Gly Ser Ile
305 310 315 320
Asn Lys Ala Leu Val Gly Lys Asp Ala Ala Phe Ile Leu Asn Lys Ala
325 330 335
Gly Leu Lys Ala Lys Ser Glu Asp Leu Arg Leu Val Ile Val Asp Val
340 345 350
Asp Leu Arg His Pro Phe Val Gln Lys Glu Gln Leu Met Pro Val Ile
355 360 365
Pro Leu Val Arg Ala Lys Asn Phe Asn Glu Ala Met Glu Met Gly Val
370 375 380
Asp Ile Glu Glu Gly Asn Arg His Thr Ala Ile Ile His Ser Lys Asn
385 390 395 400
Val Asp Asn Leu Thr Lys Phe Ala Lys Lys Ile Glu Thr Thr Ile Tyr
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Ala Gly Gly Glu Gly
420 425 430
Tyr Ala Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser
435 440 445
Ala Arg Ser Phe Thr Arg Lys Arg Arg Cys Val Leu Val Asp Gly Phe
450 455 460
Ser Ile Ile
465
<210> 113
<211> 469
<212> PRT
<213> 粘液真杆菌（Eubacterium limosum）
<400> 113
Met Asn Ile Asp Thr Thr Gly Ile Glu Tyr Ile Val Lys Lys Val Met
1 5 10 15
Asp Gln Ile Asp Tyr Ala Glu Glu Thr Gly Ala Pro Val Val Asp Gly
20 25 30
Lys Asp Gly Val Phe Gln Thr Met Asp Ala Ala Ile Glu Ala Ala Ala
35 40 45
Val Ala Gln Lys Glu Tyr Met Lys Lys Pro Leu Ala Leu Arg Arg Gln
50 55 60
Met Ile Ala Ala Met Arg Glu Ile Met Leu Lys Lys Glu Asn Ile Glu
65 70 75 80
Thr Ile Cys Ala Met Val Val Glu Glu Ser Gly Met Gly Asn Tyr Glu
85 90 95
His Lys Leu Ala Lys His Arg Leu Ala Thr Thr Gly Thr Pro Gly Val
100 105 110
Glu Asp Leu Leu Thr Glu Ala Trp Ala Gly Asp Asp Gly Cys Thr Leu
115 120 125
Leu Glu Leu Ser Pro Phe Gly Val Ile Gly Ala Ile Thr Pro Thr Thr
130 135 140
Asn Pro Asn Glu Thr Ile Val Asn Asn Ser Ile Gly Met Leu Ala Ala
145 150 155 160
Gly Asn Ala Val Val Phe Ser Pro His Pro Lys Ala Leu Lys Thr Ser
165 170 175
Phe Leu Cys Ile Lys Leu Leu Asn Glu Ala Ile Val Ser Val Gly Gly
180 185 190
Pro Arg Asn Leu Ile Val Thr Cys Ala Asn Pro Thr Ile Glu Ala Ala
195 200 205
Asn Glu Met Met Val His Pro Lys Ile Arg Met Leu Val Ala Thr Gly
210 215 220
Gly Pro Gly Val Val Lys Ala Val Leu Ser Ser Gly Lys Lys Ala Ile
225 230 235 240
Gly Ala Gly Ala Gly Asn Pro Pro Ala Leu Val Asp Glu Thr Ala Asp
245 250 255
Ile Glu Lys Ala Ala Lys Asp Ile Ile Asp Gly Cys Ser Phe Asp Asn
260 265 270
Asn Leu Pro Cys Ile Ala Glu Lys Glu Val Val Val Val Asp Gln Val
275 280 285
Ala Asp Tyr Leu Ile Phe Asn Met Lys Lys Asn Gly Ala Tyr Glu Ile
290 295 300
Thr Asp Lys Lys Ala Ile Asp Ala Leu Ala Asp Leu Val Cys Pro Glu
305 310 315 320
Gly Arg Leu Ser Arg Asp Phe Val Gly Lys Ser Ala Lys Tyr Ile Ala
325 330 335
Ala Ala Ala Gly Leu Asp Val Pro Glu Asp Thr Arg Val Leu Ile Cys
340 345 350
Glu Thr Ser Lys Asp His Leu Leu Ala Val Glu Glu Leu Met Met Pro
355 360 365
Ile Leu Pro Ile Val Arg Val Ala Asn Val Asp Glu Gly Ile Asp Val
370 375 380
Ala Val Glu Leu Glu His Gly Asn Arg His Thr Ala Ile Met His Ser
385 390 395 400
Lys Asn Val Asp Lys Leu Thr Glu Met Ala Lys Arg Ile Gln Thr Thr
405 410 415
Ile Phe Val Lys Asn Gly Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly
420 425 430
Glu Gly Tyr Pro Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu
435 440 445
Thr Ser Ala Lys Ser Phe Ala Arg Arg Arg Arg Cys Val Leu Val Gly
450 455 460
Gly Phe Asp Ile Lys
465
<210> 114
<211> 466
<212> PRT
<213> 嗜热厌氧杆菌属（Thermoanaerobacter sp.）
<400> 114
Met Ile Asp Glu Asn Leu Val Val Thr Ile Thr Lys Lys Ile Leu Asn
1 5 10 15
Glu Ile Asn Leu Lys Glu Ala Glu Glu Lys Lys Glu Lys Asp Asn Pro
20 25 30
Asp Leu Gly Ile Phe Asn Asp Val Asn Glu Ala Val Glu Cys Ala Lys
35 40 45
Glu Ala Gln Lys Lys Phe Ala Leu Met Asp Leu Glu Lys Arg Glu Glu
50 55 60
Ile Ile Ala Ala Ile Arg Glu Ala Cys Val Asn Asn Ala Arg Leu Leu
65 70 75 80
Ala Glu Ile Ala Cys Ser Glu Thr Gly Arg Gly Arg Val Glu Asp Lys
85 90 95
Val Ala Lys Asn Ile Leu Ala Ala Lys Lys Thr Pro Gly Thr Glu Asp
100 105 110
Leu Lys Pro Thr Ala Trp Thr Gly Asp Arg Gly Leu Thr Leu Val Glu
115 120 125
Met Ala Pro Val Gly Val Ile Ala Ser Ile Thr Pro Val Thr Asn Pro
130 135 140
Thr Ala Thr Ile Ile Asn Asn Thr Ile Ser Met Leu Ala Ala Gly Asn
145 150 155 160
Ala Val Val Phe Asn Pro His Pro Ser Ala Lys Lys Thr Ser Asn Lys
165 170 175
Ala Val Glu Ile Ile Asn Glu Ala Ile Leu Lys Val Gly Ala Pro Asn
180 185 190
Gly Leu Val Cys Ser Ile Asn Asn Pro Thr Ile Gln Thr Ala Gln Lys
195 200 205
Leu Met Glu His Pro Glu Val Asn Met Val Val Val Thr Gly Gly Lys
210 215 220
Ala Val Val Gln Thr Ala Leu Arg Cys Gly Lys Lys Val Ile Gly Ala
225 230 235 240
Gly Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Val
245 250 255
Lys Ala Ala His Asp Ile Ala Cys Gly Ala Ser Phe Asp Asn Asn Leu
260 265 270
Pro Cys Ile Ala Glu Lys Glu Ile Ile Ala Val Glu Arg Ile Ala Asp
275 280 285
Thr Leu Leu Glu Arg Met Lys Arg Glu Gly Ala Tyr Val Leu His Gly
290 295 300
Lys Asp Ile Asp Arg Met Thr Glu Leu Ile Phe Gln Gly Gly Ala Ile
305 310 315 320
Asn Lys Asp Leu Ile Gly Arg Asp Ala His Phe Ile Leu Ser Gln Ile
325 330 335
Gly Ile Glu Thr Gly Lys Asp Ile Arg Leu Val Val Met Pro Val Asp
340 345 350
Val Ser His Pro Leu Val Tyr His Glu Gln Leu Met Pro Val Ile Pro
355 360 365
Phe Val Thr Val Pro Thr Val Glu Glu Ala Ile Asn Leu Ala Val Lys
370 375 380
Ala Glu Gly Gly Asn Arg His Thr Ala Met Met His Ser Lys Asn Val
385 390 395 400
Glu Asn Met Thr Ala Phe Ala Arg Ala Ile Gln Thr Thr Ile Phe Val
405 410 415
Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Phe Gly Gly Glu Gly Tyr
420 425 430
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala
435 440 445
Arg Thr Phe Thr Arg Gln Arg Arg Cys Val Leu Val Asp Ala Phe Arg
450 455 460
Ile Val
465
<210> 115
<211> 529
<212> PRT
<213> 深红螺菌（Rhodospirillum rubrum）
<400> 115
Met Asn Asp Gly Gln Ile Ala Ala Ala Val Ala Lys Val Leu Glu Ala
1 5 10 15
Tyr Gly Val Pro Ala Asp Pro Ser Ala Ala Ala Pro Ala Pro Ala Ala
20 25 30
Pro Val Ala Pro Ala Ala Pro Thr Ala Gly Ser Val Ser Glu Met Ile
35 40 45
Ala Arg Gly Ile Ala Lys Ala Ser Ser Asp Asp Gln Ile Ala Gln Ile
50 55 60
Val Ala Lys Val Val Gly Asp Tyr Ser Ala Gln Ala Ala Lys Pro Ala
65 70 75 80
Val Val Pro Gly Ala Ala Ala Ser Thr Glu Ala Gly Asp Gly Val Phe
85 90 95
Asp Thr Met Asp Ala Ala Val Asp Ala Ala Val Leu Ala Gln Gln Gln
100 105 110
Tyr Leu Leu Cys Ser Met Thr Asp Arg Gln Arg Phe Val Asp Gly Ile
115 120 125
Arg Glu Val Ile Leu Gln Lys Asp Thr Leu Glu Leu Ile Ser Arg Met
130 135 140
Ala Ala Glu Glu Thr Gly Met Gly Asn Tyr Glu His Lys Leu Ile Lys
145 150 155 160
Asn Arg Leu Ala Ala Glu Lys Thr Pro Gly Thr Glu Asp Leu Thr Thr
165 170 175
Glu Ala Phe Ser Gly Asp Asp Gly Leu Thr Leu Val Glu Tyr Ser Pro
180 185 190
Phe Gly Ala Ile Gly Ala Val Ala Pro Thr Thr Asn Pro Thr Glu Thr
195 200 205
Ile Ile Cys Asn Ser Ile Gly Met Leu Ala Ala Gly Asn Ser Val Ile
210 215 220
Phe Ser Pro His Pro Arg Ala Thr Lys Val Ser Leu Leu Thr Val Lys
225 230 235 240
Leu Ile Asn Gln Lys Leu Ala Cys Leu Gly Ala Pro Ala Asn Leu Val
245 250 255
Val Thr Val Ser Lys Pro Ser Val Glu Asn Thr Asn Ala Met Met Ala
260 265 270
His Pro Lys Ile Arg Met Leu Val Ala Thr Gly Gly Pro Gly Ile Val
275 280 285
Lys Ala Val Met Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly
290 295 300
Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala Ala
305 310 315 320
Leu Asp Ile Ile Asn Gly Cys Ser Phe Asp Asn Asn Leu Pro Cys Ile
325 330 335
Ala Glu Lys Glu Ile Ile Ala Val Ala Gln Ile Ala Asp Tyr Leu Ile
340 345 350
Phe Ser Met Lys Lys Gln Gly Ala Tyr Gln Ile Thr Asp Pro Ala Val
355 360 365
Leu Arg Lys Leu Gln Asp Leu Val Leu Thr Ala Lys Gly Gly Pro Gln
370 375 380
Thr Ser Cys Val Gly Lys Ser Ala Val Trp Leu Leu Asn Lys Ile Gly
385 390 395 400
Ile Glu Val Asp Ser Ser Val Lys Val Ile Leu Met Glu Val Pro Lys
405 410 415
Glu His Pro Phe Val Gln Glu Glu Leu Met Met Pro Ile Leu Pro Leu
420 425 430
Val Arg Val Ser Asp Val Asp Glu Ala Ile Ala Val Ala Ile Glu Val
435 440 445
Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Thr Asn Val Arg
450 455 460
Lys Leu Thr Lys Met Ala Lys Leu Ile Gln Thr Thr Ile Phe Val Lys
465 470 475 480
Asn Gly Pro Ser Tyr Ala Gly Leu Gly Val Gly Gly Glu Gly Tyr Thr
485 490 495
Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Ala Lys
500 505 510
Ser Phe Ala Arg Lys Arg Lys Cys Val Met Val Glu Ala Leu Asn Ile
515 520 525
Arg
<210> 116
<211> 466
<212> PRT
<213> 尤氏真杆菌（Eubacterium yurii）
<400> 116
Met Asn Pro Glu Leu Leu Glu Asp Val Val Arg Gln Val Leu Ser Glu
1 5 10 15
Met Lys Leu Glu Ser Ser Lys Met Val Asp Ile Tyr Asn Tyr Gly Ile
20 25 30
Phe Asp Ser Val Asp Asp Ala Ile Asn Ala Ser Glu Ile Ala Gln Arg
35 40 45
Gln Leu Phe Glu Cys Ser Val Gln Lys Arg Asn Glu Tyr Val Asn Ala
50 55 60
Ile Arg Gln Ile Ile Leu Lys Lys Asp Asn Leu Glu Met Met Ser Arg
65 70 75 80
Asp Ala Val Glu Glu Thr Gly Ile Gly Arg Tyr Glu Asp Lys Ile Leu
85 90 95
Lys Asn Lys Leu Ala Ala Glu Lys Thr Pro Gly Met Glu Asp Leu Ile
100 105 110
Thr Arg Ala Val Ser Gly Gln Asp Gly Leu Thr Leu Glu Glu Tyr Cys
115 120 125
Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Phe Ile Ser Asn Ser Ile Ser Met Ile Val Gly Gly Asn Thr Val
145 150 155 160
Val Phe Ser Pro His Pro Arg Ala Lys Asn Thr Ser Ile Lys Leu Val
165 170 175
Lys Leu Met Asn Lys Ala Leu Glu Gln Val Gly Ala Pro Arg Asn Leu
180 185 190
Ile Ser Met Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Leu Met Met
195 200 205
Asn His Pro Lys Ile Lys Met Leu Val Ala Thr Gly Gly Pro Ala Ile
210 215 220
Val Lys Thr Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Val Ala Gly Ser Ser Phe Asp Asn Asn Val Pro Cys
260 265 270
Ile Ala Glu Lys Glu Val Phe Ala Val Glu Ser Ile Cys Asp Gln Leu
275 280 285
Ile Tyr His Met Lys Lys Asn Gly Ala Tyr Glu Ile Thr Ser Tyr Glu
290 295 300
Met Ile Glu Lys Leu Asp Lys Leu Val Ser Gln Glu Asn Gly Lys Pro
305 310 315 320
Asn Thr Asp Phe Val Gly Lys Ser Ala Lys Tyr Ile Leu Glu Lys Leu
325 330 335
Gly Ile Ser Val Asp Asp Ser Ile Arg Leu Ile Ile Cys Arg Thr Asn
340 345 350
Lys Asp His His Leu Val Gln Glu Glu Met Leu Met Pro Ile Leu Pro
355 360 365
Ile Val Ser Val Ser Asp Val Asp Val Ala Ile Glu Tyr Ala Tyr Glu
370 375 380
Ala Glu His Arg Asn Arg His Thr Ala Ile Met His Ser Arg Asn Val
385 390 395 400
Glu Lys Leu Ser Lys Met Ala Lys Lys Leu Glu Ala Thr Ile Phe Val
405 410 415
Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Tyr
420 425 430
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Pro
435 440 445
Lys Ser Phe Cys Arg Val Arg Arg Cys Thr Met Ser Asp Ser Phe Ser
450 455 460
Ile Arg
465
<210> 117
<211> 466
<212> PRT
<213> 真杆菌属（Eubacterium sp.）
<400> 117
Met Asn Pro Glu Leu Leu Glu Asp Val Val Arg Gln Val Leu Ser Glu
1 5 10 15
Met Lys Leu Glu Ser Ser Lys Met Val Asp Ile Tyr Asn Tyr Gly Ile
20 25 30
Phe Asp Ser Val Asp Asp Ala Ile Asn Ala Ser Glu Ile Ala Gln Arg
35 40 45
Gln Leu Phe Glu Cys Ser Val Gln Lys Arg Asn Glu Tyr Val Asn Ala
50 55 60
Ile Arg Gln Ile Ile Leu Lys Lys Asp Asn Leu Glu Met Met Ser Arg
65 70 75 80
Asp Ala Val Glu Glu Thr Gly Ile Gly Arg Tyr Glu Asp Lys Ile Leu
85 90 95
Lys Asn Lys Leu Ala Ala Glu Lys Thr Pro Gly Met Glu Asp Leu Ile
100 105 110
Thr Arg Ala Val Ser Gly Gln Asp Gly Leu Thr Leu Glu Glu Tyr Cys
115 120 125
Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr Glu
130 135 140
Thr Phe Ile Ser Asn Ser Ile Ser Met Ile Val Gly Gly Asn Thr Val
145 150 155 160
Val Phe Ser Pro His Pro Arg Ala Lys Asn Thr Ser Ile Lys Leu Val
165 170 175
Lys Leu Met Asn Lys Ala Leu Glu Gln Val Gly Ala Pro Arg Asn Leu
180 185 190
Ile Ser Met Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Leu Met Met
195 200 205
Asn His Pro Lys Ile Lys Met Leu Val Ala Thr Gly Gly Pro Ala Ile
210 215 220
Val Lys Thr Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly Ala
225 230 235 240
Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys Ala
245 250 255
Ala Lys Asp Ile Val Ala Gly Ser Ser Phe Asp Asn Asn Val Pro Cys
260 265 270
Ile Ala Glu Lys Glu Val Phe Ala Val Glu Ser Ile Cys Asp Gln Leu
275 280 285
Ile Tyr His Met Lys Lys Asn Gly Ala Tyr Glu Ile Thr Ser Tyr Glu
290 295 300
Met Ile Glu Lys Leu Asp Lys Leu Val Ser Gln Glu Asn Gly Lys Pro
305 310 315 320
Asn Thr Asp Phe Val Gly Lys Ser Ala Lys Tyr Ile Leu Glu Lys Leu
325 330 335
Gly Ile Asn Val Asp Asp Ser Ile Arg Leu Ile Ile Cys Arg Thr Asn
340 345 350
Lys Asp His His Leu Val Gln Glu Glu Met Leu Met Pro Ile Leu Pro
355 360 365
Ile Val Ser Val Ser Asp Val Asp Val Ala Ile Glu Tyr Ala Tyr Glu
370 375 380
Ala Glu His Arg Asn Arg His Thr Ala Ile Met His Ser Arg Asn Val
385 390 395 400
Glu Lys Leu Ser Lys Met Ala Lys Lys Leu Glu Ala Thr Ile Phe Val
405 410 415
Lys Asn Ala Pro Ser Tyr Ala Gly Ile Gly Val Gly Gly Glu Gly Tyr
420 425 430
Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Leu Thr Ser Pro
435 440 445
Lys Ser Phe Cys Arg Val Arg Arg Cys Thr Met Ser Asp Ser Phe Ser
450 455 460
Ile Arg
465
<210> 118
<211> 532
<212> PRT
<213> 弧菌属（Vibrio sp.）
<400> 118
Met Asn Glu Gln Glu Ile Ala His Ala Val Glu Asn Val Leu Ser Lys
1 5 10 15
Tyr Thr Asn Val Thr Ala Gln Asn Ala Glu Pro Val Ser Tyr Ser Ser
20 25 30
Asn Ala Ser Leu Glu Asn Ile Val Ser Gln Ala Leu Ala Gly Asn Met
35 40 45
Val Lys Gln Pro Glu Thr Gln Thr Ala Pro Asp Leu Asn Ser Asn Ile
50 55 60
Glu Asn Ile Val Ser Gln Ile Leu Ala Glu Asn Gln Ala Lys Pro Gln
65 70 75 80
Ser Val Gln Cys Gln Ser Ala Asn His Gly Thr Thr Glu Tyr Leu Gly
85 90 95
Cys Phe Ala Ser Met Glu Glu Ala Ile Ser Ala Ala Ser His Ala Gln
100 105 110
Val Gln Tyr Arg His Cys Thr Met Gly Asp Arg Ala Ser Phe Val Lys
115 120 125
Gly Ile Arg Glu Val Phe Thr Gln Asp Asp Val Leu Glu Lys Ile Ser
130 135 140
Arg Met Ala Val Glu Glu Thr Gly Met Gly Asn Tyr Ala Asp Lys Leu
145 150 155 160
Thr Lys Asn Arg Ile Ala Ala Thr Lys Thr Pro Gly Ile Glu Asp Leu
165 170 175
Thr Thr Ser Ala Leu Ser Gly Asp Ser Gly Leu Thr Leu Thr Glu Phe
180 185 190
Ser Ala Tyr Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr
195 200 205
Glu Thr Ile Ile Asn Asn Ser Ile Gly Met Leu Ala Ala Gly Asn Thr
210 215 220
Val Val Tyr Ser Pro His Pro Arg Ser Arg Asn Val Ser Leu Val Ala
225 230 235 240
Val Asp Leu Ile Asn Arg Lys Leu Ala Glu Leu Gly Ala Pro Ala Asn
245 250 255
Leu Val Val Thr Val Leu Glu Pro Ser Ile Asp Asn Thr Asn Ala Met
260 265 270
Met Asn Asp Pro Arg Val Asn Met Leu Val Ala Thr Gly Gly Pro Ser
275 280 285
Ile Val Lys Thr Val Met Ser Thr Gly Lys Lys Ala Ile Gly Ala Gly
290 295 300
Ala Gly Asn Pro Pro Ala Val Val Asp Glu Thr Ala Asn Ile Glu Lys
305 310 315 320
Ala Ala Lys Asp Ile Ile Asn Gly Cys Ala Phe Asp Asn Asn Leu Pro
325 330 335
Cys Ile Ala Glu Lys Glu Val Ile Val Val Asn Glu Val Ala Asp Tyr
340 345 350
Leu Ile His Cys Met Lys Lys Ser Gly Ala Tyr Leu Leu Cys Asp Lys
355 360 365
Gln Lys Ile Gln Gln Leu Gln Ser Leu Val Leu Asn Glu Lys Gly Thr
370 375 380
Gly Pro Asn Thr Ser Phe Val Gly Lys Gly Ala Arg Tyr Ile Leu Asp
385 390 395 400
Lys Leu Asn Ile Gln Val Ser Asp Asp Ile Lys Val Ile Leu Ile Glu
405 410 415
Thr Glu Arg Asn His Pro Phe Val Val His Glu Leu Met Met Pro Ile
420 425 430
Leu Pro Val Val Arg Val Glu Asn Val Asp Glu Ala Ile Asp Leu Ala
435 440 445
Ile Lys Val Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Thr
450 455 460
Asn Val Glu Lys Leu Ser Lys Met Ala Arg Leu Ile Gln Thr Thr Ile
465 470 475 480
Phe Val Lys Asn Gly Pro Ser Tyr Ser Gly Ile Gly Val Gly Gly Glu
485 490 495
Gly His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr
500 505 510
Ser Ala Arg Ser Phe Ala Arg Tyr Arg Arg Cys Val Met Val Glu Ala
515 520 525
Leu Asn Ile Arg
530
<210> 119
<211> 467
<212> PRT
<213> 优杆菌科细菌（Eubacteriaceae bacterium）
<400> 119
Met Asn Ala Glu Leu Leu Gln Asp Val Val Arg Gln Val Leu Ser Glu
1 5 10 15
Met Lys Leu Glu Ser Ser Asn Ile Leu Ser Asn Glu Tyr Asn Tyr Gly
20 25 30
Ile Phe Asp Asp Met Glu Ala Ala Ile Asn Ala Ser Glu Thr Ala Gln
35 40 45
Arg Lys Leu Phe Glu Cys Ser Val Gln Gln Arg Asn Glu Phe Ala Asn
50 55 60
Val Ile Arg Lys Glu Ile Leu Lys Lys Asp Asn Leu Glu Met Ile Ser
65 70 75 80
Arg Asp Ala Val Glu Glu Thr Glu Ile Gly Arg Phe Glu Asp Lys Ile
85 90 95
Leu Lys Asn Lys Val Ala Ala Glu Lys Thr Pro Gly Met Glu Asp Leu
100 105 110
Thr Thr Arg Ala Ile Ser Gly Lys Asp Gly Leu Met Ile Glu Glu Tyr
115 120 125
Cys Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr
130 135 140
Glu Thr Leu Ile Asn Asn Ser Ile Ser Met Ile Val Gly Gly Asn Thr
145 150 155 160
Val Val Phe Ser Pro His Pro Arg Ala Lys Asn Val Ser Ile Lys Leu
165 170 175
Val Lys Met Met Asn Lys Ala Leu Glu Glu His Gly Ala Pro Arg Asn
180 185 190
Met Ile Thr Met Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Leu Met
195 200 205
Met Ser Asn Pro Lys Val Lys Leu Leu Val Ala Thr Gly Gly Pro Phe
210 215 220
Ile Val Asn Thr Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys
245 250 255
Ala Ala Ile Asp Ile Val Ser Gly Ala Ser Phe Asp Asn Asn Val Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser Ile Ser Asp Met
275 280 285
Leu Ile Tyr His Met Lys Lys Asn Gly Ala Tyr Glu Ile Val Ser Gln
290 295 300
Asp Met Ile Glu Lys Leu Asp Lys Leu Val Ser Gln Glu Asn Gly Lys
305 310 315 320
Pro Lys Thr Glu Phe Val Gly Lys Ser Ala Lys Tyr Ile Leu Glu Lys
325 330 335
Leu Gly Ile Tyr Val Asp Asp Ser Ile Arg Leu Ile Ile Cys Arg Thr
340 345 350
Ser Lys Asn His His Leu Val Gln Glu Glu Met Leu Met Pro Ile Leu
355 360 365
Pro Ile Val Ser Val Ser Asp Val Asp Ile Ala Ile Glu Tyr Ala Tyr
370 375 380
Glu Ala Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys Asn
385 390 395 400
Val Glu Lys Leu Ser Lys Met Ala Lys Lys Leu Glu Ala Thr Ile Phe
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ser Gly Ile Gly Val Gly Gly Glu Gly
420 425 430
His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser
435 440 445
Ala Lys Ser Phe Cys Arg Ile Arg Arg Cys Val Met His Asp Ser Phe
450 455 460
Ser Ile Arg
465
<210> 120
<211> 473
<212> PRT
<213> 丙酸丙酸杆菌（Propionibacterium propionicum）
<400> 120
Met Lys Ile Asp Pro Ala Gln Leu Glu Ala Thr Ile Arg Glu Val Leu
1 5 10 15
Ala Ala Met Leu Pro Gly Asn Asp Asn Gln Thr Glu Ala Pro Ala Thr
20 25 30
Gln Gln Glu Ala Pro Gly Asp Gly Val Phe Ala Asp Met Asp Ser Ala
35 40 45
Val Glu Ala Ala His Leu Ala Gln Arg Glu Tyr Leu Ser His Pro Met
50 55 60
Ala Asp Arg Arg Arg Tyr Val Ala Ala Ile Arg Glu Ala Met Leu Ala
65 70 75 80
Pro Glu Ala Leu Asp Tyr Met Ser Glu Gln Ala Val Ala Gln Ser Gly
85 90 95
Met Gly Asp Val Gly His Lys Tyr Leu Lys Asn Lys Val Ala Ala Ala
100 105 110
Glu Thr Pro Gly Val Glu Asp Leu Val Thr Glu Ala Trp Ser Gly Asp
115 120 125
Asp Gly Leu Thr Thr Ile Glu Tyr Ser Pro Tyr Gly Val Ile Gly Ala
130 135 140
Ile Thr Pro Thr Thr Asn Pro Thr Glu Thr Ile Thr Cys Asn Ser Ile
145 150 155 160
Gly Met Leu Ala Ala Gly Asn Ala Val Val Phe Ser Pro His Pro Arg
165 170 175
Val Ala Lys Leu Ser Cys Trp Gln Val Arg Arg Ile Asn Arg Ala Leu
180 185 190
Arg Ala Ala Gly Ala Pro Asp Asn Leu Val Val Thr Val Thr Ala Pro
195 200 205
Ser Leu Glu Asn Thr Asn Ala Met Met Ala His Pro Lys Val Arg Met
210 215 220
Leu Val Ala Thr Gly Gly Pro Gly Ile Val Lys Ala Val Leu Ser Ser
225 230 235 240
Gly Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Ala Val Val
245 250 255
Asp Glu Thr Ala Asp Ile Glu His Ala Ala Lys Cys Ile Val Asp Gly
260 265 270
Ala Ser Phe Asp Asn Asn Leu Pro Cys Thr Ala Glu Lys Glu Ile Ile
275 280 285
Ala Val Asp Ser Ile Ala Asp Met Leu Lys Phe Cys Met Ile Lys His
290 295 300
Gly Ala Tyr Glu Ala Thr Ala Ser Glu Val Ala Glu Leu Glu Lys Leu
305 310 315 320
Leu Val Asn Gly Asp Lys Pro Arg Thr Glu Trp Val Gly Lys Pro Ala
325 330 335
Ala Lys Ile Leu Glu Ala Ile Gly Val Thr Pro Pro Pro Gly Val Arg
340 345 350
Leu Ile Val Cys Glu Ala Ser Ala Thr His Pro Phe Val Val His Glu
355 360 365
Leu Met Met Pro Val Leu Gly Leu Val Arg Val Pro Asp Val Asp Ala
370 375 380
Ala Ile Asp Leu Ala Val Glu Leu Glu His Gly Asn Arg His Thr Ala
385 390 395 400
Val Met His Ser Leu Asn Val Ser Lys Leu Thr Lys Met Gly Lys Leu
405 410 415
Ile Gln Thr Thr Ile Phe Val Lys Asn Gly Pro Ser Tyr Asn Gly Ile
420 425 430
Gly Ile Gly Gly Glu Gly Tyr Pro Thr Phe Thr Ile Ala Gly Pro Thr
435 440 445
Gly Glu Gly Leu Thr Ser Ala Arg Ser Phe Thr Arg Lys Arg Arg Cys
450 455 460
Val Leu Val Gly Asp Leu Asn Val Arg
465 470
<210> 121
<211> 467
<212> PRT
<213> 优杆菌科细菌（Eubacteriaceae bacterium）
<400> 121
Met Asn Ala Glu Leu Leu Gln Asp Val Val Arg Gln Val Leu Ser Glu
1 5 10 15
Met Lys Leu Glu Ser Ser Asn Ile Leu Ser Asn Glu Tyr Asn Tyr Gly
20 25 30
Ile Phe Asp Asp Met Glu Ala Ala Ile Asn Ala Ser Glu Thr Ala Gln
35 40 45
Arg Lys Leu Phe Glu Cys Ser Val Gln Gln Arg Asn Glu Phe Ala Asn
50 55 60
Val Ile Arg Lys Glu Ile Leu Lys Lys Asp Asn Leu Glu Met Ile Ser
65 70 75 80
Arg Asp Ala Val Glu Glu Thr Glu Ile Gly Arg Phe Glu Asp Lys Ile
85 90 95
Leu Lys Asn Lys Val Ala Ala Glu Lys Thr Pro Gly Met Glu Asp Leu
100 105 110
Thr Thr Arg Ala Leu Thr Gly Lys Asp Gly Leu Met Ile Glu Glu Tyr
115 120 125
Cys Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr
130 135 140
Glu Thr Leu Ile Asn Asn Ser Ile Ser Met Ile Val Gly Gly Asn Thr
145 150 155 160
Val Val Phe Ser Pro His Pro Arg Ala Lys Asn Val Ser Ile Lys Leu
165 170 175
Val Lys Met Met Asn Lys Ala Leu Glu Glu Tyr Gly Ala Pro Arg Asn
180 185 190
Met Ile Thr Met Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Leu Met
195 200 205
Met Ser Asn Pro Lys Val Lys Leu Leu Val Ala Thr Gly Gly Pro Phe
210 215 220
Ile Val Asn Thr Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys
245 250 255
Ala Ala Ile Asp Ile Val Ser Gly Ala Ser Phe Asp Asn Asn Val Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser Ile Ser Asp Met
275 280 285
Leu Ile Tyr His Met Lys Lys Asn Gly Ala Tyr Glu Ile Val Ser Gln
290 295 300
Asp Met Ile Glu Lys Leu Asp Lys Leu Val Ser Gln Glu Asn Gly Lys
305 310 315 320
Pro Lys Thr Glu Phe Val Gly Lys Ser Ala Lys Tyr Ile Leu Glu Lys
325 330 335
Leu Gly Ile Tyr Val Asp Asp Ser Ile Arg Leu Ile Ile Cys Arg Thr
340 345 350
Ser Lys Asn His His Leu Val Gln Glu Glu Met Leu Met Pro Ile Leu
355 360 365
Pro Ile Val Ser Val Ser Asp Val Asp Ile Ala Ile Glu Tyr Ala Tyr
370 375 380
Glu Ala Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys Asn
385 390 395 400
Val Glu Lys Leu Ser Lys Met Ala Lys Lys Leu Glu Ala Thr Ile Phe
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ser Gly Ile Gly Val Gly Gly Glu Gly
420 425 430
His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser
435 440 445
Ala Lys Ser Phe Cys Arg Ile Arg Arg Cys Val Met His Asp Ser Phe
450 455 460
Ser Ile Arg
465
<210> 122
<211> 467
<212> PRT
<213> 优杆菌科细菌（Eubacteriaceae bacterium）
<400> 122
Met Asn Ala Glu Leu Leu Gln Asp Val Val Arg Gln Val Leu Ser Glu
1 5 10 15
Met Lys Leu Glu Ser Ser Asn Ile Leu Ser Asn Glu Tyr Asn Tyr Gly
20 25 30
Ile Phe Asp Asp Met Glu Ala Ala Ile Asn Ala Ser Glu Thr Ala Gln
35 40 45
Arg Lys Leu Phe Glu Cys Ser Val Gln Gln Arg Asn Glu Phe Ala Asn
50 55 60
Val Ile Arg Arg Glu Val Leu Lys Lys Asp Asn Leu Glu Met Ile Ser
65 70 75 80
Arg Asp Ala Val Glu Glu Thr Glu Ile Gly Arg Phe Glu Asp Lys Ile
85 90 95
Leu Lys Asn Lys Val Ala Ala Glu Lys Thr Pro Gly Met Glu Asp Leu
100 105 110
Thr Thr Arg Ala Leu Thr Gly Lys Asp Gly Leu Met Ile Glu Glu Tyr
115 120 125
Cys Pro Phe Gly Val Ile Gly Ser Ile Thr Pro Thr Thr Asn Pro Thr
130 135 140
Glu Thr Leu Ile Asn Asn Ser Ile Ser Met Ile Val Gly Gly Asn Thr
145 150 155 160
Val Val Phe Ser Pro His Pro Arg Ala Lys Asn Val Ser Ile Lys Leu
165 170 175
Val Lys Met Met Asn Lys Ala Leu Glu Glu Tyr Gly Ala Pro Arg Asn
180 185 190
Met Ile Thr Met Val Lys Glu Pro Ser Ile Glu Asn Thr Asn Leu Met
195 200 205
Met Ser Asn Pro Lys Val Lys Leu Leu Val Ala Thr Gly Gly Pro Phe
210 215 220
Ile Val Asn Thr Val Leu Ser Ser Gly Lys Lys Ala Ile Gly Ala Gly
225 230 235 240
Ala Gly Asn Pro Pro Val Val Val Asp Glu Thr Ala Asp Ile Glu Lys
245 250 255
Ala Ala Ile Asp Ile Val Ser Gly Ala Ser Phe Asp Asn Asn Val Pro
260 265 270
Cys Ile Ala Glu Lys Glu Val Phe Ala Val Asp Ser Ile Ser Asp Met
275 280 285
Leu Ile Tyr His Met Lys Lys Asn Gly Ala Tyr Glu Ile Val Ser Gln
290 295 300
Asp Met Ile Glu Lys Leu Asp Lys Leu Val Ser Gln Glu Asn Gly Lys
305 310 315 320
Pro Lys Thr Glu Phe Val Gly Lys Ser Ala Lys Tyr Ile Leu Glu Lys
325 330 335
Leu Gly Ile Tyr Val Asp Asp Ser Ile Arg Leu Ile Ile Cys Arg Thr
340 345 350
Ser Lys Asn His His Leu Val Gln Glu Glu Met Leu Met Pro Ile Leu
355 360 365
Pro Ile Val Ser Val Ser Asp Val Asp Ile Ala Ile Glu Tyr Ala Tyr
370 375 380
Glu Ala Glu His Gly Asn Arg His Thr Ala Ile Met His Ser Lys Asn
385 390 395 400
Val Glu Lys Leu Ser Lys Met Ala Lys Lys Leu Glu Ala Thr Ile Phe
405 410 415
Val Lys Asn Ala Pro Ser Tyr Ser Gly Ile Gly Val Gly Gly Glu Gly
420 425 430
His Thr Thr Phe Thr Ile Ala Gly Pro Thr Gly Glu Gly Ile Thr Ser
435 440 445
Ala Lys Ser Phe Cys Arg Ile Arg Arg Cys Val Met His Asp Ser Phe
450 455 460
Ser Ile Arg
465
<210> 123
<211> 468
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 123
Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Val Lys
1 5 10 15
Thr Asn Gly Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser
20 25 30
Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Ser Ala Val
35 40 45
His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu
50 55 60
Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Gln Asn Lys Glu Val
65 70 75 80
Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp
85 90 95
Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu
100 105 110
Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val
115 120 125
Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn
130 135 140
Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly
145 150 155 160
Asn Ala Val Val Phe Asn Gly His Pro Cys Ala Lys Lys Cys Val Ala
165 170 175
Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro
180 185 190
Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp
195 200 205
Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly
210 215 220
Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly
225 230 235 240
Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile
245 250 255
Glu Lys Ala Gly Arg Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn
260 265 270
Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala
275 280 285
Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn
290 295 300
Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn
305 310 315 320
Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala
325 330 335
Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Val Lys
340 345 350
Cys Ile Ile Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu
355 360 365
Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu
370 375 380
Ala Ile Lys Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala
385 390 395 400
Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu
405 410 415
Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val
420 425 430
Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr
435 440 445
Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys
450 455 460
Val Leu Ala Gly
465
<210> 124
<211> 20
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 124
Leu Gln Lys Asn Asn Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp
1 5 10 15
Val Gly Lys Asp
20
<210> 125
<211> 15
<212> PRT
<213> 短乳酸杆菌（Lactobacillus brevis）
<400> 125
Ile Gly Pro Lys Gly Ala Pro Asp Arg Lys Phe Val Gly Lys Asp
1 5 10 15
<210> 126
<211> 14
<212> PRT
<213> 植物发酵梭菌（Clostridium phytofermentans）
<400> 126
Ile Thr Pro Lys Gly Leu Asn Arg Asn Cys Val Gly Lys Asp
1 5 10
<210> 127
<211> 18
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 127
Ser Phe Ala Gly Val Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr
1 5 10 15
Ile Ala
<210> 128
<211> 22
<212> PRT
<213> 短乳酸杆菌（Lactobacillus brevis）
<400> 128
Thr Tyr Cys Gly Thr Gly Val Ala Thr Asn Gly Ala His Ser Gly Ala
1 5 10 15
Ser Ala Leu Thr Ile Ala
20
<210> 129
<211> 18
<212> PRT
<213> 植物发酵梭菌（Clostridium phytofermentans）
<400> 129
Ser Tyr Ala Ala Ile Gly Phe Gly Gly Glu Gly Phe Cys Thr Phe Thr
1 5 10 15
Ile Ala

Claims (89)

1.分离的核酸分子，其选自：
(a)编码被称为SEQ ID NO：1、2或3的或表4中的氨基酸序列的核酸分子，其中所述氨基酸序列包含对应于位置I66的氨基酸替换；
(b)在高严格杂交条件下与(a)所述的核酸杂交并且包含编码对应于位置I66的氨基酸替换的核酸序列的核酸分子；和
(c)与(a)或(b)互补的核酸分子。
2.根据权利要求1所述的分离的核酸分子，其中位置I66处的所述氨基酸替换是如表1、2和/或3中所示的氨基酸替换。
3.根据权利要求1或2所述的分离的核酸分子，其中除位置I66处的所述替换之外，所述氨基酸序列包含位于表1、2和/或3中所示的其它氨基酸变体位置的一个或多个氨基酸替换。
4.根据权利要求1-3中任一项所述的分离的核酸分子，其中除位置I66处所述的替换之外，所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。
5.根据权利要求1-4中任一项所述的分离的核酸分子，其中除所述一个或多个氨基酸替换以外，所述氨基酸序列与SEQ ID NO：1、2或3或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性或者是相同的。
6.根据权利要求1-5中任一项所述的分离的核酸分子，其中所述氨基酸序列包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个表1、2和/或3中所示的氨基酸替换。
7.根据权利要求1-6中任一项所述的分离的核酸分子，其中所述氨基酸序列包含如表1、2和/或3中所示的氨基酸替换。
8.含有根据权利要求1-7中任一项所述的核酸分子的载体。
9.根据权利要求8所述的载体，其中所述载体是表达载体。
10.根据权利要求8或9所述的载体，其中所述载体包含双链DNA。
11.包含被称为SEQ ID NO：1、2或3的或表4中所提及的氨基酸序列的分离的多肽，其中所述氨基酸序列包含对应于位置I66的氨基酸替换。
12.根据权利要求11所述的分离的多肽，其中位置I66处的所述氨基酸替换是如表1、2和/或3中所示的氨基酸替换。
13.根据权利要求11或12所述的分离的多肽，其中除对应于氨基酸位置I66处的所述替换之外，所述氨基酸序列包含位于表1、2和/或3中所示的其它氨基酸变体位置的一个或多个氨基酸替换。
14.根据权利要求11-13中任一项所述的分离的多肽，其中除位置I66处所述的替换之外，所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。
15.包含被称为SEQ ID NO：1、2或3的或表4中所提及的氨基酸序列的分离的多肽，其中所述氨基酸序列包含对应于位置I66的氨基酸替换，其中除对应于位置I66的所述氨基酸替换外，所述氨基酸序列与被称为SEQ ID NO：1、2或3的或表4中所提及的氨基酸序列具有至少65％、70％、75％、80％、85％、90％、95％、98％或99％的序列同一性或者是相同的。
16.根据权利要求15所述的分离的多肽，其中位置I66处的所述氨基酸替换是如表1、2和/或3中所示的氨基酸替换。
17.根据权利要求15或16所述的分离的多肽，其中除对应于氨基酸位置I66处的所述替换之外，所述氨基酸序列包含位于表1、2和/或3中所示的其它氨基酸变体位置的一个或多个氨基酸替换。
18.根据权利要求15-17中任一项所述的分离的多肽，其中除位置I66处所述的替换之外，所述氨基酸序列包含表1、2和/或3中所示的一个或多个氨基酸替换。
19.根据权利要求15-18中任一项所述的分离的多肽，其中所述氨基酸序列还包含1至100个氨基酸位置中的保守氨基酸替换，其中所述位置是除表1、2和/或3中所示的一个或多个氨基酸替换以外的。
20.根据权利要求11-19中任一项所述的分离的多肽，其中除表1、2和/或3中所示的一个或多个氨基酸替换以外，与亲代序列相比，所述氨基酸序列在2至300个氨基酸位置处不包含修饰，其中所述位置选自被称为SEQ ID NO：1、2或3的或表4中所提及的2、3、4或5条氨基酸序列之间相同的那些。
21.根据权利要求11-20中任一项所述的分离的多肽，其中所述氨基酸序列包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个表1、2和/或3中所示的氨基酸替换。
22.根据权利要求11-21中任一项所述的分离的多肽，其中所述氨基酸序列包含如表1、2和/或3中所示的变体的氨基酸替换。
23.根据权利要求11-22中任一项所述的分离的多肽，其中所述多肽编码醛脱氢酶。
24.根据权利要求11-23中任一项所述的分离的多肽，其中所述多肽可以将3-羟基丁酰基-CoA转化为3-羟基丁醛。
25.根据权利要求11-23中任一项所述的分离的多肽，其中所述多肽可以将4-羟基丁酰基-CoA转化为4-羟基丁醛。
26.根据权利要求11-25中任一项所述的分离的多肽，其中所述多肽相对于亲代多肽具有更高的活力。
27.根据权利要求11-24中任一项所述的分离的多肽，其中所述多肽对于3-羟基-(R)-丁酰基-CoA具有高于3-羟基-(S)-丁酰基-CoA的活力。
28.根据权利要求11-24中任一项所述的分离的多肽，其中所述多肽对于3-羟基丁酰基-CoA具有高于乙酰-CoA的特异性。
29.根据权利要求11-23或25中任一项所述的分离的多肽，其中所述多肽对于4-羟基丁酰基-CoA具有高于乙酰-CoA的特异性。
30.根据权利要求11-25中任一项所述的分离的多肽，其中所述多肽在细胞或细胞提取物中产生减少的副产品。
31.根据权利要求30所述的分离的多肽，其中所述副产品是乙醇或4-羟基-2-丁酮。
32.根据权利要求11-25中任一项所述的分离的多肽，其中所述多肽相对于亲代多肽具有更高的kcat。
33.细胞，其包含根据权利要求8-10中任一项所述的载体。
34.细胞，其包含根据权利要求1-7中任一项所述的核酸。
35.根据权利要求34所述的细胞，其中所述核酸分子被整合到所述细胞的染色体中。
36.根据权利要求35所述的细胞，其中所述整合是位点-特异的。
37.根据权利要求33-36中任一项所述的细胞，其中表达了所述核酸分子。
38.细胞，其包含根据权利要求11-32中任一项所述的多肽。
39.根据权利要求33-38中任一项所述的细胞，其中所述细胞是微生物。
40.根据权利要求39所述的细胞，其中所述微生物是细菌、酵母或真菌。
41.根据权利要求33-38中任一项所述的细胞，其中所述细胞是分离的真核细胞。
42.根据权利要求33-41中任一项所述的细胞，其中所述细胞包含产生3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或其酯或酰胺的途径。
43.根据权利要求33-41中任一项所述的细胞，其中所述细胞包含产生4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)或其酯或酰胺的途径。
44.根据权利要求33-43中任一项所述的细胞，其中所述细胞能够发酵。
45.根据权利要求33-44中任一项所述的细胞，还包含所述多肽的至少一种底物。
46.根据权利要求45所述的细胞，其中所述底物是3-羟基丁酰基-CoA。
47.根据权利要求46所述的细胞，其中所述底物是3-羟基-(R)-丁酰基-CoA。
48.根据权利要求46或47所述的细胞，其中所述细胞对于3-羟基-(R)-丁酰基-CoA具有高于3-羟基-(S)-丁酰基-CoA的活力。
49.根据权利要求45所述的细胞，其中所述底物是4-羟基丁酰基-CoA。
50.根据权利要求11-32中任一项所述的多肽作为生物催化剂的用途。
51.组合物，其包含根据权利要求11-32中任一项所述的多肽和所述多肽的至少一种底物。
52.根据权利要求51所述的组合物，其中所述多肽可以与所述底物在体外条件下反应。
53.根据权利要求51或52所述的组合物，其中所述底物是3-羟基丁酰基-CoA。
54.根据权利要求53所述的组合物，其中所述底物是3-羟基-(R)-丁酰基-CoA。
55.根据权利要求51或52所述的组合物，其中所述底物是4-羟基丁酰基-CoA。
56.培养基，其包含根据权利要求33-49中任一项所述的细胞。
57.构建宿主株的方法，其包括将根据权利要求8-10中任一项所述的载体引入能够发酵的细胞中。
58.用于生产3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或其酯或酰胺的方法，其包括培养根据权利要求33-49中任一项所述的细胞以产生3-HBal和/或1,3-BDO或其酯或酰胺。
59.用于生产4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)或其酯或酰胺的方法，其包括培养根据权利要求33-49中任一项所述的细胞以产生4-HBal和/或1,4-BDO或其酯或酰胺。
60.根据权利要求58或59所述的方法，其中所述细胞处于基本厌氧的培养基中。
61.根据权利要求58-60中任一项所述的方法，其进一步包括分离或纯化3-HBal和/或1,3-BDO，或者4-HBal和/或1,4-BDO或其酯或酰胺。
62.根据权利要求61所述的方法，其中所述分离或纯化包括蒸馏。
63.包含生物衍生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的培养基，其中所述生物衍生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO具有反映大气二氧化碳吸收源的碳-12、碳-13和碳-14同位素比，并且其中所述生物衍生的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO是通过根据权利要求33-49中任一项所述的细胞或者根据权利要求58-62中任一项所述的方法所产生的。
64.根据权利要求63所述的培养基，其中所述培养基与所述细胞分离。
65.3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或者4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)，其具有反映大气二氧化碳吸收源的碳-12、碳-13和碳-14同位素比，其中所述3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO是通过根据权利要求33-49中任一项所述的细胞或者根据权利要求58-62中任一项所述的方法所产生的。
66.根据权利要求65所述的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO，其中所述3-HBal和/或1,3-BDO或者所述4-HBal和/或1,4-BDO具有至少80％，至少85％，至少90％，至少95％或者至少98％的Fm值。
67.3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或者4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)，其通过根据权利要求33-49中任一项所述的细胞或者根据权利要求58-62中任一项所述的方法产生。
68.3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)，其具有反映大气二氧化碳吸收源的碳-12、碳-13和碳-14同位素比，其中所述3-HBal和/或1,3-BDO是通过根据权利要求33-49中任一项所述的细胞或者根据权利要求58-62中任一项所述的方法所产生的，其中所述3-HBal和/或1,3-BDO是对R形式对映体富集的。
69.根据权利要求68所述的3-HBal和/或1,3-BDO，其中所述3-HBal和/或1,3-BDO具有至少80％，至少85％，至少90％，至少95％或者至少98％的Fm值。
70.通过根据权利要求33-49中任一项所述的细胞或者根据权利要求58-62中任一项所述的方法产生的3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)，其中所述3-HBal和/或1,3-BDO是对R形式对映体富集的。
71.根据权利要求70所述的3-HBal和/或1,3-BDO，其中所述R形式是大于95％、96％、97％、98％、99％、99.5％或99.9％的3-HBal和/或1,3-BDO。
72.组合物，其包含根据权利要求68-71中任一项所述的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO和分别除3-HBal和/或1,3-BDO或者4-HBal或1,4-BDO以外的化合物。
73.根据权利要求62所述的组合物，其中除所述3-HBal和/或1,3-BDO或者所述4-HBal和/或1,4-BDO以外的化合物是分别产生3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO或者表达根据权利要求11-32中任一项所述的多肽的细胞的一部分。
74.组合物，其包含根据权利要求65-71中任一项所述的3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO，或者产生所述3-HBal和/或1,3-BDO或者所述4-HBal和/或1,4-BDO的细胞的细胞裂解液或培养上清液。
75.根据权利要求65-71中任一项所述的包含所述3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的产品，其中所述产品是塑料、弹性纤维、聚氨脂、聚酯，聚羟基脂肪酸酯，聚-4-羟基丁酸酯(P4HB)或其共聚物、聚(四亚甲基醚)二醇(PTMEG)、聚对苯二甲酸丁二醇酯(PBT)、聚氨脂-聚脲共聚物、尼龙、有机溶剂、聚氨酯树脂、聚酯树脂、降血糖剂、丁二烯和/或丁二烯-基产品。
76.根据权利要求75所述的产品，其中所述产品是化妆品产品或食品添加剂。
77.根据权利要求75或76所述的产品，其包含至少5％，至少10％，至少20％，至少30％，至少40％或至少50％的生物衍生的3-HBal和/或1,3-BDO，或者生物衍生的4-HBal和/或1,4-BDO。
78.根据权利要求75-77中任一项所述的产品，其中所述产品作为重复单元包含所产生的3-HBal和/或1,3-BDO，或者所产生的4-HBal和/或1,4-BDO的部分。
79.通过模制根据权利要求75、77或78中任一项所述的产品所获得的模制产品。
80.用于生产根据权利要求75-78中任一项所述的产品的方法，其包括将3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO与自身或另一种化合物在生产所述产品的反应中化学反应。
81.用于生产根据权利要求79所述的产品的方法，其包括将3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO与自身或另一种化合物在生产所述产品的反应中化学反应。
82.用于生产3-羟基丁醛(3-HBal)和/或1,3-丁二醇(1,3-BDO)或其酯或酰胺的方法，其包括向根据权利要求11-32中任一项所述的多肽提供底物并将所述底物转化为3-HBal和/或1,3-BDO，其中所述底物是1,3-羟基丁酰基-CoA的外消旋混合物。
83.根据权利要求82所述的方法，其中所述3-HBal和/或1,3-BDO是对R形式对映体富集的。
84.用于生产4-羟基丁醛(4-HBal)和/或1,4-丁二醇(1,4-BDO)或其酯或酰胺的方法，其包括向根据权利要求11-32中任一项所述的多肽提供底物并将所述底物转化为4-HBal和/或1,4-BDO，其中所述底物是1,4-羟基丁酰基-CoA。
85.根据权利要求82-84中任一项所述的方法，其中所述多肽存在于细胞中，细胞裂解液中，或者分离自细胞或细胞裂解液。
86.用于生产3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的方法，其包括培育根据权利要求33-49中任一项所述的的细胞裂解液以生产3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO。
87.根据权利要求86所述的方法，其中将所述细胞裂解液与第二细胞裂解液混合，其中所述第二细胞裂解液包含酶活力以生产根据权利要求11-32中任一项所述的多肽的底物或者3-HBal和/或1,3-BDO或者4-HBal和/或1,4-BDO的下游产品。
88.用于生产根据权利要求11-32中任一项所述的多肽的方法，其包括在细胞中表达所述多肽。
89.用于生产根据权利要求11-32中任一项所述的多肽的方法，其包括体外转录和翻译根据权利要求1-7中任一项所述的核酸或者根据权利要求4-6的载体以产生所述多肽。
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