使用新的酶将甲基乙二醛转化为羟基丙酮及其应用的制作方法

本发明涉及新的甲基乙二醛还原酶(mgr)酶，其用于将甲基乙二醛有效转化为羟基丙酮。本发明更特别地涉及一种用于使用所述酶将甲基己二醛有效转化为羟基丙酮的方法，涉及一种用于使用过表达所述酶的微生物产生1,2-丙二醇的方法，且涉及所述微生物。1,2-丙二醇或丙二醇，具有式c3h8o2或ho-ch2-choh-ch3的c3二醇，是广泛使用的化学制品，以其cas号57-55-6公知，其已经具有大量工业应用，举例来说，例如在制药、化妆品、航空学、食品业、烟草和纺织品中的应用。它是无色、几乎无味、清澈的粘性液体，具有微弱的甜味、吸湿性且可与水、丙酮和氯仿混溶，主要用作不饱和聚酯树脂的成分，但也用作用于航空器的液体去垢剂、冷却剂、防冻和除冰液的成分。它还可以用作食品中的润湿剂(e1520)、溶剂和防腐剂以及用于烟草产品。乙烯衍生物被认为比丙烯衍生物具有更大的毒性，自从1993-1994年以来，丙二醇已经越来越多地用作乙烯衍生物的替代品。1,2-丙二醇目前主要通过化学方法使用氧化丙烯水合作用工艺产生，这消耗大量的水、采用高毒性的物质且产生副产物例如叔丁醇和1-苯基乙醇。这样的化学工艺还有代表性地导致产生(r)-1,2-丙二醇和(s)-1,2-丙二醇的混合物。微生物中产生1,2-丙二醇的天然的或合成的代谢途径代表了有吸引力的替代选择，因为它缓解了以上所述的许多问题。迄今为止，两个天然生物途径已经经表征用于在微生物中从糖开始发酵产生1,2-丙二醇。在第一个途径中，该第一个途径在厌氧条件下在大肠杆菌中起作用，将6-脱氧糖(例如l-鼠李糖或l-岩藻糖)切割为磷酸二羟丙酮和(s)-乳醛，(s)-乳醛进一步由1,2-丙二醇氧化还原酶还原为(s)-1,2-丙二醇，1,2-丙二醇氧化还原酶也被称为乳醛还原酶(lar)且由fuco基因编码(badia等,1985)。但是，由于脱氧己糖底物的成本提高，因此依赖于该途径的发酵工艺在经济上是不可行的。第二个天然途径涉及常见糖(例如葡萄糖和木糖)的代谢，更具体地包括糖酵解途径随后是丙酮醛途径。该途径将磷酸二羟丙酮转化为甲基乙二醛，然后甲基乙二醛可以被还原为(r)-乳醛或羟丙酮(即丙酮醇)，这取决于还原酶的特性。这两个化合物然后被转变成(r)-1,2-丙二醇。该途径典型地在天然产生(r)-1,2-丙二醇的微生物例如楔形梭状芽胞杆菌(clostridiumsphenoides)和热解糖热厌氧杆菌(thermoanaerobacterthermosaccharolyticum)中观察到。但是，这些生物表现出的生产性能非常有限。考虑到甲基乙二醛途径在肠杆菌科(enterobacteriaceae)中起作用，已经进行了一些研究以工程化改造合成途径，用于在所述微生物(更特别在大肠杆菌)中使用简单碳源来提高1,2-丙二醇的产量(wo98/37204；cameron等,1998；altaras和cameron,1999；huang等,1999；altaras和cameron,2000；berrios-rivera等,2003)。在经重组工程化产生1,2-丙二醇的大肠杆菌菌株中，可以以三步从中心代谢衍生出1,2-丙二醇。甲基乙二醛合成酶将磷酸二羟丙酮转化为甲基乙二醛是强制性的第一步，随后是通过甲基乙二醛还原酶将甲基乙二醛转化为(r)-乳醛或羟丙酮的第二步(cameron等，1998；bennet等,2001；ko等,2005)。已经表明nadph依赖性醛还原酶yqhd更特别地将甲基乙二醛转化为羟丙酮(wo2008/116853)，同时已经表明甘油脱氢酶glda将甲基乙二醛转化为(r)-乳醛(subedi等,2008)。在最后一步中，羟丙酮或乳醛通过不同的酶学活性(特别是甘油脱氢酶(由glda基因编码)或1,2-丙二醇氧化还原酶(由fuco基因编码))转化为1,2-丙二醇(altaras和cameron,2000)。为了进一步提高所述大肠杆菌菌株中1,2-丙二醇的产量，已经用新的突变yqhd酶代替天然的yqhd酶，该新的突变yqhd酶展示出更高的催化效率(即增加的kcat/km)和对甲基乙二醛和nadph的亲和力，特别是突变酶yqhd*(g149e)(yqhd:kcat/km＝0.4mm-1.s-1和km＝2.09mm,相对于yqhd*(g149e):kcat/km＝0.8mm-1s-1和km＝2.92mm)(wo2011/012697)。但是，发明人已经观察到，yqhd*(g149e)必须在微生物内高表达以便允许产生1,2-丙二醇，这导致代谢负荷，因此由于剥夺碳和能源而对微生物产生压力。因此，本领域中存在提供可供选择的甲基乙二醛还原酶(mgr)的需求，其能够在更低表达水平下以更高的催化效率还原甲基乙二醛代谢，以便有效地产生(r)-1,2-丙二醇。本发明解决了以上所述的本领域的需求。发明人事实上已经惊人地发现，目前被称为是使用多种底物例如己醛、甘油醛或丁醛的还原酶的酶还能够使用甲基乙二醛作为底物，由此能够将所述底物转化为羟丙酮。预料不到的是，所述酶展示出比yqhd*(g149e)高至少7倍的对甲基乙二醛和nadph的催化功效。基于该发现，发明人已经修饰了现有的产生1,2-丙二醇的大肠杆菌菌株，且观察到所述菌株能够产生更多以克计1,2-丙二醇/生物量，同时保持比yqhd*(g149e)更低表达水平的这些新的甲基乙二醛还原酶。因此本发明在本文中提供了一种改进的通过使用所述酶在微生物中将甲基乙二醛有效转化为羟丙酮的方法，一种在微生物中产生1,2-丙二醇的方法，以及过表达所述酶的产生1,2-丙二醇的微生物。发明详述应理解，以下详细描述不是限制性的，并且可以在不偏离本发明范围的情况下进行多种修饰、替换、省略和改变。还应理解，本文所用的术语是为了描述本发明的具体实施方案的目的，并且不意欲进行限制。本文在上下文中引用的所有公开、专利和专利申请，以其整体通过引用并入本文。此外，除非另有说明，本文所用的所有技术和科学术语具有与本领域技术人员通常理解的相同的含义。常规微生物和分子生物学技术也是本领域公知且常用的那些。这样的技术对本领域技术人员来说是熟知的，并且在文献中有完整的解释。除非上下文清楚地另有说明，否则单数形式“一”(“a”和“an”)和“该”包括复数提及。术语“包含”、“含有”、“涉及”或“包括”或变化例如“包含”(“comprises,”“comprising”)、“涉及的”(“involved”)、“包括”(“includes”、“including”)在本文中以包括的意思使用，即用于指定所指特征存在但不排除本发明的多个实施方案中又一特征的存在或添加。术语酶的“活性”、“催化活性”或“功能”在本发明的上下文中是指由所述酶催化的用于将其一种或多种相应的底物转化成一种或多种另外分子(产物)的反应。如本领域公知的，酶的活性可以通过测量其催化效率和/或米氏常数来评估。酶的“催化效率”或“专一性常数”提供了其性能的直接度量，或者换句话说，提供了酶如何有效地将其一种或多种底物转化为一种或多种产物的直接度量。事实上，催化效率越高，将给定量的一种或多种底物转化成一种或多种产物所需的酶越少。专一性常数的比较也可用作酶对于不同底物的偏爱的度量(即底物专一性)。被称为米-曼二氏(michaelis–menten)模型的以下等式可用于描述酶的动力学：其中，e、s、es和p分别代表酶、底物、酶-底物复合物和产物。符号kf、kr和kcat分别表示“正向”结合底物、“反向”释放底物、以及将一种或多种底物“催化”转化为一种或多种产物的速率常数。催化效率等于kcat/km比率。“催化常数”(kcat)是当酶被底物饱和时产物形成的速率，以m-1s-1表示。换言之，它是指每单位时间经酶转化为产物的底物分子的数目。“米氏常数”(km)转而是酶对于其底物的亲和力的度量(km越低，亲和力越高)，以m表示。更具体地其定义如下：，且等于酶以其最大速率一半的速率将一种或多种底物转化为一种或多种产物的底物浓度。km越低，亲和力越高。测量酶的上述参数在本领域技术人员的技能范围内(segel，1993)。术语“甲基乙二醛”或“mgr”是指活性为将羰基官能团例如醛基或酮基官能团还原为羧基官能团，即在本文中将甲基乙二醛转化成羟丙酮或乳醛的酶。所述活性可以是nadph依赖性或nadh依赖性的(即辅因子依赖性的)，且可以在需氧和/或厌氧情况下发生。在本发明的上下文中，优选的甲基乙二醛还原酶是将甲基乙二醛转化为羟丙酮的甲基乙二醛还原酶。本文所使用的术语“微生物”是指活的微生物生物体，其可以是单细胞或多细胞生物体，并且一般可在自然界中发现。在本发明的上下文中，微生物优选是细菌、酵母或真菌。更优选地，本发明的微生物属于细菌科肠杆菌科(enterobacteriaceae)、梭菌科(clostridiaceae)、芽胞杆菌科(bacillaceae)、链霉菌科(streptomycetaceae)和棒状杆菌科(corynebacteriaceae)的或酵母科酵母科(saccharomycetaceae)。甚至更优选地，根据本发明的微生物是肠杆菌科细菌大肠杆菌(escherichiacoli)或肺炎克雷伯杆菌(klebsiellapneumoniae)，梭菌科楔形梭状芽胞杆菌(clostridiumsphenoides)或热解糖热厌氧杆菌(thermoanaerobacteriumthermosaccharolyticum)，棒状杆菌科细菌谷氨酸棒杆菌(corynebacteriumglutamicum)，或酵母科酵母酿酒酵母(saccharomycescerevisiae)。最优选地，本发明的微生物是大肠杆菌。术语“遗传修饰的微生物”和“重组微生物”可互换，且是指在自然界中未发现并因此在遗传上不同于其天然相似物的如上定义的微生物。换言之，它是指通过引入和/或通过缺失和/或通过修饰其遗传元件而修饰的微生物。这样的修饰可以例如通过基因工程化改造或通过在特定选择压力下通过将定点诱变与进化相结合来强迫新代谢途径发展和进化来进行(参见例如通过引用并入本文的wo2005/073364或wo2008/116852)。可以通过调节一种或多种内源基因的表达水平来对微生物进行遗传修饰。通过“调节”，在本文中是指该基因的表达水平可以上调(即过表达)、下调(即低表达或弱化)。通过“上调”或“过表达”感兴趣的基因，在本文中意指与未修饰的微生物相比增加微生物中所述基因的表达水平。与之相反，“下调”、“低表达”或“弱化”感兴趣的基因意指与未修饰的微生物相比降低微生物中所述基因的表达水平。感兴趣的基因的表达还可以被完全废除，这意指该基因的表达水平是零。因此上述调节可以导致基因产物的活性增强或者基因产物的较低或零活性。“基因”在本文中意指包含编码感兴趣的蛋白质的至少一个区域的核苷酸序列。所述区域可以进一步在各个5’和/或3’末端侧接有非翻译区(utr，称为5’utr和/或3’utr)，非翻译区可以含有控制蛋白质合成的调控元件。为了有利于本发明的理解，本申请所述的基因根据其标准命名法命名(、demerec等，1966)；但是考虑到本文提供了由每个基因编码的蛋白质的氨基酸序列，不可以将这些名称解释为限制性的，特别是关于所述基因的起源物种。术语“内源基因”在本文中是指在微生物中天然存在的如上文定义的基因。内源基因尤其可以通过以下方式过表达：通过除了内源调控元件以外另外引入有助于上调的异源序列，或通过用这样的异源序列替换那些内源调控元件，或通过在微生物内在染色体上(引入至染色体中)或在染色体外(例如引入至质粒或载体中)引入一个或多个补充拷贝的内源基因。在这方面，可以通过本领域公知的方法例如通过遗传重组引入若干拷贝的基因。与之相比，当基因在染色体外表达时，其可以由多种类型的质粒携带，这些质粒可以在其复制起点和其在细胞中的拷贝数方面有所不同，这取决于该质粒能够在其中复制的微生物。例如，质粒转化的微生物可以含有1个至5个拷贝的该质粒，或者约20拷贝的该质粒，或者甚至高达500拷贝的该质粒，这取决于所选择的质粒的性质。在其复制起点和其在细胞中的拷贝数方面有所不同的多种质粒是本领域熟知的，并且可以由熟练的专业人员出于这样的目的容易地选择。可以在大肠杆菌中复制的低拷贝数的质粒的实例包括但不限于psc101质粒(严紧复制)、rk2质粒(严紧复制)、以及pacyc和prsf1010质粒，而可以在大肠杆菌中复制的高拷贝数质粒的实例是pskbluescriptii。调节内源基因表达的另一方式是用一个更强或更弱的启动子交换其启动子(即野生型启动子)以上调或下调其表达水平。适用于这样目的的启动子可以是同源(起源于同一物种)或异源(起源于不同物种)或人工(从头设计合成)的，并且是本领域熟知的。选择适当的用于调节内源基因表达的启动子是在本领域技术人员的技能内的。最便于增加基因表达水平的启动子是本领域技术人员熟知的：其中这些包括启动子ptrc、ptac、plac，和λ启动子pr和pl。这些启动子可以是可以被特定化合物或被具体外部条件例如温度或光诱导的，和/或可以是同源的或异源的。内源基因表达水平还可以通过向其编码序列中引入突变来增加或降低。可以通过使用例如聚合酶链式反应(pcr)的定点诱变、通过例如经由诱变剂(紫外线或化学试剂例如亚硝基胍(ntg)或甲磺酸乙酯(ems))或dna改组或易错pcr的随机诱变技术来引入突变。或者，可以进行一个内源基因的全部或部分的缺失，以完全抑制其在微生物内的表达。另外，或替代性地，微生物可以经遗传修饰以过表达一种或多种外源基因，条件是将所述基因引入具有对于其在宿主微生物中表达所必需的所有调控元件的微生物中。用外源dna遗传修饰或转化微生物是本领域技术人员的常规作业。“外源基因”在本文中意指在一种微生物中非天然存在的基因。为了在微生物中表达(即过表达)外源基因，可将此类基因可以直接整合到微生物染色体中，或在微生物内在染色体外表达，如上文所解释的。有利地，根据本发明的外源基因是同源基因。在本发明的上下文中，术语“同源基因”或“同源物”不仅指由两个物种(即微生物物种)从一个理论上共同遗传祖先继承的基因，还包括可以是遗传上不相关的但是已经进化成编码进行相似功能和/或具有相似结构(即功能同源物)的蛋白质的基因。因此，术语“功能同源物”在本文中是指编码功能同源的蛋白质的基因。使用可在数据库例如uniprot(对于蛋白质)、genbank(对于基因)或ncbi(对于蛋白质或基因)中获得的信息，熟练的专业人员能够容易地确定微生物的特定蛋白质和/或基因的序列，并且基于该序列鉴定在另一微生物中的等同蛋白质或基因或其同源物的序列。可以例如通过将一个微生物的特定基因(或蛋白质)序列与可在上述数据库中发现的其它微生物的基因(或蛋白质)序列或基因组(或蛋白质组)比对来进行该常规工作。有利地，这样的序列比对可以使用altschul等(1990)开发的blast算法进行。一旦确定了那些序列之间的序列同源性，可以衍生出共有序列，并将其用于设计简并探针用于克隆相关微生物的对应的同源基因(和由此产生的同源蛋白质)。分子生物学的这些常规方法是本领域技术人员熟知的。还应理解的是，在本发明的上下文中，编码感兴趣的蛋白质的外源基因应该在特定微生物中表达，此基因的合成版本优选通过用所述微生物的编码相同氨基酸的偏爱密码子代替非偏爱密码子或较不偏爱的密码子来构建。事实上，本领域公知的是密码子使用在微生物物种之间是不同的，这可以影响感兴趣的蛋白质的重组表达水平。为了克服此问题，已经开发了密码子优化法，其由graf等(2000)、deml等(2001)和davis&olsen(2011)广泛描述。尤其是已经开发了用于密码子优化确定的一些软件程序，例如软件(lifetechnologies)或(genscript)的optimumgenetm软件。换言之，编码感兴趣的蛋白质的外源基因优选是经密码子优化以在特定微生物中表达。微生物还可以经遗传修饰以增加或降低在该微生物中天然或非天然表达的一种或多种蛋白质的活性。增加此类活性可以通过提高蛋白质催化效率(如果该蛋白质是酶)和/或降低蛋白质周转(turnover)来实现。提高蛋白质催化效率意指增加kcat和/或降低对给定底物和/或给定辅因子的km，和/或增加对给定抑制剂的ki。ki也是本领域技术人员能够确定的一种米-曼二氏(michaelis-menten)常数(segel，1993)。降低蛋白质周转意指稳定蛋白质。提高蛋白质催化效率和/或降低蛋白质周转的方法是本领域技术人员熟知的。那些方法包括用序列合理地工程化和/或结构分析和定向诱变，以及随机诱变和筛选。稳定蛋白质还可以通过在蛋白质的n末端或c末端添加“标签”肽序列来实现。这样的标签是本领域熟知的，且其中包括谷胱甘肽-s-转移酶(gst)。如本文所使用的术语“突变体”是指其序列在至少一个位置上被修饰的功能蛋白质或功能基因(即所述蛋白质的至少一个氨基酸或所述基因的至少一个核苷酸分别被修饰)。应理解，此至少一个序列修饰产生与野生型或亲本蛋白质或者野生型或亲本基因相比具有提高的生物学活性的功能蛋白突变体或功能基因突变体。增加蛋白质活性还可以通过提高蛋白质表达来实现，例如通过降低蛋白质翻转、降低信使rna(mrna)翻转、增加编码所述蛋白质的基因的转录、或增加mrna翻译。降低mrna翻转可以通过修饰5’-非翻译区(5’-utr)和/或编码区和/或3’-utr的基因序列来实现(carrier和keasling,1999)。根据上文所述的方法，增加基因的转录(无论是内源基因还是外源基因)可以通过增加微生物中该基因的拷贝数和/或通过将所述基因置于较强的启动子的控制下来实现。增加mrna的翻译可以通过修饰核糖体结合位点(rbs)来实现。rbs是mrna上的当起始蛋白质翻译时由核糖体结合的序列。其可以是真核细胞中的mrna的5’帽、原核细胞中的起始密码子aug的上游6-7个核苷酸区(也被称为shine-dalgarno序列)、或病毒中的内部核糖体进入位点(ires)。通过修饰此序列，可能改变蛋白翻译起始速率以成比例地改变其产生速率，并且控制其在细胞内部的活性。还可能通过使用软件rbscalculator(salis,2011)使rbs序列的强度优化以实现目标的翻译起始速率。基于mrna的性质选择rbs序列是在本领域技术人员的技能范围内的。与之相反，降低蛋白质的活性可以意指通过突变编码所述蛋白质的基因来降低其特定催化活性，或者通过缺失所述基因的编码区来降低其表达。术语“发酵工艺”、“发酵”或“培养”在本文中可互换使用以表示微生物的生长。术语“发酵条件”是指允许给定微生物生长的实验条件。微生物生长通常在具有合适的生长培养基的发酵罐中进行，所述培养基适合正在使用的微生物并且可以由本领域技术人员容易地确定。在本发明的上下中，“发酵转化”意指当在适当的发酵条件下培养微生物时发生甲基乙二醛转化成羟丙酮。“培养基”在本文中意指包含对微生物的维持和/或生长至关重要或有利的营养素的培养基(例如无菌液体培养基)，所述营养素例如碳源或碳基质；氮源，例如蛋白胨、酵母提取物、肉类提取物、麦芽提取物、尿素、硫酸铵、氯化铵、硝酸铵和磷酸铵；磷源，例如磷酸二氢钾或磷酸氢二钾；微量元素(例如金属盐)，例如镁盐、钴盐和/或锰盐；以及生长因子，例如氨基酸和维生素。根据本发明的术语“碳的来源”、“碳源”或“碳基质”是指微生物能够代谢并且含有至少一个碳原子的任何分子。根据本发明的优选的碳源的实例包括但不限于碳水化合物。术语“碳水化合物”是如上文定义的并且进一步包含两个原子的氢和一个原子的氧的碳源。co2不是碳水化合物，因为其不含有氢。碳水化合物的实例包括但不限于单糖例如葡萄糖、果糖、甘露糖、木糖、阿拉伯糖、半乳糖等，二糖例如蔗糖、纤维二糖、麦芽糖、乳糖等，寡糖例如棉子糖、水苏糖、麦芽糊精等，多糖例如纤维素、半纤维素、淀粉等，甲醇，甲醛和甘油。根据本发明的特别优选的碳水化合物是阿拉伯糖、果糖、半乳糖、葡萄糖、乳糖、麦芽糖、蔗糖、木糖和其任意混合物。更优选地，碳水化合物选自葡萄糖、木糖、蔗糖或其混合物。甚至更优选地，优选的碳水化合物是葡萄糖和木糖的混合物。在本发明的一个优选实施方案中，碳源衍生自可再生原料。可再生原料定义为某些工业工艺所需的原材料，其能够以短暂的延迟和足以允许其转化为所需产物的量再生。预处理或未预处理的植物生物质是特别优选的可再生碳源。其它定义在本说明书全文中提供。可以参考包括本发明的优选实施方案和本文中包含的实施例在内的以下详细描述更容易地理解本发明。在第一方面，本发明涉及一种用于将甲基乙二醛发酵转化成羟丙酮的方法，包括在微生物中表达至少一种甲基乙二醛还原酶的步骤，所述甲基乙二醛还原酶的催化效率kcat/km等于或高于5mm-1s-1，米氏常数km大于0mm且等于或低于11mm。具体地，本发明涉及将甲基乙二醛有效地发酵转化为羟丙酮的方法，包括在微生物中表达至少一种甲基乙二醛还原酶的步骤，所述甲基乙二醛还原酶的催化效率kcat/km等于或高于5mm-1s-1，米氏常数km大于0mm且等于或低于11mm。因此，本发明涉及至少一种具有以上所列特性的酶通过微生物发酵而将甲基乙二醛有效转化为羟丙酮的用途。因此，当甲基乙二醛被至少一种甲基乙二醛还原酶转化成羟丙酮时，实现了本发明的上下文中理解的有效发酵转化，所述甲基乙二醛还原酶的催化效率kcat/km等于或高于5mm-1s-1，米氏常数km大于0mm且等于或低于11mm。事实上，发明人已经发现，与常规甲基乙二醛还原酶相比，特别是与由yqhd或yqhd*基因编码的甲基乙二醛还原酶(其km和kcat/km分别为2.09mm、0.40mm-1s-1以及2.92mm、0.80mm-1s-1)相比，展示出以上活性的酶大大提高了甲基乙二醛到羟丙酮的转化率。因此，根据本发明的酶大大降低了用于进行所述转化的代谢负荷，并因此能够促进微生物的生长。发明人已经更加具体地鉴定了具有进行以上转化的能力的特定酶。根据优选的实施方案，所述甲基乙二醛还原酶选自序列seqidno:1的yjgb酶及其突变体、序列seqidno:3的yahk酶及其突变体、序列seqidno:5的yhdn酶及其突变体、序列seqidno:7的gld酶及其突变体，和其组合。优选的yjgb突变体的实例是序列seqidno:9的yjgb*(n240y)、序列seqidno:125的yjgb*(i165v)和序列seqidno:127的yjgb*(q39r/i165v/a296v)。最优选地，所述甲基乙二醛还原酶是序列seqidno:1的yjgb酶。甚至更优选地，序列seqidno:1的yjgb酶与序列seqidno:3的yahk酶、序列seqidno:5的yhdn酶、序列seqidno:7的gld酶、序列seqidno:11的yafb酶、或序列seqidno:13的yqhd酶或序列seqidno:15的其突变yqhd*(g149e)组合表达。下表1中提供了关于所述酶的相应的氨基酸和核苷酸序列和催化活性的信息。该表明显表明，这些酶表现出甲基乙二醛还原酶活性是未知的。在本发明的一个优选实施方案中，微生物单独过表达甲基乙二醛还原酶或联合过表达甲基乙二醛还原酶与上文所述的另一个甲基乙二醛还原酶。更准确地，在一个优选的实施方案中，以上方法涉及在发酵条件下在包含碳水化合物作为碳源的培养基中培养微生物，所述微生物过表达至少一种编码所述酶的基因并将甲基乙二醛有效转化为羟丙酮。为了这个目的，因此碳源优选被所述微生物通过中心碳代谢的方式使用合适的途径和酶(例如在通过引用并入本文的neidhardt等(1996)中所述的途径和酶)还原为中间代谢产物磷酸二羟丙酮(dhap)。然后通过甲基乙二醛合成酶(ec4.2.3.3)的作用将dhap转化为甲基乙二醛(mg)。如上文所述，在微生物中过表达编码所述酶的基因是在本领域技术人员的能力范围内的。优选地，此过表达能够通过过表达核苷酸序列例如编码每种酶的已知基因或其突变体来实现。所述核苷酸序列可以是已经存在于感兴趣的微生物中的，在这种情况中其被称为内源基因，且可以根据上文所述的任一种方法被过表达。与之相比，当微生物天然地不包含编码这类酶的基因时，所述微生物可以有利地用一种或多种外源核苷酸序列转化，所述外源核苷酸序列例如来自其它微生物的基因或其突变体，其根据上文所述的任一方法编码所述一种或多种酶：所述外源核苷酸序列也被称为过表达的。可以由熟练的专业人员通过将例如所述蛋白质的氨基酸序列上载到uniprot或ncbi数据库中并通过搜索可以在特定微生物中表达的相应编码核苷酸序列而容易地检索到编码特定蛋白质的基因。此外，如何从给定氨基酸序列中推断得到人工核苷酸序列以合成编码感兴趣的特定蛋白质的人工基因对于本领域技术人员来说是可能且熟知的。本领域技术人员能够容易地确定对于培养根据本发明的微生物所必需的培养条件。具体地，细菌可以在20℃至55℃，优选25℃至40℃的温度发酵是公知的。更具体地，大肠杆菌能够在约30℃至约37℃的温度培养。此培养过程可以在分批过程、分批补料过程或连续过程中在需氧、微氧或厌氧条件下进行。“在需氧条件下”发酵意指通过将气体溶解于培养物的液相中将氧气提供至培养物中。这可以通过以下方式实现：(1)将含有氧气的气体(例如空气)鼓泡到液相中，或(2)振荡含有培养基的容器以将顶部空间中含有的氧气转移到液相中。在需氧条件下发酵的主要优势在于作为电子受体的氧气的存在提高了菌株产生atp形式的更多能量用于细胞过程的能力，从而提高菌株的一般代谢。微氧条件可以在本文中使用，被定义为其中将低百分比的氧气(使用含有0.1至10％的氧气并用氮气补充至100％的气体混合物)溶解到液相中的培养条件。与之相比，“厌氧条件”定义为其中在培养基中未提供氧气的培养条件。严格厌氧条件可以通过将惰性气体例如氮气鼓泡到培养基中以去除痕量的其它气体来实现。硝酸盐可以用作电子受体以提高菌株的atp产生和提高其代谢。当应用于涉及产生1,2-丙二醇特别是涉及产生(r)-1,2-丙二醇的微生物发酵过程中时，以上方法更加特别有用。事实上，发明人已经发现，用根据本发明的甲基乙二醛还原酶替代能够产生1,2-丙二醇的大肠杆菌菌株中的yqhd酶(天然或突变的)，在非常低的表达水平下大大增强1,2-丙二醇的产生。因此，在另一方面，本发明涉及一种经遗传修饰用于产生1,2-丙二醇的微生物，其中所述微生物过表达至少一种编码根据本发明的甲基乙二醛还原酶的基因。关于所述甲基乙二醛还原酶的优选实施方案如上文所述。因此，由于根据本发明的甲基乙二醛还原酶直接用于将甲基乙二醛转化为羟丙酮，微生物还优选包含编码序列seqidno:13或seqidno:15的甲基乙二醛还原酶的yqhd或yqhd*基因的缺失。用根据本发明的一种或多种甲基乙二醛还原酶替代或缺失编码天然或突变的yqhd酶的yqhd或yqhd*基因是特别有利的，因为这减轻了与微生物表达yqhd或yqhd*相关的代谢负荷。事实上，由于天然或突变的yqhd酶具有比根据本发明的甲基乙二醛还原酶更低的催化效率，所以其必须被过表达，并且可能占微生物中总蛋白的40％，这在细胞机器上施加了显著水平的压力。术语“经遗传修饰用于产生1,2-丙二醇的微生物”在本文中是指通过引入或缺失遗传元件或通过在专利申请wo2005/073364中所述的进化步骤修饰的微生物。特别地，其是指与未修饰的微生物(即无遗传修饰的)相比，表现出提高的1,2-丙二醇产量的遗传修饰的微生物。这样的微生物是本领域公知的，且尤其已经被广泛描述，例如在全部通过引用并入本文的专利申请wo2008/116848、wo2008/116853、wo2011/012693、wo2011/012697、wo2011/012702或ep2532751中描述。优选的用于产生1,2-丙二醇更特别用于产生(r)1,2-丙二醇的遗传修饰是以下修饰：-过表达选自以下的至少一种基因：编码序列seqidno:17的甲基乙二醛合成酶或其突变体例如seqidno:19的mgsa*(h21q)的mgsa基因、编码序列seqidno:21的甘油脱氢酶或其突变体例如序列seqidno:23的glda*(a160t)(此突变体也是nadh依赖性的)的glda基因、和编码序列seqidno:25的乳醛还原酶的fuco基因；-缺失编码序列seqidno:27的磷酸葡糖酸脱水酶的edd基因和/或编码序列seqidno:29的2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶的eda基因；-通过缺失以下基因中的至少一种来弱化不想要的副产物的合成：编码参与从甲基乙二醛合成乳酸的酶的基因(例如编码序列seqidno:31的乙二醛酶i的gloa基因、编码序列seqidno:33的醛脱氢酶a的alda基因、编码序列seqidno:35的乙醛脱氢酶b的aldb基因)、编码参与从丙酮酸合成乳酸的酶的基因(编码序列seqidno:37的乳酸脱氢酶的ldha基因)、编码参与从甲酸合成乳酸的酶的基因(编码序列seqidno:39的丙酮酸甲酸裂解酶的pfla基因、编码序列seqidno:41的丙酮酸甲酸裂解酶的pflb基因)、编码参与从乙醇合成乳酸的酶的基因(编码序列seqidno:43的醛醇脱氢酶的adhe基因)和编码参与从乙酸合成乳酸的酶的基因(编码序列seqidno:45的乙酸激酶的acka基因、编码序列seqidno:47的磷酸转乙酰酶的pta基因、编码序列seqidno:49的丙酮酸氧化酶的poxb基因)；-消除消耗磷酸烯醇式丙酮酸(pep)的途径例如序列seqidno:51和seqidno:53的丙酮酸激酶(由pyka和pykf基因编码)和/或通过例如通过过表达编码序列seqidno:55的pep合成酶的ppsa基因来提高pep的合成；-编码序列seqidno:57的硫辛酰胺脱氢酶的lpd基因的特定突变；-可以缺失编码序列seqidno:59的arca双重转录调节子的arca基因和编码序列seqidno:61的nadh:泛醌氧化还原酶ii的ndh基因；-编码序列seqidno:63的甘油醛3-磷酸脱氢酶的gapa基因可以在温度诱导型启动子的控制下；-过表达参与输入和代谢蔗糖的基因(编码序列seqidno:65的蔗糖透性酶的cscb基因、编码序列seqidno:67的蔗糖水解酶的csca基因、编码序列seqidno:69的果糖激酶的csck基因、编码序列seqidno:71的磷酸烯醇丙酮酸依赖性磷酸转移酶体系的scra基因、编码序列seqidno:73的atp依赖性果糖激酶的scrk基因、编码序列seqidno:75的蔗糖6-磷酸水解酶(转化酶)的scrb基因、编码序列seqidno:77的蔗糖porine的scry基因)；和-其组合。最优选的用于产生1,2-丙二醇更特别用于产生(r)1,2-丙二醇的遗传修饰是过表达mgsa基因。应理解，这些优选的遗传修饰，特别是过表达mgsa基因，能够优选地与以下描述的实施方案结合。更准确地，为了将羟丙酮转化为1,2-丙二醇，根据本发明的微生物优选进一步过表达编码序列seqidno:21的nadh依赖性甘油脱氢酶的glda基因或编码序列seqidno:23的nadh依赖性甘油脱氢酶的其突变体。后者相对于野生型glda基因特别有优势，因为其编码受反应的底物(羟丙酮)和产物(nad+和1,2-乙二醇)较小抑制的甘油脱氢酶。然而，使用序列seqidno:21和23的酶将羟丙酮不完全还原为1,2-丙二醇，特别是由于在厌氧条件下细胞的内部氧化还原状态。在该上下文中，因此可以特别优选增加nadph依赖性丙酮醇还原酶活性以及nadph供应。因此，在本发明的一个优选实施方案中，为了增强羟丙酮转化为1,2-丙二醇，根据本发明的微生物优选还过表达编码nadph依赖性丙酮醇还原酶的至少一种基因，所述nadph依赖性丙酮醇还原酶与序列seqidno:79、seqidno:81、seqidno:83、seqidno:85、seqidno:87和seqidno:5具有至少60％的氨基酸同一性。优选地，所述nadph依赖性丙酮醇还原酶与上述序列具有至少70％、75％、80％、85％、90％、95％序列同一性，更优选与所述序列具有至少96％、97％、98％、99％或99,999％序列同一性，条件是保持该酶的活性，尽管可能具有不同效力。在一些情况中，所述nadph依赖性丙酮醇还原酶可以对应于以上序列中的任一个(即具有100％序列同一性)。氨基酸序列之间的序列同一性可以通过本领域熟知的方法确定，例如通过用needleman和wunsch(1970)的总体同源性比对算法通过最佳比对、通过此算法的计算机实现(例如clustalw)或通过目测确定。甚至更优选地，根据本发明的nadph依赖性丙酮醇还原酶属于序列seqidno:79。nadph依赖性丙酮醇还原酶活性可通过降低nadh依赖性har活性而被进一步增强。因此，在另一个优选的实施方案中，为了增强羟丙酮转化为1,2-丙二醇，根据本发明的微生物可以进一步包含编码序列seqidno:21或seqidno:23的nadh依赖性甘油脱氢酶的glda或glda*基因的缺失和/或过表达编码nadph依赖性甘油脱氢酶的其突变体。应理解，该实施方案可以优选与上文所述的实施方案结合，其中nadph依赖性丙酮醇还原酶活性增加。如上文所述，所述功能性突变体具有与野生型glda酶相比不同的辅因子特异性，因为其是nadph依赖性的。这可以由本领域技术人员通过辅因子工程改造(katzberg等，2010)轻易实现。更准确地，glda辅因子特异性的改变可以由位置d37处的至少一个突变介导。因此，在一个优选的实施方案中，位置d37处的氨基酸残基可以由甘氨酸(d37g)、丙氨酸(d37a)或缬氨酸(d37v)取代。在一个最优选的实施方案中，位置d37处的氨基酸残基由甘氨酸(d37g)取代。在一个优选的实施方案中，glda辅因子特异性的改变可以通过将位置d37处的突变与位置p161处的至少一个突变结合而提高。优选地，位置p161处的氨基酸残基可以由丝氨酸(p161s)或苏氨酸(p161t)取代。更优选地，位置p161处的氨基酸残基由丝氨酸(p161s)取代。在一个最优选的实施方案中，glada辅因子特异性的改变可以通过将位置d37和p161处的突变与位置l164处的至少一个突变结合而提高。优选地，位置l164处的氨基酸残基可以由丙氨酸(l164a)、甘氨酸(l164g)或缬氨酸(l164v)取代。更优选地，位置l164处的氨基酸残基由丙氨酸(l164a)取代。在一个特别优选的实施方案中，本发明的微生物过表达编码nadph依赖性甘油脱氢酶的突变glda*基因，所述nadph依赖性甘油脱氢酶至少包含以下突变：d37g、p161s和l164a，例如序列seqidno:87的酶。1,2-丙二醇的产量可以通过将上文所述的增加nadph依赖性丙酮醇还原酶活性与增加细胞中nadph可用性结合来进一步提高。用于增加细胞中nadph可用性的策略是熟练的专业人员熟知的(lee等,2013)因此，在又一优选的实施方案中，为了增加nadph可用性，本发明的微生物可以进一步包含以下遗传修饰中的至少一种：-过表达编码序列seqidno:89和seqidno:91的烟酰胺核苷酸转氢酶的pntab基因操纵子，如wo2012/055798a1所描述的(通过引用并入本文)；-弱化编码序列seqidno:93的磷酸葡糖异构酶的pgi基因；-减弱编码序列seqidno:95的磷酸果糖激酶的pfka基因，如wo2005/047498所描述的(通过引用并入本文)；-过表达编码序列seqidno:97的葡萄糖-6-磷酸脱氢酶的zwf基因，如lim等,2002所记载的(通过引用并入本文)；-过表达编码序列seqidno:99的adp-依赖性脱水酶的yjef基因，如marbaix等,2011所描述的(通过引用并入本文)；-过表达编码序列seqidno:101的nadp-依赖性甘油醛-3-磷酸脱氢酶的gapn基因；-过表达编码seqidno:103的nadp-依赖性硫辛酰胺脱氢酶的突变lpd*基因；和-其组合。本发明的微生物可以经进一步遗传改造，以专有地将碳源转化为羟丙酮，由此弱化或废除不想要的副产物特别是乳酸的合成。因此，本发明的一个优选实施方案是提供上文所述的微生物，其进一步包含编码序列seqidno:31的乙二醛酶i的gloa基因的缺失。根据一个特别优选的实施方案，本发明的微生物至少包含：-过表达编码seqidno:17或19的甲基乙二醛合成酶的mgsa或mgsa*基因和编码选自seqidno:21或23的nadh依赖性甘油脱氢酶和seqidno:79、81、83、85、87和5的酶的酶的基因，和-缺失编码seqidno:31的乙二醛酶i的基因gloa、编码seqidno:39和41的丙酮酸甲酸裂解酶的pflab、编码seqidno:43的醛醇脱氢酶的adhe、编码seqidno:37的乳酸脱氢酶的ldha、编码seqidno:33和35的乳醛脱氢酶的alda和aldb、编码seqidno:27的磷酸葡萄糖酸脱水酶的edd、编码seqidno:59的双重转录调节子的arca、以及编码seqidno:61的nadh脱氢酶的ndh。根据一个甚至更优选的实施方案，本发明的微生物至少包含：-过表达seqidno:18或20的mgsa或mgsa*基因和选自seqidno:22或24和seqidno:80、82、84、86、88和6的基因的基因，和-缺失seqidno:32的gloa、seqidno:40和42的pflab、seqidno:44的adhe、seqidno:37的ldha、seqidno:34和36的alda和aldb、seqidno:28的edd、seqidno:60的arca、以及seqidno:62的ndh。关于所述微生物的科、属和/或种的优选实施方案如上文所述。表1：根据本发明的酶和基因(n/a:不可用)如上所述，本发明的微生物可用于产生1,2-丙二醇，特别是(r)-1,2-丙二醇。因此，在又一的方面中，本发明涉及用于发酵产生1,2-丙二醇的方法，包括以下步骤：a)在发酵条件下在包含碳水化合物作为碳源的培养基中培养微生物，所述微生物经遗传修饰用于产生1,2-丙二醇；和b)从所述培养基中回收1,2-丙二醇，其中所述微生物过表达至少一种编码上文所述的甲基乙二醛还原酶的基因，且将甲基乙二醛有效转化为羟丙酮。碳源可以优选通过所述微生物还原以提供中间代谢物甲基乙二醇。上文所述的微生物和碳源的优选实施方案在本文中可以在细节上作必要的变更来应用。根据一个优选的实施方案，上述方法进一步包括步骤c)纯化从步骤b)回收的1,2-丙二醇。使用本领域的常规方法从培养基中纯化所需的产物是在熟练的专业人员的能力范围内，所述方法例如通过引用并入本文的专利申请wo2011/076690和wo2012/130316中所述的方法。附图图1.携带不同mgr酶的菌株的甲基乙二醛还原酶(mgr)特定活性和相关菌株中相关mgr酶表达的百分数。实施例在目前可得的产生1,2-丙二醇的大肠杆菌菌株中，甲基乙二醛通过甲基乙二醛还原酶(mgr)酶yqhd*(g149e)转化为羟丙酮。但是，yqhd*(g149e)表现出低催化效率并且必须经高度表达才能允许产生1,2-丙二醇(其占菌株中表达的总蛋白的高达40％)。该高水平表达导致对微生物的代谢负荷，因此由于损失碳和能量而对细胞产生压力。此外，即使yqhd*(g149e)的表达水平被推向较高表达水平，其催化效率也不足以达到最大1,2-丙二醇产量的性能。因此，为了增加1,2-丙二醇产量，必须使用具有比yqhd*(g149e)更高的催化效率的甲基乙二醛还原酶。为此，通过测量其体外催化效率来评估了未已知用于还原甲基乙二醛的若干候选酶。然后，筛选表现出在体内使甲基乙二醛(mg)解毒的最佳能力的酶。然后将表现出对甲基乙二醛最高抗性的酶引入1,2-丙二醇产生大肠杆菌菌株中。材料和方法：在以下给出的实施例中，本领域熟知的方法用于构建大肠杆菌菌株，所述大肠杆菌菌株含有复制载体和/或多种染色体缺失以及使用由datsenko&wanner,(2000)详细描述的用于大肠杆菌的同源重组的替换。以相同方式，使用质粒或载体在重组微生物中表达或过表达一种或多种基因是本领域技术人员熟知的。合适的大肠杆菌表达载体的实例包括ptrc、pacyc184、pbr322、puc18、puc19、pkc30、prep4、phs1、phs2、pplc236等(studier等,1990；和pouwels等,1985)。一些方案已经用于以下实施例中。本发明中使用的方案1(通过同源重组进行染色体修饰，选择重组子)、方案2(噬菌体p1转导)和方案3(抗生素盒切除，必要时去除抗性基因)已经在通过引用并入本文的专利申请ep2532751中详尽描述。通过pcr分析使用本领域技术人员能够设计的适当寡核苷酸验证染色体修饰。方案4：构建重组质粒重组dna技术描述于molecularcloning:sambrook和russell(2001)中。简言之，使用寡核苷酸和适当的基因组dna作为基质(本领域技术人员能够限定的)pcr扩增dna片段。将dna片段和选择的质粒用兼容的限制性酶消化，然后连接并转化到感受态细胞中。分析转化子并通过dna测序验证感兴趣的重组质粒。实施例1：鉴定新的甲基乙二醛还原酶(mgr)1.1测定多种候选酶的甲基乙二醛还原酶活性1.1.1.构建菌株1至16为了确定多种醛还原酶候选者的动力学参数，构建了以下菌株：表2：经构建并用于确定16种醛还原酶候选者的动力学参数的菌株将编码不同的假定的甲基乙二醛还原酶的基因克隆到表达载体ppal7中，除了菌株2和7，将获得的质粒转化到菌株bl21(de3)star中。对于菌株2，将质粒克隆到如下所获得的缺失yqhd的bl21(de3)star菌株中。使用同源重组策略(根据方案1)将菌株mg1655中的yqhd基因失活。dyqhd:seqidn°119和120的寡核苷酸用于pcr扩增抗性盒。保留的菌株指定为mg1655dyqhd::cm。最后，通过p1噬菌体转导(根据方案2)将dyqhd::cm缺失转移到菌株bl21(de3)star中并且引入ppal7-yqhd*(g149e)质粒得到菌株2。对于菌株7，将质粒克隆到如下获得的缺失ydjg的bl21(de3)star菌株中。使用同源重组策略(根据方案1)将菌株mg1655中的ydjg基因失活。dydjg:seqidn°121和122的寡核苷酸用于pcr扩增抗性盒。保留的菌株指定为g1655dydjg::km。最后，通过p1噬菌体转导(根据方案2)将dydjg::km缺失转移到菌株bl21(de3)star中并且引入ppal7-ydjg*(d232e)质粒得到菌株7。携带glda*(a160t)酶的菌株14与通过引用并入本文的专利申请ep14305691中所述的菌株号20相同。1.1.2.过量产生蛋白质用于过量产生蛋白质的培养物在2l爱伦美氏烧瓶中使用补充有2.5g/l葡萄糖和100mg/l氨苄青霉素的lb肉汤(bertani,1951)实现。过夜预培养物用于接种500ml培养物，至od600nm为约0.15。此预培养在500ml爱伦美氏烧瓶中进行，该烧瓶装有补充有2.5g/l葡萄糖和100mg/l氨苄青霉素的50mllb肉汤。首先将培养物以37℃和200rpm保持在振荡器上，直至od600nm为0.6–0.8(约1小时)，然后用500μmiptg诱导。将培养物保持在25℃和200rpm直至od600nm为约4，然后停止。将细胞在4℃以7000rpm离心5分钟，然后保存在-20℃。1.1.3.蛋白质纯化步骤1：制备无细胞提取物将约400mg的大肠杆菌生物质悬浮于60ml的ph7.6的100mm磷酸钾和蛋白酶抑制剂的混合物中。将细胞悬浮液(每个锥形管15ml)在50ml锥形管中在冰上超声处理(bandelinsonoplus,70w)，30秒超声-30秒间隔，8个循环。超声后，将细胞与5mmmgcl2和1ui/ml的dnasei在室温孵育30分钟。通过在4℃以12000g离心30分钟去除细胞碎片。步骤2：亲和纯化除了菌株14之外，通过在profinity柱(biorad,bio-scaleminiprofinityexactcartridge5ml)上根据制造商推荐的方案通过亲和性从粗细胞提取物中纯化蛋白质。将粗提取物装载到用ph7.6的100mm磷酸钾平衡的5mlprofinity精确柱体上。将柱用10个柱体积的同一缓冲液洗涤，并且用ph7.6的100mm磷酸钾、100mm氟化物在室温孵育30分钟。使用2个柱体积的ph7.6的100mm磷酸钾将蛋白质从柱上洗脱。标签保持牢固地结合到树脂上，且纯化的蛋白质被释放。将含有蛋白质的级分合并、浓缩并且装载到用不同贮存缓冲液(表3)平衡的凝胶过滤柱(superdex20010/300glcolumn,gehealthcare)上。使用bradford蛋白质测定法测量蛋白质浓度。对于菌株14，纯化步骤以前描述于通过引用并入本文的专利申请wo2015/173247中。表3：蛋白质贮存缓冲液1.1.4.确定纯化的假定的甲基乙二醛还原酶的动力学参数通过在30℃在分光光度计上在340nm处(λ340＝6290m-1cm-1)测量nad(p)h的消耗来确定甲基乙二醛还原酶活性(mgr)。将含有测定缓冲液和纯化的酶的反应混合物(1ml)在30℃孵育5分钟。然后，添加0.1-40mm甲基乙二醛以开始反应。将一个单位的酶活性定义为每分钟催化1μmol的nad(p)h减少的酶的量。用sigmaplot确定动力学参数。表4中提供了纯化的酶的动力学参数。1.1.5.确定反应产物与甲基苯并噻唑啉酮-2-腙(mbth)和fecl3反应后，通过gc-ms(agilenttechnologies)测量通过不同假定的酶从甲基乙二醛(mg)得到的反应产物羟丙酮(ha)，通过uhplc-ms/ms测量产物乳醛(la)。将含有测定缓冲液、10mm甲基乙二醛、5mmnadph和5-10μg的纯化酶的反应混合物(1ml)在30℃孵育30分钟。注射1μl的反应产物。制备不含有mg的反应混合物作为对照。表4中提供了纯化酶的反应产物。表4：纯化酶的动力学参数和反应产物ha：羟丙酮，la：乳醛，nd：未确定选择产生羟丙酮并且具有为yqhd*(g149e)(本身具有为天然yqhd酶的催化效率的2倍的催化效率的突变酶)的催化效率的至少2倍的催化效率的五种酶用于进一步的表征和筛选：gld、yhdn、yafb、yahk和yjgb。1.2.选择最佳甲基乙二醛还原酶1.2.1.构建菌株17至23随后通过将相应基因克隆到修饰的大肠杆菌菌株15中来筛选选择的mgr酶，所述修饰的大肠杆菌菌株15为如以下构建的mg1655dgloadedddpflabdldhadadhedgldadyqhd。为了使由gloa编码的乙二醛酶i、由edd编码的磷酸葡糖酸脱水酶、分别由pfla和pflb编码的丙酮酸甲酸裂解酶激活酶和丙酮酸甲酸裂解酶、由ldha编码的乳酸脱氢酶和由adhe编码的醇脱氢酶失活，通过p1噬菌体(根据方案2)将在专利申请wo2008/116852(通过引用并入本文)中所述的dgloa、dedd、dpflab、dldha和dadhe缺失转移到菌株mg1655中，并且根据方案3去除抗性基因。为了使由glda编码的甘油脱氢酶失活，通过p1噬菌体(根据方案2)将在专利申请wo2015/173247(通过引用并入本文)中所述的dglda缺失转移到以前的菌株中。最后，为了使由yqhd编码的醛还原酶失活，通过p1噬菌体转导(根据方案2)将上文所述的dyqhd::cm缺失转到以前的菌株中，产生菌株17。然后，在明确的rbs下在专利申请ep2532751(通过引用并入本文)中所述的pme101vb06质粒上表达下表5中所述的基因，将每种质粒引入菌株17中，产生菌株18至23。表5：甲基乙二醛还原酶菌株18至23的描述菌株酶18gld19yhdn20yafb21yqhd*(g149e)22yahk23yjgb1.2.2.粗提取物的甲基乙二醛还原酶测定通过在30℃在分光光度计上在340nm处(λ340＝6290m-1cm-1)测量nad(p)h的消耗来测定甲基乙二醛还原酶活性(mgr)。将含有测定缓冲液和粗提取物的反应混合物(1ml)在30℃孵育5分钟。然后添加10mm甲基乙二醛以开始反应。将一个单位的酶活性定义为每分钟催化1μmol的nad(p)h减少的酶的量。特定的酶活性表述为每毫克蛋白质的酶活性单位。减去了测定中不使用底物确定的活性值。1.2.3.确定甲基乙二醛还原酶的表达水平与所有菌株中的特定活性平行，通过sds-page分析定量不同mgr的表达水平。将相同量的粗提取物装载到sds-page上，且使用bioradimagelabtm软件，将表达水平确定为mgr的条带体积相对于总泳道体积的比率。这些不同酶的特定活性是非常不同的，并且与表达水平无关。例如，菌株21表现出高表达且低特定活性，而菌株22表现出低5倍的表达水平，但是高4倍的特定活性(图1)。1.2.4.甲基乙二醛(mg)筛选由于体外活性可能不反映真实的体内活性，因此在lb琼脂板上关于对mg的抗性对菌株17至23进行筛选。在37℃在补充有50μg/ml大观霉素的lb丰富培养基中培养菌株，直至od600nm约为1。然后将100μl的0、10-1或10-2稀释液涂板到补充有50μg/ml大观霉素和0、2、3或4mm的mg的lb琼脂板上。将板在37℃孵育48小时。下表6指示一些克隆生长所处的最低稀释和最高mg浓度，这给出了菌株抗性水平的指标(mg浓度越高以及对于给定浓度的越低稀释，抗性越高)。表6：菌株17至23的甲基乙二醛筛选菌株17181920212223mgrxgldyhdnyafbyqhd*yahkyjgbmmmg0223233稀释10-210-210-210-110-210-210-2抗性水平-+++++++++++-：无抗性；+：中度抗性；++：高度抗性；+++：非常高度抗性yahk和yjgb酶允许较好的mg抗性，这些候选mgr酶在mpg产生菌株中保持替换yqhd*(g149e)。尽管如此，熟练的专业人员会容易地理解酶gld、yafb和yhdn也适合在所述mpg产生菌株中替换yqhd*(g149e)。实施例2：使用根据本发明的新的甲基乙二醛还原酶(mgr)产生1,2-丙二醇2.1.构建菌株24至26为了使ptsg基因失活，使用同源重组策略(根据方案1)。使用用于dptsg的寡核苷酸seqidn°123和124来pcr扩增抗性盒。保留的菌株被指定为mg1655dptsg::km。通过p1噬菌体转导(根据方案2)将dptsg::km缺失转移到在专利申请wo2015/173247(通过引用并入本文)中所述的大肠杆菌mpg产生菌株5中，产生菌株24。此后，将yahk和yjgb在ptrc启动子下和明确的rbs下在染色体中过表达。通过p1噬菌体(根据方案2)将每个构建体转移到实施例1中所述的通过缺失yqhd进一步修饰的菌株24中，对于yahk产生菌株25，对于yjgb产生菌株26。2.2.评估mpg产生菌株将1,2-丙二醇产生菌株培养在摇瓶(如通过引用并入本文的专利申请ep2532751中所述的，除了将葡萄糖和木糖用作唯一碳并且为了在整个发酵过程中保持ph在6.0以上而将40g.l-1的mops添加到培养基中)和在2l发酵罐中，如下：在37℃在含有补充有10％的lb培养基(w:w)的50ml基本培养基(m1)的三角瓶中实现24小时预培养后获得接种物。随后，将700ml的基本培养基(m2)填充到2.5l发酵罐(pierreguerin)中，然后用范围在55至80ml之间的预培养体积接种至生物量浓度为0.2g.l-1。对于菌株26，将锌添加到分批培养基中，最终浓度为4mg.l-1。培养温度保持恒定在37℃，通过自动添加nh4oh溶液(10％)将ph保持至工作值(6.8)。将初始搅拌速率设置在200rpm，将初始气流速度设置在40nl.h-1。通过增加搅拌并且如果需要的话通过增加充气，将溶解氧浓度值保持在20％-40％优选30％饱和。需要时，添加抗生素，对于大观霉素浓度为50mg.l-1。分批进料培养基包含糖(葡萄糖/木糖；比率2:1)，通过测量co2产生而自动调整营养速率，以保持发酵肉汤中糖浓度为30g.l-1。54小时后终止培养。表7：m1和m2培养基的组成通过hplc-rid用bioradhpx-87h柱定量1,2-丙二醇(pg)和其前体羟丙酮(ha)。在摇瓶中，与具有yqhd*(g149e)过表达的菌株相比，具有yahk或yjgb过表达的产生菌株产生每克生物质更多克的pg+ha。在2l发酵罐中，仅具有yjgb过表达的菌株更好。表8：pg产生菌株的以每克生物质的以克数计的pg+ha的pg+ha产率菌株24菌株25菌株26摇瓶1.882.152.142l发酵罐3.253.635.422l发酵罐中菌株25的行为归因于ha对yahk的抑制。参考文献-altarasneandcamerondc(1999),appl.environ.microbiol.,65:1180-1185-altarasneandcamerondc(2000),biotechnol.prog.,16:940-946-altschuls,gishw,millerw,myerse,lipmandj(1990).j.mol.biol；215(3):403–410-badiaj,rosj,aguilarj(1985),j.bacteriol.161:435-437-bennettgnandsanky(2001),appl.microbiol.biotechnol.55:1-9-berrios-riverasj,sanky,bennettgn(2003),j.ind.microbiol.biotechnol.,30:34-40-bertanietal.,1951.jbacteriol.62:293-300,-bocanegraj,scruttonn,perhamr(1993)biochemistry,32(11):2737-2740-camerondc,altarasne,hoffmanml,shawaj(1998),biotechnol.prog.,14:116-125-carriert&keaslingj(1999),biotechnolprog.,15(1):58-64-datsenkoka&wannerbl,(2000),procnatlacadsciusa.,97:6640-6645-davisjj&olsengj.(2011).mol.biol.evol.；28(1):211-221-demerecm,adelbergea,clarkaj,hartmenpe(1966),genetics,54:61-76-demll,bojaka,stecks,grafm,wildj,schirmbeckr,wolfh,wagnerr.(2011)-grafm,bojaka,demll,bielerk,wolfh,wagnerr.(2000).j.virol.；74(22):10/22-10826-huangk,rudolphfb,bennettgn(1999),appl.environ.microbiol.,65:3244-3247-katzbergm,skorupa-parachinn,gorwa-grauslundm,bertaum(2010),int.j.mol.sci.,11(4):1735–1758-koj,kimi,yoos,minb,kimk,parkc(2005),j.bact.,187(16):5782-5789-lees,mccormickm,lippards,chou(2013),nature,494:380-384-lims,jungy,shinh,leey(2002),jbioscibioeng.,93(6):543-549-marbaixa,noelg,detrouxa,vertommend,schaftingene,linsterc(2011),jbiolchem.,286(48):,41246-41252-needlemanandwunsch(1970),j.mol.biol.,48(3),443-453-neidhardt(1996)ed.,escherichiacoliandsalmonella:cellularandmolecularbiology,2nded.,vol2-pouwelsetal.eds.(1985).cloningvectors.elsevier:newyork-salish(2011),methodsenzymol.,498:19-42-sambrookandrussell,(2001),molecularcloning:3rdedition,coldspringharborlaboratorypress,ny,vol1,2,3-segeli(1993),enzymekinetics,johnwiley&sons,pp.44-54and100-112-studieretal.(1990).geneexpressiontechnology:methodsinenzymology；185,academicpress,sandiego,california-subedikp,kimi,kimj,minb,parkc(2008),femsmicrobiol.letters,279(2):180-187。序列表<110>metabolicexplorer<120>使用新的酶将甲基乙二醛转化成羟基丙酮及其应用<130>b373619pctd36845<150>ep16306848.9<160>2016-12-29<160>128<170>patentinversion3.5<210>1<211>339<212>prt<213>大肠杆菌(菌株k12)<400>1metsermetilelyssertyralaalalysglualaglyglygluleu151015gluvaltyrglutyraspproglygluleuargproglnaspvalglu202530valglnvalasptyrcysglyilecyshisseraspleusermetile354045aspasnglutrpglypheserglntyrproleuvalalaglyhisglu505560valileglyargvalvalalaleuglyseralaalaglnasplysgly65707580leuglnvalglyglnargvalglyileglytrpthralaargsercys859095glyhiscysaspalacysileserglyasnglnileasncysglugln100105110glyalavalprothrilemetasnargglyglyphealaglulysleu115120125argalaasptrpglntrpvalileproleuprogluasnileaspile130135140gluseralaglyproleuleucysglyglyilethrvalphelyspro145150155160leuleumethishisilethralathrserargvalglyvalilegly165170175ileglyglyleuglyhisilealailelysleuleuhisalametgly180185190cysglu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argtyr245250255glylysservalilehisleualavalargtrpileleuaspglnpro260265270glyalaaspilealaleutrpglyalaarglysproglyglnleuglu275280285alaleusergluilethrglytrpthrleuasnsergluaspglnlys290295300aspileasnthrileleugluasnthrileseraspprovalglypro305310315320gluphemetalaproprothrargglugluile325330<210>6<211>996<212>dna<213>枯草芽孢杆菌(菌株168)<400>6atggaatataccagtatagcagatacaggaatagaagcctccagaatcggcctcggcaca60tgggccattggcggaacgatgtggggaggcactgacgaaaaaacatcgattgaaacaatc120cgcgccgctcttgatcaggggattacactgattgacaccgcaccggcttacggcttcggg180cagtccgaggaaattgtcggaaaggcaatcaaagagtacggcaaaagagaccaggtgatt240ctcgcaacgaaaacggctctggactggaagaacaaccagctgttccgccatgcgaacaga300gcgagaattgtagaggaagttgagaattctttgaagcggcttcaaacagactatattgat360ctttatcaggtgcattggcccgatccgcttgtgccaattgaagaaacggctgaagtcatg420aaggaattatatgatgcgggaaaaatccgggcgattggcgtcagcaatttttcaattgag480caaatggatacatttcgcgccgtcgcacctctccatacgattcagcctccatataatctg540tttgaaagagagatggaagagagtgtccttccttatgcgaaagataacaagataacaaca600ttattatacggcagtttatgcagagggctgttaacaggcaaaatgactgaagaatataca660tttgagggcgatgatctgcgtaatcacgatccaaaattccagaagccccgctttaaagag720tatctttctgctgtgaatcaattggataagctggcgaagacacgttatggaaaatcagtg780attcacttggctgtcagatggatcttagatcagccgggagcggatatcgctctttgggga840gcaagaaagcctgggcagcttgaggccctatctgagattacaggctggacgctgaacagt900gaagatcagaaagatatcaatactatattggaaaatacgatatcagaccctgtcggaccg960gagtttatggccccgccgaccagagaggaaatataa996<210>7<211>332<212>prt<213>氧化葡糖杆菌(菌株621h)<400>7metalaseraspthrileargileproglyileaspthrproleuser151015argvalalaleuglythrtrpalaileglyglytrpmettrpglygly202530proaspaspaspasnglyvalargthrilehisalaalaleuaspglu354045glyileasnleuileaspthralaprovaltyrglypheglyhisser505560glugluilevalglyargalaleualaglulysproasnlysalahis65707580valalathrlysleuglyleuhistrpvalglygluaspglulysasn859095metlysvalpheargaspserargproalaargilearglysgluval100105110gluaspserleuargargleuargvalgluthrileaspleuglugln115120125ilehistrpproaspasplysthrproileaspgluseralaargglu130135140leuglnlysleuhisglnaspglylysileargalaleuglyvalser145150155160asnpheserprogluglnmetaspilephearggluvalalaproleu165170175alathrileglnproproleuasnleuphegluargthrileglulys180185190aspileleuprotyralaglulyshisasnalavalvalleualatyr195200205glyalaleucysargglyleuleuthrglylysmetasnargaspthr210215220thrpheprolysaspaspleuargserasnaspprolyspheglnlys225230235240proasnpheglulystyrleualaalametaspglupheglulysleu245250255alaglulysargglylysservalmetalaphealavalargtrpval260265270leuaspglnglyprovalilealaleutrpglyalaarglysprogly275280285glnvalserglyvallysaspvalpheglytrpserleuthraspglu290295300glulyslysalavalaspaspileleualaarghisvalproasnpro305310315320ileaspprothrphemetalaproproalaargasp325330<210>8<211>999<212>dna<213>人工的<220><223>密码子优化的基因gld<400>8atggcaagcgataccattcgtattccgggtattgatacaccgctgagccgtgttgcactg60ggcacctgggcaattggtggttggatgtggggtggtccggatgatgataatggtgttcgt120accattcatgcagcactggatgaaggtattaatctgattgataccgctccggtttatggt180tttggtcatagcgaagaaattgttggtcgtgcactggcagaaaaaccgaataaagcacat240gttgcaaccaaactgggtctgcattgggttggtgaagatgagaaaaacatgaaagtgttt300cgtgatagccgtccggcacgtattcgtaaagaagttgaagatagcctgcgtcgtctgcgt360gttgaaaccattgatctggaacaaattcattggcctgatgataaaaccccgattgatgaa420agcgcacgtgaactgcagaaactgcatcaggatggtaaaattcgtgccctgggtgttagc480aattttagtccggaacaaatggatatctttcgtgaagttgcaccgctggcaaccattcag540cctccgctgaacctgtttgaacgtaccattgaaaaagatattctgccgtatgccgaaaaa600cataatgcagttgttctggcatatggtgcactgtgtcgtggtctgctgaccggcaaaatg660aatcgtgataccacctttccgaaagatgatctgcgtagcaatgatccgaaatttcagaaa720ccgaacttcgagaaatatctggctgcaatggatgagtttgaaaaactggccgagaaacgt780ggtaaaagcgttatggcatttgcagttcgttgggttctggatcagggtccggttattgca840ctgtggggtgcacgtaaaccgggtcaggttagcggtgttaaagatgtttttggttggagc900ctgaccgacgaagaaaaaaaagcagttgatgatattctggcacgtcatgttccgaatccg960attgatccgacctttatggcaccgcctgcacgtgattaa999<210>9<211>339<212>prt<213>人工的<220><223>yjgb*(n240y)<400>9metsermetilelyssertyralaalalysglualaglyglygluleu151015gluvaltyrglutyraspproglygluleuargproglnaspvalglu202530valglnvalasptyrcysglyilecyshisseraspleusermetile354045aspasnglutrpglypheserglntyrproleuvalalaglyhisglu505560valileglyargvalvalalaleuglyseralaalaglnasplysgly65707580leuglnvalglyglnargvalglyileglytrpthralaargsercys859095glyhiscysaspalacysileserglyasnglnileasncysglugln100105110glyalavalprothrilemetasnargglyglyphealaglulysleu115120125argalaasptrpglntrpvalileproleuprogluasnileaspile130135140gluseralaglyproleuleucysglyglyilethrvalphelyspro145150155160leuleumethishisilethralathrserargvalglyvalilegly165170175ileglyglyleuglyhisilealailelysleuleuhisalametgly180185190cysgluvalthralapheserserasnproalalysgluglngluval195200205leualametglyalaasplysvalvalasnserargaspproglnala210215220leulysalaleualaglyglnpheaspleuileileasnthrvaltyr225230235240valserleuasptrpglnprotyrpheglualaleuthrtyrglygly245250255asnphehisthrvalglyalavalleuthrproleuservalproala260265270phethrleuilealaglyaspargservalserglyseralathrgly275280285thrprotyrgluleuarglysleumetargphealaalaargserlys290295300valalaprothrthrgluleupheprometserlysileasnaspala305310315320ileglnhisvalargaspglylysalaargtyrargvalvalleulys325330335alaaspphe<210>10<211>1020<212>dna<213>人工的<220><223>yjgb*(n240y)<400>10atgtcgatgataaaaagctatgccgcaaaagaagcgggcggcgaactggaagtttatgag60tacgatcccggtgagctgaggccacaagatgttgaagtgcaggtggattactgcgggatc120tgccattccgatctgtcgatgatcgataacgaatggggattttcacaatatccgctggtt180gccgggcatgaggtgattgggcgcgtggtggcactcgggagcgccgcgcaggataaaggt240ttgcaggtcggtcagcgtgtcgggattggctggacggcgcgtagctgtggtcactgcgac300gcctgtattagcggtaatcagatcaactgcgagcaaggtgcggtgccgacgattatgaat360cgcggtggctttgccgagaagttgcgtgcggactggcaatgggtgattccactgccagaa420aatattgatatcgagtccgccgggccgctgttgtgcggcggtatcacggtctttaaacca480ctgttgatgcaccatatcactgctaccagccgcgttggggtaattggtattggcgggctg540gggcatatcgctataaaacttctgcacgcaatgggatgcgaggtgacagcctttagttct600aatccggcgaaagagcaggaagtgctggcgatgggtgccgataaagtggtgaatagccgc660gatccgcaggcactgaaagcactggcggggcagtttgatctcattatcaacaccgtctac720gtcagcctcgactggcagccctattttgaggcgctgacctatggcggtaatttccatacg780gtcggtgcggttctcacgccgctgtctgttccggcctttacgttaattgcgggcgatcgc840agcgtctctggttctgctaccggcacgccttatgagctgcgtaagctgatgcgttttgcc900gcccgcagcaaggttgcgccgaccaccgaactgttcccgatgtcgaaaattaacgacgcc960atccagcatgtgcgcgacggtaaggcgcgttaccgcgtggtgttgaaagccgatttttga1020<210>11<211>267<212>prt<213>大肠杆菌(菌株k12)<400>11metalaileproalapheglyleuglythrpheargleulysaspasp151015valvalileserservalilethralaleugluleuglytyrargala202530ileaspthralaglniletyraspasnglualaalavalglyglnala354045ilealagluserglyvalproarghisgluleutyrilethrthrlys505560iletrpilegluasnleuserlysasplysleuileproserleulys65707580gluserleuglnlysleuargthrasptyrvalaspleuthrleuile859095histrpproserproasnaspgluvalservalglugluphemetgln100105110alaleuleuglualalyslysglnglyleuthrarggluileglyile115120125serasnphethrileproleumetglulysalailealaalavalgly130135140alagluasnilealathrasnglnilegluleuserprotyrleugln145150155160asnarglysvalvalalatrpalalysglnhisglyilehisilethr165170175sertyrmetthrleualatyrglylysalaleulysaspgluvalile180185190alaargilealaalalyshisasnalathrproalaglnvalileleu195200205alatrpalametglygluglytyrservalileproserserthrlys210215220arglysasnleugluserasnleulysalaglnasnleuglnleuasp225230235240alagluasplyslysalailealaalaleuaspcysasnaspargleu245250255valserprogluglyleualaproglutrpasp260265<210>12<211>804<212>dna<213>大肠杆菌(菌株k12)<400>12atggctatccctgcatttggtttaggtactttccgtctgaaagacgacgttgttatttca60tctgtgataacggcgcttgaacttggttatcgcgcaattgataccgcacaaatctatgat120aacgaagccgcagtaggtcaggcgattgcagaaagtggcgtgccacgtcatgaactctac180atcaccactaaaatctggattgaaaatctcagcaaagacaaattgatcccaagtctgaaa240gagagcctgcaaaaattgcgtaccgattatgttgatctgacgctaatccactggccgtca300ccaaacgatgaagtctctgttgaagagtttatgcaggcgctgctggaagccaaaaaacaa360gggctgacgcgtgagatcggtatttccaacttcacgatcccgttgatggaaaaagcgatt420gctgctgttggtgctgaaaacatcgctactaaccagattgaactctctccttatctgcaa480aaccgtaaagtggttgcctgggctaaacagcacggcatccatattacttcctatatgacg540ctggcgtatggtaaggccctgaaagatgaggttattgctcgtatcgcagctaaacacaat600gcgactccggcacaagtgattctggcgtgggctatgggggaaggttactcagtaattcct660tcttctactaaacgtaaaaacctggaaagtaatcttaaggcacaaaatttacagcttgat720gccgaagataaaaaagcgatcgccgcactggattgcaacgaccgcctggttagcccggaa780ggtctggctcctgaatgggattaa804<210>13<211>387<212>prt<213>大肠杆菌(菌株k12)<400>13metasnasnpheasnleuhisthrprothrargileleupheglylys151015glyalailealaglyleuarggluglnileprohisaspalaargval202530leuilethrtyrglyglyglyservallyslysthrglyvalleuasp354045glnvalleuaspalaleulysglymetaspvalleuglupheglygly505560ilegluproasnproalatyrgluthrleumetasnalavallysleu65707580valarggluglnlysvalthrpheleuleualavalglyglyglyser859095valleuaspglythrlyspheilealaalaalaalaasntyrproglu100105110asnileaspprotrphisileleuglnthrglyglylysgluilelys115120125seralaileprometglycysvalleuthrleuproalathrglyser130135140gluserasnalaglyalavalileserarglysthrthrglyasplys145150155160glnalaphehisseralahisvalglnprovalphealavalleuasp165170175provaltyrthrtyrthrleuproproargglnvalalaasnglyval180185190valaspalaphevalhisthrvalgluglntyrvalthrlysproval195200205aspalalysileglnaspargphealagluglyileleuleuthrleu210215220ilegluaspglyprolysalaleulysgluprogluasntyraspval225230235240argalaasnvalmettrpalaalathrglnalaleuasnglyleuile245250255glyalaglyvalproglnasptrpalathrhismetleuglyhisglu260265270leuthralamethisglyleuasphisalaglnthrleualaileval275280285leuproalaleutrpasnglulysargaspthrlysargalalysleu290295300leuglntyralagluargvaltrpasnilethrgluglyseraspasp305310315320gluargileaspalaalailealaalathrargasnphepheglugln325330335leuglyvalprothrhisleuserasptyrglyleuaspglyserser340345350ileproalaleuleulyslysleuglugluhisglymetthrglnleu355360365glygluasnhisaspilethrleuaspvalserargargiletyrglu370375380alaalaarg385<210>14<211>1164<212>dna<213>大肠杆菌(菌株k12)<400>14atgaacaactttaatctgcacaccccaacccgcattctgtttggtaaaggcgcaatcgct60ggtttacgcgaacaaattcctcacgatgctcgcgtattgattacctacggcggcggcagc120gtgaaaaaaaccggcgttctcgatcaagttctggatgccctgaaaggcatggacgtgctg180gaatttggcggtattgagccaaacccggcttatgaaacgctgatgaacgccgtgaaactg240gttcgcgaacagaaagtgactttcctgctggcggttggcggcggttctgtactggacggc300accaaatttatcgccgcagcggctaactatccggaaaatatcgatccgtggcacattctg360caaacgggcggtaaagagattaaaagcgccatcccgatgggctgtgtgctgacgctgcca420gcaaccggttcagaatccaacgcaggcgcggtgatctcccgtaaaaccacaggcgacaag480caggcgttccattctgcccatgttcagccggtatttgccgtgctcgatccggtttatacc540tacaccctgccgccgcgtcaggtggctaacggcgtagtggacgcctttgtacacaccgtg600gaacagtatgttaccaaaccggttgatgccaaaattcaggaccgtttcgcagaaggcatt660ttgctgacgctaatcgaagatggtccgaaagccctgaaagagccagaaaactacgatgtg720cgcgccaacgtcatgtgggcggcgactcaggcgctgaacggtttgattggcgctggcgta780ccgcaggactgggcaacgcatatgctgggccacgaactgactgcgatgcacggtctggat840cacgcgcaaacactggctatcgtcctgcctgcactgtggaatgaaaaacgcgataccaag900cgcgctaagctgctgcaatatgctgaacgcgtctggaacatcactgaaggttccgatgat960gagcgtattgacgccgcgattgccgcaacccgcaatttctttgagcaattaggcgtgccg1020acccacctctccgactacggtctggacggcagctccatcccggctttgctgaaaaaactg1080gaagagcacggcatgacccaactgggcgaaaatcatgacattacgttggatgtcagccgc1140cgtatatacgaagccgcccgctaa1164<210>15<211>387<212>prt<213>人工的<220><223>yqhd*(g149e)<400>15metasnasnpheasnleuhisthrprothrargileleupheglylys151015glyalailealaglyleuarggluglnileprohisaspalaargval202530leuilethrtyrglyglyglyservallyslysthrglyvalleuasp354045glnvalleuaspalaleulysglymetaspvalleuglupheglygly505560ilegluproasnproalatyrgluthrleumetasnalavallysleu65707580valarggluglnlysvalthrpheleuleualavalglyglyglyser859095valleuaspglythrlyspheilealaalaalaalaasntyrproglu100105110asnileaspprotrphisileleuglnthrglyglylysgluilelys115120125seralaileprometglycysvalleuthrleuproalathrglyser130135140gluserasnalaglualavalileserarglysthrthrglyasplys145150155160glnalaphehisseralahisvalglnprovalphealavalleuasp165170175provaltyrthrtyrthrleuproproargglnvalalaasnglyval180185190valaspalaphevalhisthrvalgluglntyrvalthrlysproval195200205aspalalysileglnaspargphealagluglyileleuleuthrleu210215220ilegluaspglyprolysalaleulysgluprogluasntyraspval225230235240argalaasnvalmettrpalaalathrglnalaleuasnglyleuile245250255glyalaglyvalproglnasptrpalathrhismetleuglyhisglu260265270leuthralamethisglyleuasphisalaglnthrleualaileval275280285leuproalaleutrpasnglulysargaspthrlysargalalysleu290295300leuglntyralagluargvaltrpasnilethrgluglyseraspasp305310315320gluargileaspalaalailealaalathrargasnphepheglugln325330335leuglyvalprothrhisleuserasptyrglyleuaspglyserser340345350ileproalaleuleulyslysleuglugluhisglymetthrglnleu355360365glygluasnhisaspilethrleuaspvalserargargiletyrglu370375380alaalaarg385<210>16<211>1164<212>dna<213>人工的<220><223>yqhd*(g149e)<400>16atgaacaactttaatctgcacaccccaacccgcattctgtttggtaaaggcgcaatcgct60ggtttacgcgaacaaattcctcacgatgctcgcgtattgattacctacggcggcggcagc120gtgaaaaaaaccggcgttctcgatcaagttctggatgccctgaaaggcatggacgtgctg180gaatttggcggtattgagccaaacccggcttatgaaacgctgatgaacgccgtgaaactg240gttcgcgaacagaaagtgactttcctgctggcggttggcggcggttctgtactggacggc300accaaatttatcgccgcagcggctaactatccggaaaatatcgatccgtggcacattctg360caaacgggcggtaaagagattaaaagcgccatcccgatgggctgtgtgctgacgctgcca420gcaaccggttcagaatccaacgcagaagcggtgatatcccgtaaaaccacaggcgacaag480caggcgttccattctgcccatgttcagccggtatttgccgtgctcgatccggtttatacc540tacaccctgccgccgcgtcaggtggctaacggcgtagtggacgcctttgtacacaccgtg600gaacagtatgttaccaaaccggttgatgccaaaattcaggaccgtttcgcagaaggcatt660ttgctgacgctaatcgaagatggtccgaaagccctgaaagagccagaaaactacgatgtg720cgcgccaacgtcatgtgggcggcgactcaggcgctgaacggtttgattggcgctggcgta780ccacaggactgggcaacgcatatgctgggccacgaactgactgcgatgcacggtctggat840cacgcgcaaacactggctatcgtcctgcctgcactgtggaatgaaaaacgcgataccaag900cgcgctaagctgctgcaatatgctgaacgcgtctggaacatcactgaaggttccgatgat960gagcgtattgacgccgcgattgccgcaacccgcaatttctttgagcaattaggcgtgccg1020acccacctctccgactacggtctggacggcagctccatcccggctttgctgaaaaaactg1080gaagagcacggcatgacccaactgggcgaaaatcacgacattacgttggatgtcagccgc1140cgtatatacgaagccgcccgctaa1164<210>17<211>152<212>prt<213>大肠杆菌(菌株k12)<400>17metgluleuthrthrargthrleuproalaarglyshisilealaleu151015valalahisasphiscyslysglnmetleumetsertrpvalgluarg202530hisglnproleuleugluglnhisvalleutyralathrglythrthr354045glyasnleuileserargalathrglymetasnvalasnalametleu505560serglyprometglyglyaspglnglnvalglyalaleuileserglu65707580glylysileaspvalleuilephephetrpaspproleuasnalaval859095prohisaspproaspvallysalaleuleuargleualathrvaltrp100105110asnileprovalalathrasnvalalathralaasppheileilegln115120125serprohispheasnaspalavalaspileleuileproasptyrgln130135140argtyrleualaaspargleulys145150<210>18<211>459<212>dna<213>大肠杆菌(菌株k12)<400>18atggaactgacgactcgcactttacctgcgcggaaacatattgcgctggtggcacacgat60cactgcaaacaaatgctgatgagctgggtggaacggcatcaaccgttactggaacaacac120gtactgtatgcaacaggcactaccggtaacttaatttcccgcgcgaccggcatgaacgtc180aacgcgatgttgagtggcccaatggggggtgaccagcaggttggcgcattgatctcagaa240gggaaaattgatgtattgattttcttctgggatccactaaatgccgtgccgcacgatcct300gacgtgaaagccttgctgcgtctggcgacggtatggaacattccggtcgccaccaacgtg360gcaacggcagacttcataatccagtcgccgcatttcaacgacgcggtcgatattctgatc420cccgattatcagcgttatctcgcggaccgtctgaagtaa459<210>19<211>152<212>prt<213>人工的<220><223>mgsa*(h21q)<400>19metgluleuthrthrargthrleuproalaarglyshisilealaleu151015valalahisaspglncyslysglnmetleumetsertrpvalgluarg202530hisglnproleuleugluglnhisvalleutyralathrglythrthr354045glyasnleuileserargalathrglymetasnvalasnalametleu505560serglyprometglyglyaspglnglnvalglyalaleuileserglu65707580glylysileaspvalleuilephephetrpaspproleuasnalaval859095prohisaspproaspvallysalaleuleuargleualathrvaltrp100105110asnileprovalalathrasnvalalathralaasppheileilegln115120125serprohispheasnaspalavalaspileleuileproasptyrgln130135140argtyrleualaaspargleulys145150<210>20<211>459<212>dna<213>人工的<220><223>mgsa*(h21q)<400>20atggaactgacgactcgcactttacctgcgcggaaacatattgcgctggtggcacacgat60caatgcaaacagatgctgatgagctgggtggaacggcatcaaccgttactggaacaacac120gtactgtatgcaacaggcactaccggtaacttaatttcccgcgcgaccggcatgaacgtc180aacgcgatgttgagtggcccaatggggggtgaccagcaggttggcgcattgatctcagaa240gggaaaattgatgtattgattttcttctgggatccactaaatgccgtgccgcacgatcct300gacgtgaaagccttgctgcgtctggcgacggtatggaacattccggtcgccaccaacgtg360gcaacggcagacttcataatccagtcgccgcatttcaacgacgcggtcgatattctgatc420cccgattatcagcgttatctcgcggaccgtctgaagtaa459<210>21<211>367<212>prt<213>大肠杆菌(菌株k12)<400>21metaspargileileglnserproglylystyrileglnglyalaasp151015valileasnargleuglyglutyrleulysproleualagluargtrp202530leuvalvalglyasplysphevalleuglyphealaglnserthrval354045glulysserphelysaspalaglyleuvalvalgluilealaprophe505560glyglyglucysserglnasngluileaspargleuargglyileala65707580gluthralaglncysglyalaileleuglyileglyglyglylysthr859095leuaspthralalysalaleualahisphemetglyvalprovalala100105110ilealaprothrilealaserthraspalaprocysseralaleuser115120125valiletyrthraspgluglyglupheaspargtyrleuleuleupro130135140asnasnproasnmetvalilevalaspthrlysilevalalaglyala145150155160proalaargleuleualaalaglyileglyaspalaleualathrtrp165170175pheglualaargalacysserargserglyalathrthrmetalagly180185190glylyscysthrglnalaalaleualaleualagluleucystyrasn195200205thrleuleuglugluglyglulysalametleualaalagluglnhis210215220valvalthrproalaleugluargvalileglualaasnthrtyrleu225230235240serglyvalglyphegluserglyglyleualaalaalahisalaval245250255hisasnglyleuthralaileproaspalahishistyrtyrhisgly260265270glulysvalalapheglythrleuthrglnleuvalleugluasnala275280285provalglugluilegluthrvalalaalaleuserhisalavalgly290295300leuproilethrleualaglnleuaspilelysgluaspvalproala305310315320lysmetargilevalalaglualaalacysalagluglygluthrile325330335hisasnmetproglyglyalathrproaspglnvaltyralaalaleu340345350leuvalalaaspglntyrglyglnargpheleuglnglutrpglu355360365<210>22<211>1104<212>dna<213>大肠杆菌(菌株k12)<400>22atggaccgcattattcaatcaccgggtaaatacatccagggcgctgatgtgattaatcgt60ctgggcgaatacctgaagccgctggcagaacgctggttagtggtgggtgacaaatttgtt120ttaggttttgctcaatccactgtcgagaaaagctttaaagatgctggactggtagtagaa180attgcgccgtttggcggtgaatgttcgcaaaatgagatcgaccgtctgcgtggcatcgcg240gagactgcgcagtgtggcgcaattctcggtatcggtggcggaaaaaccctcgatactgcc300aaagcactggcacatttcatgggtgttccggtagcgatcgcaccgactatcgcctctacc360gatgcaccgtgcagcgcattgtctgttatctacaccgatgagggtgagtttgaccgctat420ctgctgttgccaaataacccgaatatggtcattgtcgacaccaaaatcgtcgctggcgca480cctgcacgtctgttagcggcgggtatcggcgatgcgctggcaacctggtttgaagcgcgt540gcctgctctcgtagcggcgcgaccaccatggcgggcggcaagtgcacccaggctgcgctg600gcactggctgaactgtgctacaacaccctgctggaagaaggcgaaaaagcgatgcttgct660gccgaacagcatgtagtgactccggcgctggagcgcgtgattgaagcgaacacctatttg720agcggtgttggttttgaaagtggtggtctggctgcggcgcacgcagtgcataacggcctg780accgctatcccggacgcgcatcactattatcacggtgaaaaagtggcattcggtacgctg840acgcagctggttctggaaaatgcgccggtggaggaaatcgaaaccgtagctgcccttagc900catgcggtaggtttgccaataactctcgctcaactggatattaaagaagatgtcccggcg960aaaatgcgaattgtggcagaagcggcatgtgcagaaggtgaaaccattcacaacatgcct1020ggcggcgcgacgccagatcaggtttacgccgctctgctggtagccgaccagtacggtcag1080cgtttcctgcaagagtgggaataa1104<210>23<211>367<212>prt<213>人工的<220><223>glda*(a160t)<400>23metaspargileileglnserproglylystyrileglnglyalaasp151015valileasnargleuglyglutyrleulysproleualagluargtrp202530leuvalvalglyasplysphevalleuglyphealaglnserthrval354045glulysserphelysaspalaglyleuvalvalgluilealaprophe505560glyglyglucysserglnasngluileaspargleuargglyileala65707580gluthralaglncysglyalaileleuglyileglyglyglylysthr859095leuaspthralalysalaleualahisphemetglyvalprovalala100105110ilealaprothrilealaserthraspalaprocysseralaleuser115120125valiletyrthraspgluglyglupheaspargtyrleuleuleupro130135140asnasnproasnmetvalilevalaspthrlysilevalalaglythr145150155160proalaargleuleualaalaglyileglyaspalaleualathrtrp165170175pheglualaargalacysserargserglyalathrthrmetalagly180185190glylyscysthrglnalaalaleualaleualagluleucystyrasn195200205thrleuleuglugluglyglulysalametleualaalagluglnhis210215220valvalthrproalaleugluargvalileglualaasnthrtyrleu225230235240serglyvalglyphegluserglyglyleualaalaalahisalaval245250255hisasnglyleuthralaileproaspalahishistyrtyrhisgly260265270glulysvalalapheglythrleuthrglnleuvalleugluasnala275280285provalglugluilegluthrvalalaalaleuserhisalavalgly290295300leuproilethrleualaglnleuaspilelysgluaspvalproala305310315320lysmetargilevalalaglualaalacysalagluglygluthrile325330335hisasnmetproglyglyalathrproaspglnvaltyralaalaleu340345350leuvalalaaspglntyrglyglnargpheleuglnglutrpglu355360365<210>24<211>1104<212>dna<213>人工的<220><223>glda*(a160t)<400>24atggaccgcattattcaatcaccgggtaaatacatccagggcgctgatgtgattaatcgt60ctgggcgaatacctgaagccgctggcagaacgctggttagtggtgggtgacaaatttgtt120ttaggttttgctcaatccactgtcgagaaaagctttaaagatgctggactggtagtagaa180attgcgccgtttggcggtgaatgttcgcaaaatgagatcgaccgtctgcgtggcatcgcg240gagactgcgcagtgtggcgcaattctcggtatcggtggcggaaaaaccctcgatactgcc300aaagcactggcacatttcatgggtgttccggtagcgatcgcaccgactatcgcctctacc360gatgcaccgtgcagcgcattgtctgttatctacaccgatgagggtgagtttgaccgctat420ctgctgttgccaaataacccgaatatggtcattgtcgacaccaaaatcgtcgctggcaca480cctgcacgtctgctagcggcgggtatcggcgatgcgctggcaacctggtttgaagcgcgt540gcctgctctcgtagcggcgcgaccaccatggcgggcggcaagtgcacccaggctgcgctg600gcactggctgaactgtgctacaacaccctgctggaagaaggcgaaaaagcgatgcttgct660gccgaacagcatgtagtgactccggcgctggagcgcgtgattgaagcgaacacctatttg720agcggtgttggttttgaaagtggtggtctggctgcggcgcacgcagtgcataacggcctg780accgctatcccggacgcgcatcactattatcacggtgaaaaagtggcattcggtacgctg840acgcagctggttctggaaaatgcgccggtggaggaaatcgaaaccgtagctgcccttagc900catgcggtaggtttgccaataactctcgctcaactggatattaaagaagatgtcccggcg960aaaatgcgaattgtggcagaagcggcatgtgcagaaggtgaaaccattcacaacatgcct1020ggcggcgcgacgccagatcaggtttacgccgctctgctggtagccgaccagtacggtcag1080cgtttcctgcaagagtgggaataa1104<210>25<211>382<212>prt<213>大肠杆菌(菌株k12)<400>25metalaasnargmetileleuasngluthralatrppheglyarggly151015alavalglyalaleuthraspgluvallysargargglytyrglnlys202530alaleuilevalthrasplysthrleuvalglncysglyvalvalala354045lysvalthrasplysmetaspalaalaglyleualatrpalailetyr505560aspglyvalvalproasnprothrilethrvalvallysgluglyleu65707580glyvalpheglnasnserglyalaasptyrleuilealaileglygly859095glyserproglnaspthrcyslysalaileglyileileserasnasn100105110progluphealaaspvalargserleugluglyleuserprothrasn115120125lysproservalproileleualaileprothrthralaglythrala130135140alagluvalthrileasntyrvalilethraspgluglulysargarg145150155160lysphevalcysvalaspprohisaspileproglnvalalapheile165170175aspalaaspmetmetaspglymetproproalaleulysalaalathr180185190glyvalaspalaleuthrhisalailegluglytyrilethrarggly195200205alatrpalaleuthraspalaleuhisilelysalailegluileile210215220alaglyalaleuargglyservalalaglyasplysaspalaglyglu225230235240glumetalaleuglyglntyrvalalaglymetglypheserasnval245250255glyleuglyleuvalhisglymetalahisproleuglyalaphetyr260265270asnthrprohisglyvalalaasnalaileleuleuprohisvalmet275280285argtyrasnalaaspphethrglyglulystyrargaspilealaarg290295300valmetglyvallysvalgluglymetserleugluglualaargasn305310315320alaalavalglualavalphealaleuasnargaspvalglyilepro325330335prohisleuargaspvalglyvalarglysgluaspileproalaleu340345350alaglnalaalaleuaspaspvalcysthrglyglyasnproargglu355360365alathrleugluaspilevalgluleutyrhisthralatrp370375380<210>26<211>1149<212>dna<213>大肠杆菌(菌株k12)<400>26atggctaacagaatgattctgaacgaaacggcatggtttggtcggggtgctgttggggct60ttaaccgatgaggtgaaacgccgtggttatcagaaggcgctgatcgtcaccgataaaacg120ctggtgcaatgcggcgtggtggcgaaagtgaccgataagatggatgctgcagggctggca180tgggcgatttacgacggcgtagtgcccaacccaacaattactgtcgtcaaagaagggctc240ggtgtattccagaatagcggcgcggattacctgatcgctattggtggtggttctccacag300gatacttgtaaagcgattggcattatcagcaacaacccggagtttgccgatgtgcgtagc360ctggaagggctttccccgaccaataaacccagtgtaccgattctggcaattcctaccaca420gcaggtactgcggcagaagtgaccattaactacgtgatcactgacgaagagaaacggcgc480aagtttgtttgcgttgatccgcatgatatcccgcaggtggcgtttattgacgctgacatg540atggatggtatgcctccagcgctgaaagctgcgacgggtgtcgatgcgctcactcatgct600attgaggggtatattacccgtggcgcgtgggcgctaaccgatgcactgcacattaaagcg660attgaaatcattgctggggcgctgcgaggatcggttgctggtgataaggatgccggagaa720gaaatggcgctcgggcagtatgttgcgggtatgggcttctcgaatgttgggttagggttg780gtgcatggtatggcgcatccactgggcgcgttttataacactccacacggtgttgcgaac840gccatcctgttaccgcatgtcatgcgttataacgctgactttaccggtgagaagtaccgc900gatatcgcgcgcgttatgggcgtgaaagtggaaggtatgagcctggaagaggcgcgtaat960gccgctgttgaagcggtgtttgctctcaaccgtgatgtcggtattccgccacatttgcgt1020gatgttggtgtacgcaaggaagacattccggcactggcgcaggcggcactggatgatgtt1080tgtaccggtggcaacccgcgtgaagcaacgcttgaggatattgtagagctttaccatacc1140gcctggtaa1149<210>27<211>603<212>prt<213>大肠杆菌(菌株k12)<400>27metasnproglnleuleuargvalthrasnargileilegluargser151015arggluthrargseralatyrleualaargilegluglnalalysthr202530serthrvalhisargserglnleualacysglyasnleualahisgly354045phealaalacysglnprogluasplysalaserleulyssermetleu505560argasnasnilealaileilethrsertyrasnaspmetleuserala65707580hisglnprotyrgluhistyrprogluileilearglysalaleuhis859095glualaasnalavalglyglnvalalaglyglyvalproalametcys100105110aspglyvalthrglnglyglnaspglymetgluleuserleuleuser115120125arggluvalilealametseralaalavalglyleuserhisasnmet130135140pheaspglyalaleupheleuglyvalcysasplysilevalprogly145150155160leuthrmetalaalaleuserpheglyhisleuproalavalpheval165170175proserglyprometalaserglyleuproasnlysglulysvalarg180185190ileargglnleutyralagluglylysvalaspargmetalaleuleu195200205gluserglualaalasertyrhisalaproglythrcysthrphetyr210215220glythralaasnthrasnglnmetvalvalgluphemetglymetgln225230235240leuproglyserserphevalhisproaspserproleuargaspala245250255leuthralaalaalaalaargglnvalthrargmetthrglyasngly260265270asnglutrpmetproileglylysmetileaspglulysvalvalval275280285asnglyilevalalaleuleualathrglyglyserthrasnhisthr290295300methisleuvalalametalaargalaalaglyileglnileasntrp305310315320aspasppheseraspleuseraspvalvalproleumetalaargleu325330335tyrproasnglyproalaaspileasnhispheglnalaalaglygly340345350valprovalleuvalarggluleuleulysalaglyleuleuhisglu355360365aspvalasnthrvalalaglypheglyleuserargtyrthrleuglu370375380protrpleuasnasnglygluleuasptrparggluglyalaglulys385390395400serleuaspserasnvalilealaserphegluglnpropheserhis405410415hisglyglythrlysvalleuserglyasnleuglyargalavalmet420425430lysthrseralavalprovalgluasnglnvalileglualaproala435440445valvalphegluserglnhisaspvalmetproalapheglualagly450455460leuleuaspargaspcysvalvalvalvalarghisglnglyprolys465470475480alaasnglymetprogluleuhislysleumetproproleuglyval485490495leuleuaspargcysphelysilealaleuvalthraspglyargleu500505510serglyalaserglylysvalproseralailehisvalthrproglu515520525alatyraspglyglyleuleualalysvalargaspglyaspileile530535540argvalasnglyglnthrglygluleuthrleuleuvalaspgluala545550555560gluleualaalaarggluprohisileproaspleuseralaserarg565570575valglythrglyarggluleupheseralaleuargglulysleuser580585590glyalagluglnglyalathrcysilethrphe595600<210>28<211>1812<212>dna<213>大肠杆菌(菌株k12)<400>28atgaatccacaattgttacgcgtaacaaatcgaatcattgaacgttcgcgcgagactcgc60tctgcttatctcgcccggatagaacaagcgaaaacttcgaccgttcatcgttcgcagttg120gcatgcggtaacctggcacacggtttcgctgcctgccagccagaagacaaagcctctttg180aaaagcatgttgcgtaacaatatcgccatcatcacctcctataacgacatgctctccgcg240caccagccttatgaacactatccagaaatcattcgtaaagccctgcatgaagcgaatgcg300gttggtcaggttgcgggcggtgttccggcgatgtgtgatggtgtcacccaggggcaggat360ggaatggaattgtcgctgctaagccgcgaagtgatagcgatgtctgcggcggtggggctg420tcccataacatgtttgatggtgctctgttcctcggtgtgtgcgacaagattgtcccgggt480ctgacgatggcagccctgtcgtttggtcatttgcctgcggtgtttgtgccgtctggaccg540atggcaagcggtttgccaaataaagaaaaagtgcgtattcgccagctttatgccgaaggt600aaagtggaccgcatggccttactggagtcagaagccgcgtcttaccatgcgccgggaaca660tgtactttctacggtactgccaacaccaaccagatggtggtggagtttatggggatgcag720ttgccaggctcttcttttgttcatccggattctccgctgcgcgatgctttgaccgccgca780gctgcgcgtcaggttacacgcatgaccggtaatggtaatgaatggatgccgatcggtaag840atgatcgatgagaaagtggtggtgaacggtatcgttgcactgctggcgaccggtggttcc900actaaccacaccatgcacctggtggcgatggcgcgcgcggccggtattcagattaactgg960gatgacttctctgacctttctgatgttgtaccgctgatggcacgtctctacccgaacggt1020ccggccgatattaaccacttccaggcggcaggtggcgtaccggttctggtgcgtgaactg1080ctcaaagcaggcctgctgcatgaagatgtcaatacggtggcaggttttggtctgtctcgt1140tatacccttgaaccatggctgaataatggtgaactggactggcgggaaggggcggaaaaa1200tcactcgacagcaatgtgatcgcttccttcgaacaacctttctctcatcatggtgggaca1260aaagtgttaagcggtaacctgggccgtgcggttatgaaaacctctgccgtgccggttgag1320aaccaggtgattgaagcgccagcggttgtttttgaaagccagcatgacgttatgccggcc1380tttgaagcgggtttgctggaccgcgattgtgtcgttgttgtccgtcatcaggggccaaaa1440gcgaacggaatgccagaattacataaactcatgccgccacttggtgtattattggaccgg1500tgtttcaaaattgcgttagttaccgatggacgactctccggcgcttcaggtaaagtgccg1560tcagctatccacgtaacaccagaagcctacgatggcgggctgctggcaaaagtgcgcgac1620ggggacatcattcgtgtgaatggacagacaggcgaactgacgctgctggtagacgaagcg1680gaactggctgctcgcgaaccgcacattcctgacctgagcgcgtcacgcgtgggaacagga1740cgtgaattattcagcgccttgcgtgaaaaactgtccggtgccgaacagggcgcaacctgt1800atcactttttaa1812<210>29<211>213<212>prt<213>大肠杆菌(菌株k12)<400>29metlysasntrplysthrseralagluserileleuthrthrglypro151015valvalprovalilevalvallyslysleugluhisalavalpromet202530alalysalaleuvalalaglyglyvalargvalleugluvalthrleu354045argthrglucysalavalaspalaileargalailealalysgluval505560proglualailevalglyalaglythrvalleuasnproglnglnleu65707580alagluvalthrglualaglyalaglnphealaileserproglyleu859095thrgluproleuleulysalaalathrgluglythrileproleuile100105110proglyileserthrvalsergluleumetleuglymetasptyrgly115120125leulysgluphelysphepheproalaglualaasnglyglyvallys130135140alaleuglnalailealaglypropheserglnvalargphecyspro145150155160thrglyglyileserproalaasntyrargasptyrleualaleulys165170175servalleucysileglyglysertrpleuvalproalaaspalaleu180185190glualaglyasptyraspargilethrlysleualaargglualaval195200205gluglyalalysleu210<210>30<211>642<212>dna<213>大肠杆菌(菌株k12)<400>30atgaaaaactggaaaacaagtgcagaatcaatcctgaccaccggcccggttgtaccggtt60atcgtggtaaaaaaactggaacacgcggtgccgatggcaaaagcgttggttgctggtggg120gtgcgcgttctggaagtgactctgcgtaccgagtgtgcagttgacgctatccgtgctatc180gccaaagaagtgcctgaagcgattgtgggtgccggtacggtgctgaatccacagcagctg240gcagaagtcactgaagcgggtgcacagttcgcaattagcccgggtctgaccgagccgctg300ctgaaagctgctaccgaagggactattcctctgattccggggatcagcactgtttccgaa360ctgatgctgggtatggactacggtttgaaagagttcaaattcttcccggctgaagctaac420ggcggcgtgaaagccctgcaggcgatcgcgggtccgttctcccaggtccgtttctgcccg480acgggtggtatttctccggctaactaccgtgactacctggcgctgaaaagcgtgctgtgc540atcggtggttcctggctggttccggcagatgcgctggaagcgggcgattacgaccgcatt600actaagctggcgcgtgaagctgtagaaggcgctaagctgtaa642<210>31<211>135<212>prt<213>大肠杆菌(菌株k12)<400>31metargleuleuhisthrmetleuargvalglyaspleuglnargser151015ileaspphetyrthrlysvalleuglymetlysleuleuargthrser202530gluasnproglutyrlystyrserleualaphevalglytyrglypro354045gluthrgluglualavalilegluleuthrtyrasntrpglyvalasp505560lystyrgluleuglythralatyrglyhisilealaleuservalasp65707580asnalaalaglualacysglulysileargglnasnglyglyasnval859095thrargglualaglyprovallysglyglythrthrvalilealaphe100105110valgluaspproaspglytyrlysilegluleuilegluglulysasp115120125alaglyargglyleuglyasn130135<210>32<211>408<212>dna<213>大肠杆菌(菌株k12)<400>32atgcgtcttcttcataccatgctgcgcgttggcgatttgcaacgctccatcgatttttat60accaaagtgctgggcatgaaactgctgcgtaccagcgaaaacccggaatacaaatactca120ctggcgtttgttggctacggcccggaaaccgaagaagcggtgattgaactgacctacaac180tggggcgtggataaatacgaactcggcactgcttatggtcacatcgcgcttagcgtagat240aacgccgctgaagcgtgcgaaaaaatccgtcaaaacgggggtaacgtgacccgtgaagcg300ggtccggtaaaaggcggtactacggttatcgcgtttgtggaagatccggacggttacaaa360attgagttaatcgaagagaaagacgccggtcgcggtctgggcaactaa408<210>33<211>479<212>prt<213>大肠杆菌(菌株k12)<400>33metservalprovalglnhispromettyrileaspglyglnpheval151015thrtrpargglyaspalatrpileaspvalvalasnproalathrglu202530alavalileserargileproaspglyglnalagluaspalaarglys354045alaileaspalaalagluargalaglnproglutrpglualaleupro505560alailegluargalasertrpleuarglysileseralaglyilearg65707580gluargalasergluileseralaleuilevalglugluglyglylys859095ileglnglnleualagluvalgluvalalaphethralaasptyrile100105110asptyrmetalaglutrpalaargargtyrgluglygluileilegln115120125seraspargproglygluasnileleuleuphelysargalaleugly130135140valthrthrglyileleuprotrpasnpheprophepheleuileala145150155160arglysmetalaproalaleuleuthrglyasnthrilevalilelys165170175prosergluphethrproasnasnalailealaphealalysileval180185190aspgluileglyleuproargglyvalpheasnleuvalleuglyarg195200205glygluthrvalglyglngluleualaglyasnprolysvalalamet210215220valsermetthrglyservalseralaglyglulysilemetalathr225230235240alaalalysasnilethrlysvalcysleugluleuglyglylysala245250255proalailevalmetaspaspalaaspleugluleualavallysala260265270ilevalaspserargvalileasnserglyglnvalcysasncysala275280285gluargvaltyrvalglnlysglyiletyraspglnphevalasnarg290295300leuglyglualametglnalavalglnpheglyasnproalagluarg305310315320asnaspilealametglyproleuileasnalaalaalaleugluarg325330335valgluglnlysvalalaargalavalglugluglyalaargvalala340345350pheglyglylysalavalgluglylysglytyrtyrtyrproprothr355360365leuleuleuaspvalargglnglumetserilemethisglugluthr370375380pheglyprovalleuprovalvalalapheaspthrleugluaspala385390395400ilesermetalaasnaspserasptyrglyleuthrserseriletyr405410415thrglnasnleuasnvalalametlysalailelysglyleulysphe420425430glygluthrtyrileasnarggluasnpheglualametglnglyphe435440445hisalaglytrparglysserglyileglyglyalaaspglylyshis450455460glyleuhisglutyrleuglnthrglnvalvaltyrleuglnser465470475<210>34<211>1440<212>dna<213>大肠杆菌(菌株k12)<400>34atgtcagtacccgttcaacatcctatgtatatcgatggacagtttgttacctggcgtgga60gacgcatggattgatgtggtaaaccctgctacagaggctgtcatttcccgcatacccgat120ggtcaggccgaggatgcccgtaaggcaatcgatgcagcagaacgtgcacaaccagaatgg180gaagcgttgcctgctattgaacgcgccagttggttgcgcaaaatctccgccgggatccgc240gaacgcgccagtgaaatcagtgcgctgattgttgaagaagggggcaagatccagcagctg300gctgaagtcgaagtggcttttactgccgactatatcgattacatggcggagtgggcacgg360cgttacgagggcgagattattcaaagcgatcgtccaggagaaaatattcttttgtttaaa420cgtgcgcttggtgtgactaccggcattctgccgtggaacttcccgttcttcctcattgcc480cgcaaaatggctcccgctcttttgaccggtaataccatcgtcattaaacctagtgaattt540acgccaaacaatgcgattgcattcgccaaaatcgtcgatgaaataggccttccgcgcggc600gtgtttaaccttgtactggggcgtggtgaaaccgttgggcaagaactggcgggtaaccca660aaggtcgcaatggtcagtatgacaggcagcgtctctgcaggtgagaagatcatggcgact720gcggcgaaaaacatcaccaaagtgtgtctggaattggggggtaaagcaccagctatcgta780atggacgatgccgatcttgaactggcagtcaaagccatcgttgattcacgcgtcattaat840agtgggcaagtgtgtaactgtgcagaacgtgtttatgtacagaaaggcatttatgatcag900ttcgtcaatcggctgggtgaagcgatgcaggcggttcaatttggtaaccccgctgaacgc960aacgacattgcgatggggccgttgattaacgccgcggcgctggaaagggtcgagcaaaaa1020gtggcgcgcgcagtagaagaaggggcgagagtggcgttcggtggcaaagcggtagagggg1080aaaggatattattatccgccgacattgctgctggatgttcgccaggaaatgtcgattatg1140catgaggaaacctttggcccggtgctgccagttgtcgcatttgacacgctggaagatgct1200atctcaatggctaatgacagtgattacggcctgacctcatcaatctatacccaaaatctg1260aacgtcgcgatgaaagccattaaagggctgaagtttggtgaaacttacatcaaccgtgaa1320aacttcgaagctatgcaaggcttccacgccggatggcgtaaatccggtattggcggcgca1380gatggtaaacatggcttgcatgaatatctgcagacccaggtggtttatttacagtcttaa1440<210>35<211>512<212>prt<213>大肠杆菌(菌株k12)<400>35metthrasnasnproproseralaglnilelysproglyglutyrgly151015pheproleulysleulysalaargtyraspasnpheileglyglyglu202530trpvalalaproalaaspglyglutyrtyrglnasnleuthrproval354045thrglyglnleuleucysgluvalalaserserglylysargaspile505560aspleualaleuaspalaalahislysvallysasplystrpalahis65707580thrservalglnaspargalaalaileleuphelysilealaasparg859095metgluglnasnleugluleuleualathralagluthrtrpaspasn100105110glylysproilearggluthrseralaalaaspvalproleualaile115120125asphispheargtyrphealasercysileargalaglngluglygly130135140ilesergluvalaspsergluthrvalalatyrhisphehisglupro145150155160leuglyvalvalglyglnileileprotrpasnpheproleuleumet165170175alasertrplysmetalaproalaleualaalaglyasncysvalval180185190leulysproalaargleuthrproleuservalleuleuleumetglu195200205ilevalglyaspleuleuproproglyvalvalasnvalvalasngly210215220alaglyglyvalileglyglutyrleualathrserlysargileala225230235240lysvalalaphethrglyserthrgluvalglyglnglnilemetgln245250255tyralathrglnasnileileprovalthrleugluleuglyglylys260265270serproasnilephephealaaspvalmetaspglugluaspalaphe275280285pheasplysalaleugluglyphealaleuphealapheasnglngly290295300gluvalcysthrcysproserargalaleuvalglngluseriletyr305310315320gluargphemetgluargalaileargargvalgluserileargser325330335glyasnproleuaspservalthrglnmetglyalaglnvalserhis340345350glyglnleugluthrileleuasntyrileaspileglylyslysglu355360365glyalaaspvalleuthrglyglyargarglysleuleugluglyglu370375380leulysaspglytyrtyrleugluprothrileleupheglyglnasn385390395400asnmetargvalpheglnglugluilepheglyprovalleualaval405410415thrthrphelysthrmetgluglualaleugluleualaasnaspthr420425430glntyrglyleuglyalaglyvaltrpserargasnglyasnleuala435440445tyrlysmetglyargglyileglnalaglyargvaltrpthrasncys450455460tyrhisalatyrproalahisalaalapheglyglytyrlysglnser465470475480glyileglyarggluthrhislysmetmetleugluhistyrglngln485490495thrlyscysleuleuvalsertyrserasplysproleuglyleuphe500505510<210>36<211>1539<212>dna<213>大肠杆菌(菌株k12)<400>36atgaccaataatcccccttcagcacagattaagcccggcgagtatggtttccccctcaag60ttaaaagcccgctatgacaactttattggcggcgaatgggtagcccctgccgacggcgag120tattaccagaatctgacgccggtgaccgggcagctgctgtgcgaagtggcgtcttcgggc180aaacgagacatcgatctggcgctggatgctgcgcacaaagtgaaagataaatgggcgcac240acctcggtgcaggatcgtgcggcgattctgtttaagattgccgatcgaatggaacaaaac300ctcgagctgttagcgacagctgaaacctgggataacggcaaacccattcgcgaaaccagt360gctgcggatgtaccgctggcgattgaccatttccgctatttcgcctcgtgtattcgggcg420caggaaggtgggatcagtgaagttgatagcgaaaccgtggcctatcatttccatgaaccg480ttaggcgtggtggggcagattatcccgtggaacttcccgctgctgatggcgagctggaaa540atggctcccgcgctggcggcgggcaactgtgtggtgctgaaacccgcacgtcttaccccg600ctttctgtactgctgctaatggaaattgtcggtgatttactgccgccgggcgtggtgaac660gtggtcaatggcgcaggtggggtaattggcgaatatctggcgacctcgaaacgcatcgcc720aaagtggcgtttaccggctcaacggaagtgggccaacaaattatgcaatacgcaacgcaa780aacattattccggtgacgctggagttgggcggtaagtcgccaaatatcttctttgctgat840gtgatggatgaagaagatgcctttttcgataaagcgctggaaggctttgcactgtttgcc900tttaaccagggcgaagtttgcacctgtccgagtcgtgctttagtgcaggaatctatctac960gaacgctttatggaacgcgccatccgccgtgtcgaaagcattcgtagcggtaacccgctc1020gacagcgtgacgcaaatgggcgcgcaggtttctcacgggcaactggaaaccatcctcaac1080tacattgatatcggtaaaaaagagggcgctgacgtgctcacaggcgggcggcgcaagctg1140ctggaaggtgaactgaaagacggctactacctcgaaccgacgattctgtttggtcagaac1200aatatgcgggtgttccaggaggagatttttggcccggtgctggcggtgaccaccttcaaa1260acgatggaagaagcgctggagctggcgaacgatacgcaatatggcctgggcgcgggcgtc1320tggagccgcaacggtaatctggcctataagatggggcgcggcatacaggctgggcgcgtg1380tggaccaactgttatcacgcttacccggcacatgcggcgtttggtggctacaaacaatca1440ggtatcggtcgcgaaacccacaagatgatgctggagcattaccagcaaaccaagtgcctg1500ctggtgagctactcggataaaccgttggggctgttctga1539<210>37<211>329<212>prt<213>大肠杆菌(菌株k12)<400>37metlysleualavaltyrserthrlysglntyrasplyslystyrleu151015glnglnvalasngluserpheglyphegluleugluphepheaspphe202530leuleuthrglulysthralalysthralaasnglycysglualaval354045cysilephevalasnaspaspglyserargprovalleuglugluleu505560lyslyshisglyvallystyrilealaleuargcysalaglypheasn65707580asnvalaspleuaspalaalalysgluleuglyleulysvalvalarg859095valproalatyraspproglualavalalagluhisalaileglymet100105110metmetthrleuasnargargilehisargalatyrglnargthrarg115120125aspalaasnpheserleugluglyleuthrglyphethrmettyrgly130135140lysthralaglyvalileglythrglylysileglyvalalametleu145150155160argileleulysglypheglymetargleuleualapheaspprotyr165170175proseralaalaalaleugluleuglyvalglutyrvalaspleupro180185190thrleuphesergluseraspvalileserleuhiscysproleuthr195200205progluasntyrhisleuleuasnglualaalaphegluglnmetlys210215220asnglyvalmetilevalasnthrserargglyalaleuileaspser225230235240glnalaalaileglualaleulysasnglnlysileglyserleugly245250255metaspvaltyrgluasngluargaspleuphephegluasplysser260265270asnaspvalileglnaspaspvalpheargargleuseralacyshis275280285asnvalleuphethrglyhisglnalapheleuthralaglualaleu290295300thrserileserglnthrthrleuglnasnleuserasnleuglulys305310315320glygluthrcysproasngluleuval325<210>38<211>990<212>dna<213>大肠杆菌(菌株k12)<400>38atgaaactcgccgtttatagcacaaaacagtacgacaagaagtacctgcaacaggtgaac60gagtcctttggctttgagctggaattttttgactttctgctgacggaaaaaaccgctaaa120actgccaatggctgcgaagcggtatgtattttcgtaaacgatgacggcagccgcccggtg180ctggaagagctgaaaaagcacggcgttaaatatatcgccctgcgctgtgccggtttcaat240aacgtcgaccttgacgcggcaaaagaactggggctgaaagtagtccgtgttccagcctat300gatccagaggccgttgctgaacacgccatcggtatgatgatgacgctgaaccgccgtatt360caccgcgcgtatcagcgtacccgtgatgctaacttctctctggaaggtctgaccggcttt420actatgtatggcaaaacggcaggcgttatcggtaccggtaaaatcggtgtggcgatgctg480cgcattctgaaaggttttggtatgcgtctgctggcgttcgatccgtatccaagtgcagcg540gcgctggaactcggtgtggagtatgtcgatctgccaaccctgttctctgaatcagacgtt600atctctctgcactgcccgctgacaccggaaaactatcatctgttgaacgaagccgccttc660gaacagatgaaaaatggcgtgatgatcgtcaataccagtcgcggtgcattgattgattct720caggcagcaattgaagcgctgaaaaatcagaaaattggttcgttgggtatggacgtgtat780gagaacgaacgcgatctattctttgaagataaatccaacgacgtgatccaggatgacgta840ttccgtcgcctgtctgcctgccacaacgtgctgtttaccgggcaccaggcattcctgaca900gcagaagctctgaccagtatttctcagactacgctgcaaaacttaagcaatctggaaaaa960ggcgaaacctgcccgaacgaactggtttaa990<210>39<211>246<212>prt<213>大肠杆菌(菌株k12)<400>39metservalileglyargilehisserpheglusercysglythrval151015aspglyproglyileargpheilethrphepheglnglycysleumet202530argcysleutyrcyshisasnargaspthrtrpaspthrhisglygly354045lysgluvalthrvalgluaspleumetlysgluvalvalthrtyrarg505560hisphemetasnalaserglyglyglyvalthralaserglyglyglu65707580alaileleuglnalagluphevalargasptrppheargalacyslys859095lysgluglyilehisthrcysleuaspthrasnglyphevalargarg100105110tyraspprovalileaspgluleuleugluvalthraspleuvalmet115120125leuaspleulysglnmetasnaspgluilehisglnasnleuvalgly130135140valserasnhisargthrleugluphealalystyrleualaasnlys145150155160asnvallysvaltrpileargtyrvalvalvalproglytrpserasp165170175aspaspaspseralahisargleuglygluphethrargaspmetgly180185190asnvalglulysilegluleuleuprotyrhisgluleuglylyshis195200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gccgttgaaagtgatgatgaacgtcggtaacccggaccgtgctttcgacttcgcc1500tgcctaccgaacgaaggcgtgggccttgcgcgtctggaatttatcatcaaccgtatgatt1560ggcgtccacccacgcgcactgcttgagtttgacgatcaggaaccgcagttgcaaaacgaa1620atccgcgagatgatgaaaggttttgattctccgcgtgaattttacgttggtcgtctgact1680gaagggatcgcgacgctgggtgccgcgttttatccgaagcgcgtcattgtccgtctctct1740gattttaaatcgaacgaatatgccaacctggtcggtggtgagcgttacgagccagatgaa1800gagaacccgatgctcggcttccgtggcgcgggccgctatgtttccgacagcttccgcgac1860tgtttcgcgctggagtgtgaagcagtgaaacgtgtgcgcaacgacatgggactgaccaac1920gttgagatcatgatcccgttcgtgcgtaccgtagatcaggcgaaagcggtggttgaagaa1980ctggcgcgtcaggggctgaaacgtggcgagaacgggctgaaaatcatcatgatgtgtgaa2040atcccgtccaacgccttgctggccgagcagttcctcgaatatttcgacggcttctcaatt2100ggctcaaacgatatgacgcagctggcgctcggtctggaccgtgactccggcgtggtgtct2160gaattgttcgatgagcgcaacgatgcggtgaaagcactgctgtcgatggctatccgtgcc2220gcgaagaaacagggcaaatatgtcgggatttgcggtcagggtccgtccgaccacgaagac2280tttgccgcatggttgatggaagaggggatcgatagcctgtctctgaacccggacaccgtg2340gtgcaaacctggttaagcctggctgaactgaagaaataa2379<210>57<211>474<212>prt<213>大肠杆菌(菌株k12)<400>57metserthrgluilelysthrglnvalvalvalleuglyalaglypro151015alaglytyrseralaalapheargcysalaaspleuglyleugluthr202530valilevalgluargtyrasnthrleuglyglyvalcysleuasnval354045glycysileproserlysalaleuleuhisvalalalysvalileglu505560glualalysalaleualagluhisglyilevalpheglygluprolys65707580thraspileasplysileargthrtrplysglulysvalileasngln859095leuthrglyglyleualaglymetalalysglyarglysvallysval100105110valasnglyleuglylysphethrglyalaasnthrleugluvalglu115120125glygluasnglylysthrvalileasnpheaspasnalaileileala130135140alaglyserargproileglnleupropheileprohisgluasppro145150155160argiletrpaspserthraspalaleugluleulysgluvalproglu165170175argleuleuvalmetglyglyglyileileglyleuglumetglythr180185190valtyrhisalaleuglyserglnileaspvalvalglumetpheasp195200205glnvalileproalaalaasplysaspilevallysvalphethrlys210215220argileserlyslyspheasnleumetleugluthrlysvalthrala225230235240valglualalysgluaspglyiletyrvalthrmetgluglylyslys245250255alaproalagluproglnargtyraspalavalleuvalalailegly260265270argvalproasnglylysasnleuaspalaglylysalaglyvalglu275280285valaspaspargglypheileargvalasplysglnleuargthrasn290295300valprohisilephealaileglyaspilevalglyglnprometleu305310315320alahislysglyvalhisgluglyhisvalalaalagluvalileala325330335glylyslyshistyrpheaspprolysvalileproserilealatyr340345350thrgluprogluvalalatrpvalglyleuthrglulysglualalys355360365glulysglyilesertyrgluthralathrpheprotrpalaalaser370375380glyargalailealaseraspcysalaaspglymetthrlysleuile385390395400pheasplysgluserhisargvalileglyglyalailevalglythr405410415asnglyglygluleuleuglygluileglyleualaileglumetgly420425430cysaspalagluaspilealaleuthrilehisalahisprothrleu435440445hisgluservalglyleualaalagluvalphegluglyserilethr450455460aspleuproasnprolysalalyslyslys465470<210>58<211>1425<212>dna<213>大肠杆菌(菌株k12)<400>58atgagtactgaaatcaaaactcaggtcgtggtacttggggcaggccccgcaggttactcc60gctgccttccgttgcgctgatttaggtctggaaaccgtaatcgtagaacgttacaacacc120cttggcggtgtttgcctgaacgtcggctgtatcccttctaaagcactgctgcacgtagca180aaagttatcgaagaagccaaagcgctggctgaacacggtatcgtcttcggcgaaccgaaa240accgatatcgacaagattcgtacctggaaagagaaagtgatcaatcagctgaccggtggt300ctggctggtatggcgaaaggccgcaaagtcaaagtggtcaacggtctgggtaaattcacc360ggggctaacaccctggaagttgaaggtgagaacggcaaaaccgtgatcaacttcgacaac420gcgatcattgcagcgggttctcgcccgatccaactgccgtttattccgcatgaagatccg480cgtatctgggactccactgacgcgctggaactgaaagaagtaccagaacgcctgctggta540atgggtggcggtatcatcggtctggaaatgggcaccgtttaccacgcgctgggttcacag600attgacgtggttgaaatgttcgaccaggttatcccggcagctgacaaagacatcgttaaa660gtcttcaccaagcgtatcagcaagaaattcaacctgatgctggaaaccaaagttaccgcc720gttgaagcgaaagaagacggcatttatgtgacgatggaaggcaaaaaagcacccgctgaa780ccgcagcgttacgacgccgtgctggtagcgattggtcgtgtgccgaacggtaaaaacctc840gacgcaggcaaagcaggcgtggaagttgacgaccgtggtttcatccgcgttgacaaacag900ctgcgtaccaacgtaccgcacatctttgctatcggcgatatcgtcggtcaaccgatgctg960gcacacaaaggtgttcacgaaggtcacgttgccgctgaagttatcgccggtaagaaacac1020tacttcgatccgaaagttatcccgtccatcgcctataccgaaccagaagttgcatgggtg1080ggtctgactgagaaagaagcgaaagagaaaggcatcagctatgaaaccgccaccttcccg1140tgggctgcttctggtcgtgctatcgcttccgactgcgcagacggtatgaccaagctgatt1200ttcgacaaagaatctcaccgtgtgatcggtggtgcgattgtcggtactaacggcggcgag1260ctgctgggtgaaatcggcctggcaatcgaaatgggttgtgatgctgaagacatcgcactg1320accatccacgcgcacccgactctgcacgagtctgtgggcctggcggcagaagtgttcgaa1380ggtagcattaccgacctgccgaacccgaaagcgaagaagaagtaa1425<210>59<211>238<212>prt<213>大肠杆菌(菌株k12)<400>59metglnthrprohisileleuilevalgluaspgluleuvalthrarg151015asnthrleulysserilepheglualagluglytyraspvalpheglu202530alathraspglyalaglumethisglnileleuserglutyraspile354045asnleuvalilemetaspileasnleuproglylysasnglyleuleu505560leualaarggluleuarggluglnalaasnvalalaleumetpheleu65707580thrglyargaspasngluvalasplysileleuglyleugluilegly859095alaaspasptyrilethrlyspropheasnproarggluleuthrile100105110argalaargasnleuleuserargthrmetasnleuglythrvalser115120125glugluargargservalglusertyrlyspheasnglytrpgluleu130135140aspileasnserargserleuileglyproaspglygluglntyrlys145150155160leuproargserglupheargalametleuhisphecysgluasnpro165170175glylysileglnserargalagluleuleulyslysmetthrglyarg180185190gluleulysprohisaspargthrvalaspvalthrileargargile195200205arglyshisphegluserthrproaspthrprogluileilealathr210215220ilehisglygluglytyrargphecysglyaspleugluasp225230235<210>60<211>717<212>dna<213>大肠杆菌(菌株k12)<400>60atgcagaccccgcacattcttatcgttgaagacgagttggtaacacgcaacacgttgaaa60agtattttcgaagcggaaggctatgatgttttcgaagcgacagatggcgcggaaatgcat120cagatcctctctgaatatgacatcaacctggtgatcatggatatcaatctgccgggtaag180aacggtcttctgttagcgcgtgaactgcgcgagcaggcgaatgttgcgttgatgttcctg240actggccgtgacaacgaagtcgataaaattctcggcctcgaaatcggtgcagatgactac300atcaccaaaccgttcaacccgcgtgaactgacgattcgtgcacgcaacctactgtcccgt360accatgaatctgggtactgtcagcgaagaacgtcgtagcgttgaaagctacaagttcaat420ggttgggaactggacatcaacagccgttcgttgatcggccctgatggcgagcagtacaag480ctgccgcgcagcgagttccgcgccatgcttcacttctgtgaaaacccaggcaaaattcag540tcccgtgctgaactgctgaagaaaatgaccggccgtgagctgaaaccgcacgaccgtact600gtagacgtgacgatccgccgtattcgtaaacatttcgaatctacgccggatacgccggaa660atcatcgccaccattcacggtgaaggttatcgcttctgcggtgatctggaagattaa717<210>61<211>434<212>prt<213>大肠杆菌(菌株k12)<400>61metthrthrproleulyslysilevalilevalglyglyglyalagly151015glyleuglumetalathrglnleuglyhislysleuglyarglyslys202530lysalalysilethrleuvalaspargasnhisserhisleutrplys354045proleuleuhisgluvalalathrglyserleuaspgluglyvalasp505560alaleusertyrleualahisalaargasnhisglypheglnphegln65707580leuglyservalileaspileaspargglualalysthrilethrile859095alagluleuargaspglulysglygluleuleuvalprogluarglys100105110ilealatyraspthrleuvalmetalaleuglyserthrserasnasp115120125pheasnthrproglyvallysgluasncysilepheleuaspasnpro130135140hisglnalaargargphehisglnglumetleuasnleupheleulys145150155160tyrseralaasnleuglyalaasnglylysvalasnilealaileval165170175glyglyglyalathrglyvalgluleuseralagluleuhisasnala180185190vallysglnleuhissertyrglytyrlysglyleuthrasngluala195200205leuasnvalthrleuvalglualaglygluargileleuproalaleu210215220proproargileseralaalaalahisasngluleuthrlysleugly225230235240valargvalleuthrglnthrmetvalthrseralaaspgluglygly245250255leuhisthrlysaspglyglutyrileglualaaspleumetvaltrp260265270alaalaglyilelysalaproasppheleulysaspileglyglyleu275280285gluthrasnargileasnglnleuvalvalgluprothrleuglnthr290295300thrargaspproaspiletyralaileglyaspcysalasercyspro305310315320argprogluglyglyphevalproproargalaglnalaalahisgln325330335metalathrcysalametasnasnileleualaglnmetasnglylys340345350proleulysasntyrglntyrlysasphisglyserleuvalserleu355360365serasnpheserthrvalglyserleumetglyasnleuthrarggly370375380sermetmetilegluglyargilealaargphevaltyrileserleu385390395400tyrargmethisglnilealaleuhisglytyrphelysthrglyleu405410415metmetleuvalglyserileasnargvalileargproargleulys420425430leuhis<210>62<211>1305<212>dna<213>大肠杆菌(菌株k12)<400>62ttgactacgccattgaaaaagattgtgattgtcggcggcggtgctggtgggctggaaatg60gcaacacagctggggcataagctgggacgcaagaaaaaagccaaaattacgctggtcgat120cgtaaccacagccacctgtggaaaccgctgctgcacgaagtggcgactggctcgcttgat180gaaggcgtcgatgcgttgagctatctggcccatgcgcgcaatcatggtttccagttccag240ctgggttccgtcattgatattgatcgtgaagcgaaaacaatcactattgcagaactgcgc300gacgagaaaggtgaactgctggttccggaacgtaaaatcgcctatgacaccctggtaatg360gcgctgggtagcacctctaacgatttcaatacgccaggtgtcaaagagaactgcattttc420ctcgataacccgcaccaggcgcgtcgcttccaccaggagatgctgaatttgttcctgaaa480tactccgccaacctgggcgcgaatggcaaagtgaacattgcgattgtcggcggcggcgcg540acgggtgtagaactctccgctgaattgcacaacgcggtcaagcaactgcacagctacggt600tacaaaggcctgaccaacgaagccctgaacgtaacgctggtagaagcgggagaacgtatt660ttgcctgcgttaccgccacgtatctctgctgcggcccacaacgagctaacgaaacttggc720gttcgcgtgctgacgcaaaccatggtcaccagtgctgatgaaggcggcctgcacactaaa780gatggcgaatatattgaggctgatctgatggtatgggcagccgggatcaaagcgccagac840ttcctgaaagatatcggtggtcttgaaactaaccgtatcaaccagctggtggtggaaccg900acgctgcaaaccacccgcgatccagacatttacgctattggcgactgcgcgtcatgcccg960cgtccggaagggggctttgttccgccgcgtgctcaggctgcacaccagatggcgacttgc1020gcaatgaacaacattctggcgcagatgaacggtaagccgctgaaaaattatcagtataaa1080gatcatggttcgctggtatcgctgtcgaacttctccaccgtcggtagcctgatgggtaac1140ctgacgcgcggctcaatgatgattgaaggacgaattgcgcgctttgtatatatctcgcta1200taccgaatgcatcagattgcgctgcatggttactttaaaaccggattaatgatgctggtg1260gggagtattaaccgcgttatccgtccgcgtttgaagttgcattaa1305<210>63<211>331<212>prt<213>大肠杆菌(菌株k12)<400>63metthrilelysvalglyileasnglypheglyargileglyargile151015valpheargalaalaglnlysargseraspilegluilevalalaile202530asnaspleuleuaspalaasptyrmetalatyrmetleulystyrasp354045serthrhisglyargpheaspglythrvalgluvallysaspglyhis505560leuilevalasnglylyslysileargvalthralagluargasppro65707580alaasnleulystrpaspgluvalglyvalaspvalvalalagluala859095thrglyleupheleuthraspgluthralaarglyshisilethrala100105110glyalalyslysvalvalmetthrglyproserlysaspasnthrpro115120125metphevallysglyalaasnpheasplystyralaglyglnaspile130135140valserasnalasercysthrthrasncysleualaproleualalys145150155160valileasnaspasnpheglyileilegluglyleumetthrthrval165170175hisalathrthralathrglnlysthrvalaspglyproserhislys180185190asptrpargglyglyargglyalaserglnasnileileproserser195200205thrglyalaalalysalavalglylysvalleuprogluleuasngly210215220lysleuthrglymetalapheargvalprothrproasnvalserval225230235240valaspleuthrvalargleuglulysalaalathrtyrgluglnile245250255lysalaalavallysalaalaalagluglyglumetlysglyvalleu260265270glytyrthrgluaspaspvalvalserthrasppheasnglygluval275280285cysthrservalpheaspalalysalaglyilealaleuasnaspasn290295300phevallysleuvalsertrptyraspasngluthrglytyrserasn305310315320lysvalleuaspleuilealahisileserlys325330<210>64<211>996<212>dna<213>大肠杆菌(菌株k12)<400>64atgactatcaaagtaggtatcaacggttttggccgtatcggtcgcattgttttccgtgct60gctcagaaacgttctgacatcgagatcgttgcaatcaacgacctgttagacgctgattac120atggcatacatgctgaaatatgactccactcacggccgtttcgacggtaccgttgaagtg180aaagacggtcatctgatcgttaacggtaaaaaaatccgtgttaccgctgaacgtgatccg240gctaacctgaaatgggacgaagttggtgttgacgttgtcgctgaagcaactggtctgttc300ctgactgacgaaactgctcgtaaacacatcaccgctggtgcgaagaaagtggttatgact360ggtccgtctaaagacaacactccgatgttcgttaaaggcgctaacttcgacaaatatgct420ggccaggacatcgtttccaacgcttcctgcaccaccaactgcctggctccgctggctaaa480gttatcaacgataacttcggcatcatcgaaggtctgatgaccaccgttcacgctactacc540gctactcagaaaaccgttgatggcccgtctcacaaagactggcgcggcggccgcggcgct600tcccagaacatcatcccgtcctctaccggtgctgctaaagctgtaggtaaagtactgcca660gaactgaatggcaaactgactggtatggcgttccgcgttccgaccccgaacgtatctgta720gttgacctgaccgttcgtctggaaaaagctgcaacttacgagcagatcaaagctgccgtt780aaagctgctgctgaaggcgaaatgaaaggcgttctgggctacaccgaagatgacgtagta840tctaccgatttcaacggcgaagtttgcacttccgtgttcgatgctaaagctggtatcgct900ctgaacgacaacttcgtgaaactggtatcctggtacgacaacgaaaccggttactccaac960aaagttctggacctgatcgctcacatctccaaataa996<210>65<211>415<212>prt<213>大肠杆菌(菌株w)<400>65metalaleuasnilepropheargasnalatyrtyrargphealaser151015sertyrserpheleuphepheilesertrpserleutrptrpserleu202530tyralailetrpleulysglyhisleuglyleuthrglythrgluleu354045glythrleutyrservalasnglnphethrserileleuphemetmet505560phetyrglyilevalglnasplysleuglyleulyslysproleuile65707580trpcysmetserpheileleuvalleuthrglyprophemetiletyr859095valtyrgluproleuleuglnserasnpheservalglyleuileleu100105110glyalaleuphepheglyleuglytyrleualaglycysglyleuleu115120125aspserphethrglulysmetalaargasnphehispheglutyrgly130135140thralaargalatrpglyserpheglytyralaileglyalaphephe145150155160alaglyilephepheserileserprohisileasnphetrpleuval165170175serleupheglyalavalphemetmetileasnmetargphelysasp180185190lysasphisglncysilealaalaaspalaglyglyvallyslysglu195200205asppheilealavalphelysaspargasnphetrpvalphevalile210215220pheilevalglythrtrpserphetyrasnilepheaspglnglnleu225230235240pheprovalphetyralaglyleuphegluserhisaspvalglythr245250255argleutyrglytyrleuasnserpheglnvalvalleuglualaleu260265270cysmetalaileileprophephevalasnargvalglyprolysasn275280285alaleuleuileglyvalvalilemetalaleuargileleusercys290295300alaleuphevalasnprotrpileileserleuvallysleuleuhis305310315320alailegluvalproleucysvalileservalphelystyrserval325330335alaasnpheasplysargleuserserthrilepheleuileglyphe340345350glnilealaserserleuglyilevalleuleuserthrprothrgly355360365ileleupheasphisalaglytyrglnthrvalphephealaileser370375380glyilevalcysleumetleuleupheglyilephepheleuserlys385390395400lysarggluglnilevalmetgluthrprovalproseralaile405410415<210>66<211>1248<212>dna<213>大肠杆菌(菌株w)<400>66atggcactgaatattccattcagaaatgcgtactatcgttttgcatccagttactcattt60ctcttttttatttcctggtcgctgtggtggtcgttatacgctatttggctgaaaggacat120ctaggattaacagggacggaattaggtacactttattcggtcaaccagtttaccagcatt180ctatttatgatgttctacggcatcgttcaggataaactcggtctgaagaaaccgctcatc240tggtgtatgagtttcattctggtcttgaccggaccgtttatgatttacgtttatgaaccg300ttactgcaaagcaatttttctgtaggtctaattctgggggcgctcttttttggcctgggg360tatctggcgggatgcggtttgcttgacagcttcaccgaaaaaatggcgcgaaattttcat420ttcgaatatggaacagcgcgcgcctggggatcttttggctatgctattggcgcgttcttt480gccggtatattttttagtatcagtccccatatcaacttctggttggtctcgctatttggc540gctgtatttatgatgatcaacatgcgttttaaagataaggatcaccagtgcatagcggcg600gatgcgggaggggtaaaaaaagaggattttatcgcagttttcaaggatcgaaacttctgg660gttttcgtcatatttattgtggggacgtggtctttctataacatttttgatcaacaactc720tttcctgtcttttatgcaggtttattcgaatcacacgatgtaggaacgcgcctgtatggt780tatctcaactcattccaggtggtactcgaagcgctgtgcatggcgattattcctttcttt840gtgaatcgggtagggccaaaaaatgcattacttatcggtgttgtgattatggcgttgcgt900atcctttcctgcgcgttgttcgttaacccctggattatttcattagtgaagctgttacat960gccattgaggttccactttgtgtcatatccgtcttcaaatacagcgtggcaaactttgat1020aagcgcctgtcgtcgacgatctttctgattggttttcaaattgccagttcgcttgggatt1080gtgctgctttcaacgccgactgggatactctttgaccacgcaggctaccagacagttttc1140ttcgcaatttcgggtattgtctgcctgatgttgctatttggcattttcttcctgagtaaa1200aaacgcgagcaaatagttatggaaacgcctgtaccttcagcaatatag1248<210>67<211>477<212>prt<213>大肠杆菌(菌株w)<400>67metthrglnserargleuhisalaalaglnasnalaleualalysleu151015hisgluargargglyasnthrphetyrprohisphehisleualapro202530proalaglytrpmetasnaspproasnglyleuiletrppheasnasp354045argtyrhisalaphetyrglnhishisprometsergluhistrpgly505560promethistrpglyhisalathrseraspaspmetilehistrpgln65707580hisgluproilealaleualaproglyaspgluasnasplysaspgly859095cyspheserglyseralavalaspaspasnglyvalleuserleuile100105110tyrthrglyhisvaltrpleuaspglyalaglyasnaspaspalaile115120125arggluvalglncysleualathrserargaspglyilehispheglu130135140lysglnglyvalileleuthrproprogluglyilemethisphearg145150155160aspprolysvaltrpargglualaaspthrtrptrpmetvalvalgly165170175alalysaspproglyasnthrglyglnileleuleutyrargglyser180185190serleuargglutrpthrpheaspargvalleualahisalaaspala195200205glyglusertyrmettrpglucysproaspphepheserleuglyasp210215220glnhistyrleumetpheserproglnglymetasnalagluglytyr225230235240sertyrargasnargpheglnserglyvalileproglymettrpser245250255proglyargleuphealaglnserglyhisphethrgluleuaspasn260265270glyhisaspphetyralaproglnserphevalalalysaspglyarg275280285argilevalileglytrpmetaspmettrpgluserprometproser290295300lysarggluglytrpalaglycysmetthrleualaarggluleuser305310315320gluserasnglylysleuleuglnargprovalhisglualagluser325330335leuargglnglnhisglnserileserproargthrileserasnlys340345350tyrvalleuglngluasnalaglnalavalgluileglnleuglntrp355360365alaleulysasnseraspalagluhistyrglyleuglnleuglyala370375380glymetargleutyrileaspasnglnsergluargleuvalleutrp385390395400argtyrtyrprohisgluasnleuaspglytyrargserileproleu405410415proglnglyaspmetleualaleuargilepheileaspthrserser420425430valgluvalpheileasnaspglyglualavalmetserserargile435440445tyrproglnproglugluarggluleuserleutyralaserhisgly450455460valalavalleuglnhisglyalaleutrpglnleugly465470475<210>68<211>1434<212>dna<213>大肠杆菌(菌株w)<400>68atgacgcaatctcgattgcatgcggcgcaaaacgcactagcaaaacttcacgagcgccga60ggtaacactttctatccccattttcacctcgcgcctcctgccgggtggatgaacgatcca120aacggcctgatctggtttaacgatcgttatcacgcgttttatcaacatcacccgatgagc180gaacactgggggccaatgcactggggacatgccaccagcgacgatatgatccactggcag240catgagcctattgcgctagcgccaggagacgagaatgacaaagacgggtgtttttcaggt300agtgctgtcgatgacaatggtgtcctctcacttatctacaccggacacgtctggctcgat360ggtgcaggtaatgacgatgcaattcgcgaagtacaatgtctggctaccagtcgggatggt420attcatttcgagaaacagggtgtgatcctcactccaccagaaggcatcatgcacttccgc480gatcctaaagtgtggcgtgaagccgacacatggtggatggtagtcggggcgaaagaccca540ggcaacacggggcagatcctgctttatcgcggcagttcattgcgtgaatggactttcgat600cgcgtactggcccacgctgatgcgggtgaaagctatatgtgggaatgtccggactttttc660agccttggcgatcagcattatctgatgttttccccgcagggaatgaatgccgagggatac720agttatcgaaatcgctttcaaagtggcgtaatacccggaatgtggtcgccaggacgactt780tttgcacaatccgggcattttactgaacttgataacgggcatgacttttatgcaccacaa840agctttgtagcgaaggatggtcggcgtattgttatcggctggatggatatgtgggaatcg900ccaatgccctcaaaacgtgaaggctgggcaggctgcatgacgctggcgcgcgagctatca960gagagcaatggcaaactcctacaacgcccggtacacgaagctgagtcgttacgccagcag1020catcaatctatctctccccgcacaatcagcaataaatatgttttgcaggaaaacgcgcaa1080gcagttgagattcagttgcagtgggcgctgaagaacagtgatgccgaacattacggatta1140cagctcggcgctggaatgcggctgtatattgataaccaatctgagcgacttgttttgtgg1200cggtattacccacacgagaatttagatggctaccgtagtattcccctcccgcagggtgac1260atgctcgccctaaggatatttatcgatacatcatccgtggaagtatttattaacgacggg1320gaggcggtgatgagtagccgaatatatccgcagccagaagaacgggaactgtcgctctat1380gcctcccacggagtggctgtgctgcaacatggagcactctggcaactgggttaa1434<210>69<211>307<212>prt<213>大肠杆菌(菌株w)<400>69metseralalysvaltrpvalleuglyaspalavalvalaspleuleu151015progluseraspglyargleuleuprocysproglyglyalaproala202530asnvalalavalglyilealaargleuglyglythrserglypheile354045glyargvalglyaspasppropheglyalaleumetglnargthrleu505560leuthrgluglyvalaspilethrtyrleulysglnaspglutrphis65707580argthrserthrvalleuvalaspleuasnaspglnglygluargser859095phethrphemetvalargproseralaaspleupheleugluthrthr100105110aspleuprocystrparghisglyglutrpleuhisleucysserile115120125alaleuseralagluproserargthrseralaphethralametthr130135140alailearghisalaglyglyphevalserpheaspproasnilearg145150155160gluaspleutrpglnaspgluhisleuleuargleucysleuarggln165170175alaleuglnleualaaspvalvallysleuserglugluglutrparg180185190leuileserglylysthrglnasnaspargaspilecysalaleuala195200205lysglutyrgluilealametleuleuvalthrlysglyalaglugly210215220valvalvalcystyrargglyglnvalhishisphealaglymetser225230235240valasncysvalaspserthrglyalaglyaspalaphevalalagly245250255leuleuthrglyleuserserthrglyleuserthraspgluargglu260265270metargargileileaspleualaglnargcysglyalaleualaval275280285thralalysglyalametthralaleuprocysargglngluleuglu290295300serglulys305<210>70<211>924<212>dna<213>大肠杆菌(菌株w)<400>70atgtcagccaaagtatgggttttaggggatgcggtcgtagatctcttgccagaatcagac60gggcggctactgccttgtcctggcggcgcgccagctaacgttgcggtgggaatcgccaga120ttaggcggaacaagtgggtttataggtcgggtcggtgatgatccttttggtgcgttaatg180caaagaacgctgctaactgagggtgtcgatatcacgtatctgaagcaagatgaatggcac240cggacatccacggtgcttgtcgatctgaacgatcaaggagaacgttcatttacgtttatg300gtccgccccagtgccgatctttttttagagacgacagacttgccctgctggcgacatggc360gaatggttacatctctgttcaattgcgttgtctgccgagccttcgcgtaccagcgcattt420actgcgatgacggcgatccggcatgccggaggttttgtcagcttcgatcccaatattcgt480gaagatctatggcaagacgagcatttgctccgcttgtgtttgcggcaggcgctacaactg540gcggatgtcgtcaagctctcggaagaagaatggcgacttatcagtggaaaaacacagaac600gatcgggatatatgcgccctggcaaaagagtatgagatcgccatgctgttggtgactaaa660ggtgcagaaggggtggtggtctgttatcgaggacaagtccaccattttgctggaatgtct720gtgaattgtgtcgatagcactggggcgggagatgcgttcgttgccgggttactcacaggt780ctgtcctctacgggattatctacagatgagagagaaatgcgacgaattatcgatctcgct840caacgttgcggagcgcttgcagtaacagcgaaaggggcaatgacagcgctgccatgtcga900caagaactggaaagtgagaagtaa924<210>71<211>456<212>prt<213>肠道沙门氏菌亚种肠道沙门菌鼠伤寒血清型<400>71metaspphegluglnilesercysserleuleuproleuleuglygly151015lysgluasnilealaseralaalahiscysalathrargleuargleu202530valleuvalaspaspserleualaaspglnglnalaileglylysval354045gluglyvallysglycyspheargasnalaglyglnmetglnileile505560pheglythrglyvalvalasnlysvaltyralaalaphethrglnala65707580alaglyilesergluserserlysserglualaalaaspilealaala859095lyslysleuasnpropheglnargilealaargleuleuserasnile100105110phevalproileileproalailevalalaserglyleuleumetgly115120125leuleuglymetvallysthrtyrglytrpvalaspproglyasnala130135140iletyrilemetleuaspmetcysserseralaalapheileileleu145150155160proileleuileglyphethralaalaargglupheglyglyasnpro165170175tyrleuglyalathrleuglyglyileleuthrhisproalaleuthr180185190asnalatrpglyvalalaalaglyphehisthrmetasnphephegly195200205phegluilealametileglytyrglnglythrvalpheprovalleu210215220leualavaltrpphemetserilevalglulysglnleuargargala225230235240ileproaspalaleuaspleuileleuthrpropheleuthrvalile245250255ileserglypheilealaleuleuileileglyproalaglyargala260265270leuglyaspglyileserphevalleuserthrleuileserhisala275280285glytrpleualaglyleuleupheglyglyleutyrservalileval290295300ilethrglyilehishisserphehisalavalglualaglyleuleu305310315320glyasnproserileglyvalasnpheleuleuproiletrpalamet325330335alaasnvalalaglnglyglyalacysleualavaltrpphelysthr340345350lysaspalalysilelysalailethrleuproseralapheserala355360365metleuglyilethrglualaalailepheglyileasnleuargphe370375380vallyspropheilealaalaleuileglyglyalaalaglyglyala385390395400trpvalvalservalhisvaltyrmetthralavalglyleuthrala405410415ileproglymetalailevalglnalaserserleuleuasntyrile420425430ileglymetvalilealapheglyvalalaphethrvalserleuval435440445leulystyrlysthraspalaglu450455<210>72<211>1371<212>dna<213>肠道沙门氏菌亚种肠道沙门菌鼠伤寒血清型<400>72atggattttgaacagatttcctgctcgctgcttccgcttcttggaggcaaagaaaatatc60gccagcgccgcgcactgcgccacgcgcctgcgcctggtgctggtcgatgattcgctggcc120gaccagcaggccatcggcaaagttgaaggggtgaagggctgttttcgtaatgccggacag180atgcagattattttcggcaccggggtggtaaataaggtctacgctgcctttactcaggcg240gcgggtattagcgaatccagcaaatcggaagccgccgacatcgcggcaaaaaagctcaat300ccgttccagcgcatcgcccgcctgctatcaaacatcttcgtgccgataatccctgccatc360gtcgcctctggtctgctgatgggcctgctgggaatggtcaaaacatacggctgggttgac420ccgggcaacgccatctacatcatgctggatatgtgcagctcggcggcatttatcattctg480ccgattctgattggctttaccgccgcccgcgaattcggcggtaatccttatctcggcgcg540acgcttggcggcattctgactcatccagcgctgactaacgcctggggcgtggccgcgggt600ttccacaccatgaactttttcggcttcgaaattgccatgatcggctatcagggtacggtg660ttcccggtactgctggcagtatggtttatgagcatcgttgagaagcagttgcgtcgcgca720atccccgatgccctggatttgatcctgacgccgttcctgacggtgattatatccggtttt780atcgccctgttgattatcggcccggccggtcgcgcactgggcgacggtatctcgtttgtc840ctcagcaccctgattagccacgccggctggctcgccgggttactgtttggcggtctctat900tcagttatcgtcattaccggtattcatcacagcttccatgcggttgaagccgggttgctg960ggcaatccctccatcggcgtcaacttcctgctgccgatttgggcgatggccaacgtcgct1020cagggcggagcctgtctggcggtgtggttcaaaaccaaagatgcaaaaattaaagccatt1080actctgccctcggcgttttccgccatgctgggcatcaccgaggcggcgatttttggtatt1140aacctgcgctttgtgaagccatttattgcggcgctgattggtggtgcggcgggcggcgca1200tgggtggtatctgtacacgtctacatgaccgcggtcggcttgacagcgatccccggcatg1260gccatcgtgcaggccagttcgctgttgaactacattatcgggatggttatcgcctttggc1320gtcgcctttacggtctccctggttttgaaatacaaaacggacgctgaataa1371<210>73<211>307<212>prt<213>肠道沙门氏菌亚种肠道沙门菌鼠伤寒血清型<400>73metasnalalysvaltrpvalleuglyaspalavalvalaspleuleu151015proglusergluglyargleuleuglncysproglyglyalaproala202530asnvalalavalglyvalalaargleuglyglyasnserglypheile354045glyalavalglyglyasppropheglyargtyrmetarghisthrleu505560glnglngluglnvalaspvalserhismettyrleuaspaspglnhis65707580argthrserthrvalvalvalaspleuaspaspglnglygluargthr859095phethrphemetvalargproseralaaspleupheleuvalgluglu100105110aspleuproglnphealaalaglyglntrpleuhisvalcysserile115120125alaleuseralagluproserargserthrthrphealaalametglu130135140serileargseralaglyglyargvalserpheaspproasnilearg145150155160proaspleutrpglnaspglnalaleuleuleualacysleuasparg165170175alaleuhismetalaasnvalvallysleusergluglugluleuval180185190pheileserserserasnaspleualatyrglyilealaservalthr195200205gluargtyrglnprogluleuleuleuvalthrargglylysalagly210215220valleualaalapheglnglnlysphethrhispheasnalaargpro225230235240valalaservalaspthrthrglyalaglyaspalaphevalalagly245250255leuleualaserleualaalaasnglymetprothraspmetthrala260265270leugluprothrleuthrleualaglnthrcysglyalaleualathr275280285thralalysglyalametthralaleuprotyrglnargaspleuasn290295300argglnphe305<210>74<211>924<212>dna<213>肠道沙门氏菌亚种肠道沙门菌鼠伤寒血清型<400>74atgaatgcaaaagtttgggttctgggcgacgcggtggtggacctgctgccggagagcgaa60gggcgcctgctgcagtgccctggaggcgcgccggctaacgtggcggtaggggttgcccgc120cttggcggcaacagcggatttatcggcgccgtcggcggtgacccgtttggccgctacatg180cgtcataccctgcaacaggagcaggtcgacgtcagccatatgtatctcgacgatcagcac240cgcacgtccactgtggtcgtcgaccttgacgaccagggggaacgcacctttacctttatg300gtacgccccagcgcggacctgttcctggttgaagaagacctgccacagtttgccgccgga360cagtggttgcacgtctgctccatcgcgctcagcgccgagcccagccgtagcactaccttc420gcggcgatggagagcatcaggtctgccggcggtcgggtcagctttgaccctaatattcgt480cccgatctctggcaggatcaggctttgctgctagcctgcctcgatcgcgctttgcacatg540gccaacgtggtaaagctatcggaagaggagctggtcttcatcagcagcagtaatgattta600gcatacggaatcgccagcgtaacggagcgctatcagccagaattgctactggtgacccgg660ggcaaagcgggggtgcttgccgcgtttcagcagaagtttacccatttcaacgcccggcct720gtggccagcgtggacaccaccggcgcgggagacgcatttgtcgccggactgctcgccagc780cttgcggctaacgggatgccaacggacatgaccgcactggaaccgacactcacgcttgca840cagacctgcggcgccctggccaccacagccaaaggtgcgatgaccgccttgccttatcag900cgcgatctcaaccgtcagttttaa924<210>75<211>466<212>prt<213>肠道沙门氏菌亚种肠道沙门菌鼠伤寒血清型<400>75met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cgggctttatgctcagcaacgacctg720ctgattgtgaccggtgcgctggtcggttcttcgggggctatcctttcttacattatgtgt780aaggcgatgaaccgttcctttatcagcgttattgcgggtggtttcggcaccgacggctct840tctactggcgatgatcaggaagtgggtgagcaccgcgaaatcaccgcagaagagacagcg900gaactgctgaaaaactcccattcagtgatcattactccggggtacggcatggcagtcgcg960caggcgcaatatcctgtcgctgaaattactgagaaattgcgcgctcgtggtattaatgtg1020cgtttcggtatccacccggtcgcggggcgtttgcctggacatatgaacgtattgctggct1080gaagcaaaagtaccgtatgacatcgtgctggaaatggacgagatcaatgatgactttgct1140gataccgataccgtactggtgattggtgctaacgatacggttaacccggcggcgcaggat1200gatccgaagagtccgattgctggtatgcctgtgctggaagtgtggaaagcgcagaacgtg1260attgtctttaaacgttcgatgaacactggctatgctggtgtgcaaaacccgctgttcttc1320aaggaaaacacccacatgctgtttggtgacgccaaagccagcgtggatgcaatcctgaaa1380gctctgtaa1389<210>93<211>549<212>prt<213>大肠杆菌(菌株k12)<400>93metlysasnileasnprothrglnthralaalatrpglnalaleugln151015lyshispheaspglumetlysaspvalthrilealaaspleupheala202530lysaspglyaspargpheserlyspheseralathrpheaspaspgln354045metleuvalasptyrserlysasnargilethrglugluthrleuala505560lysleuglnaspleualalysglucysaspleualaglyalailelys65707580sermetpheserglyglulysileasnargthrgluasnargalaval859095leuhisvalalaleuargasnargserasnthrproileleuvalasp100105110glylysaspvalmetprogluvalasnalavalleuglulysmetlys115120125thrpheserglualaileileserglyglutrplysglytyrthrgly130135140lysalailethraspvalvalasnileglyileglyglyseraspleu145150155160glyprotyrmetvalthrglualaleuargprotyrlysasnhisleu165170175asnmethisphevalserasnvalaspglythrhisilealagluval180185190leulyslysvalasnprogluthrthrleupheleuvalalaserlys195200205thrphethrthrglngluthrmetthrasnalahisseralaargasp210215220trppheleulysalaalaglyaspglulyshisvalalalyshisphe225230235240alaalaleuserthrasnalalysalavalglyglupheglyileasp245250255thralaasnmetphegluphetrpasptrpvalglyglyargtyrser260265270leutrpseralaileglyleuserilevalleuserileglypheasp275280285asnphevalgluleuleuserglyalahisalametasplyshisphe290295300serthrthrproalaglulysasnleuprovalleuleualaleuile305310315320glyiletrptyrasnasnphepheglyalagluthrglualaileleu325330335protyraspglntyrmethisargphealaalatyrpheglnglngly340345350asnmetgluserasnglylystyrvalaspargasnglyasnvalval355360365asptyrglnthrglyproileiletrpglygluproglythrasngly370375380glnhisalaphetyrglnleuilehisglnglythrlysmetvalpro385390395400cysasppheilealaproalailethrhisasnproleuserasphis405410415hisglnlysleuleuserasnphephealaglnthrglualaleuala420425430pheglylysserarggluvalvalgluglnglutyrargaspglngly435440445lysaspproalathrleuasptyrvalvalprophelysvalpheglu450455460glyasnargprothrasnserileleuleuarggluilethrprophe465470475480serleuglyalaleuilealaleutyrgluhislysilephethrgln485490495glyvalileleuasnilephethrpheaspglntrpglyvalgluleu500505510glylysglnleualaasnargileleuprogluleulysaspasplys515520525gluileserserhisaspserserthrasnglyleuileasnargtyr530535540lysalatrparggly545<210>94<211>1650<212>dna<213>大肠杆菌(菌株k12)<400>94atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgat60gaaatgaaagacgttacgatcgccgatctttttgctaaagacggcgatcgtttttctaag120ttctccgcaaccttcgacgatcagatgctggtggattactccaaaaaccgcatcactgaa180gagacgctggcgaaattacaggatctggcgaaagagtgcgatctggcgggcgcgattaag240tcgatgttctctggcgagaagatcaaccgcactgaaaaccgcgccgtgctgcacgtagcg300ctgcgtaaccgtagcaataccccgattttggttgatggcaaagacgtaatgccggaagtc360aacgcggtgctggagaagatgaaaaccttctcagaagcgattatttccggtgagtggaaa420ggttataccggcaaagcaatcactgacgtagtgaacatcgggatcggcggttctgacctc480ggcccatacatggtgaccgaagctctgcgtccgtacaaaaaccacctgaacatgcacttt540gtttctaacgtcgatgggactcacatcgcggaagtgctgaaaaaagtaaacccggaaacc600acgctgttcttggtagcatctaaaaccttcaccactcaggaaactatgaccaacgcccat660agcgcgcgtgactggttcctgaaagcggcaggtgatgaaaaacacgttgcaaaacacttt720gcggcgctttccaccaatgccaaagccgttggcgagtttggtattgatactgccaacatg780ttcgagttctgggactgggttggcggccgttactctttgtggtcagcgattggcctgtcg840attgttctctccatcggctttgataacttcgttgaactgctttccggcgcacacgcgatg900gacaagcatttctccaccacgcctgccgagaaaaacctgcctgtactgctggcgctgatt960ggcatctggtacaacaatttctttggtgcggaaactgaagcgattctgccgtatgaccag1020tatatgcaccgtttcgcggcgtacttccagcagggcaatatggagtccaacggtaagtat1080gttgaccgtaacggtaacgttgtggattaccagactggcccgattatctggggtgaacca1140ggcactaacggtcagcacgcgttctaccagctgatccaccagggaaccaaaatggtaccg1200tgcgatttcatcgctccggctatcacccataacccgctctctgatcatcaccagaaactg1260ctgtctaacttcttcgcccagaccgaagcgctggcgtttggtaaatcccgcgaagtggtt1320gagcaggaatatcgtgatcagggtaaagatccggcaacgcttgactacgtggtgccgttc1380aaagtattcgaaggtaaccgcccgaccaactccatcctgctgcgtgaaatcactccgttc1440agcctgggtgcgttgattgcgctgtatgagcacaaaatctttactcagggcgtgatcctg1500aacatcttcaccttcgaccagtggggcgtggaactgggtaaacagctggcgaaccgtatt1560ctgccagagctgaaagatgataaagaaatcagcagccacgatagctcgaccaatggtctg1620attaaccgctataaagcgtggcgcggttaa1650<210>95<211>320<212>prt<213>大肠杆菌(菌株k12)<400>95metilelyslysileglyvalleuthrserglyglyaspalaprogly151015metasnalaalaileargglyvalvalargseralaleuthrglugly202530leugluvalmetglyiletyraspglytyrleuglyleutyrgluasp354045argmetvalglnleuaspargtyrservalseraspmetileasnarg505560glyglythrpheleuglyseralaargpheproglupheargaspglu65707580asnileargalavalalailegluasnleulyslysargglyileasp859095alaleuvalvalileglyglyaspglysertyrmetglyalametarg100105110leuthrglumetglypheprocysileglyleupro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yrvalileasnglnalaleuthrglu130135140asnprogluprothrtrpargalametglugluleuvalglusergly145150155160leuvallysalaileglyvalserasntrpthrileproglyleulys165170175lysleuleuglnilealalysilelysproalavalasnglnileglu180185190ilehispropheleuproasnglugluleuvalalaphecyspheglu195200205asnglyileleuproglualatyrserproleuglyserglnasngln210215220valproserthrglygluargvalargaspasnprothrleulysala225230235240valalagluargserglytyrserleualaglnileleuleualatrp245250255glyleulysargglytyrvalvalleuprolysserserthrproser260265270argilegluserasnpheasnileprogluleuseraspgluaspphe275280285glualaileglnglnvalalalysglyarghisthrargphevalasn290295300metlysaspthrpheglytyrasnvaltrpproglugluglu305310315<210>114<211>957<212>dna<213>人工的<220><223>密码子优化的基因gld2<400>114atggcaagcaaaacctataccctgaatacaggtgcaaaaattccggcagttggttttggc60acctttgcaaatgaaggtgcgaaaggtgaaacctatgcagcagttaccaaagcactggat120gttggttatcgtcatctggattgtgcatggttttatcacaatgaagatgaagttggtgat180gccgttcgtgattttctggcacgtcgtccggatgttaaacgtgaggacctgtttatttgt240accaaagtgtggaatcatctgcacgaaccggaagatgttaaatggtcagcaaaaaatagc300tgcgagaacctgaaagtggattatattgacctgtttctggttcattggccgattgcagca360gaaaaaaacagcgatcgtagcgttaaactgggtccggatggcaaatatgttattaatcag420gcactgaccgaaaatccggaaccgacctggcgtgcaatggaagaactggttgaaagcggt480ctggttaaagcaattggtgttagcaattggaccattccgggtctgaaaaaactgctgcag540attgcaaaaatcaaaccggcagttaaccagattgaaatccatccgtttctgccgaatgag600gaactggtggcattttgttttgaaaatggtattctgccggaagcatatagtccgctgggt660agccagaatcaggttccgagcacaggtgaacgtgttcgtgataatccgaccctgaaagca720gttgcagaacgtagcggttatagcctggcacagattctgctggcatggggactgaaacgt780ggttatgttgtgctgccgaaaagcagcaccccgagccgtattgaaagcaatttcaatatt840ccggaactgagcgacgaagattttgaagcaattcagcaggttgcaaaaggtcgtcatacc900cgttttgtgaatatgaaagataccttcggctataacgtttggcctgaagaagaataa957<210>115<211>383<212>prt<213>大肠杆菌(菌株k12)<400>115metalaalaserthrphepheileproservalasnvalileglyala151015aspserleuthraspalametasnmetmetalaasptyrglyphethr202530argthrleuilevalthraspasnmetleuthrlysleuglymetala354045glyaspvalglnlysalaleuglugluargasnilepheservalile505560tyraspglythrglnproasnprothrthrgluasnvalalaalagly65707580leulysleuleulysgluasnasncysaspservalileserleugly859095glyglyserprohisaspcysalalysglyilealaleuvalalaala100105110asnglyglyaspileargasptyrgluglyvalaspargseralalys115120125proglnleuprometilealaileasnthrthralaglythralaser130135140glumetthrargphecysileilethraspglualaarghisilelys145150155160metalailevalasplyshisvalthrproleuleuservalasnasp165170175serserleumetileglymetprolysserleuthralaalathrgly180185190metaspalaleuthrhisalaileglualatyrvalserilealaala195200205thrproilethraspalacysalaleulysalavalthrmetileala210215220gluasnleuproleualavalgluaspglyserasnalalysalaarg225230235240glualametalatyralaglnpheleualaglymetalapheasnasn245250255alaserleuglytyrvalhisalametalahisglnleuglyglyphe260265270tyrasnleuprohisglyvalcysasnalavalleuleuprohisval275280285glnvalpheasnserlysvalalaalaalaargleuargaspcysala290295300alaalametglyvalasnvalthrglylysasnaspalagluglyala305310315320glualacysileasnalailearggluleualalyslysvalaspile325330335proalaglyleuargaspleuasnvallysglugluaspphealaval340345350leualathrasnalaleulysaspalacysglyphethrasnproile355360365glnalathrhisglugluilevalalailetyrargalaalamet370375380<210>116<211>1152<212>dna<213>大肠杆菌(菌株k12)<400>116atggcagcttcaacgttctttattccttctgtgaatgtcatcggcgctgattcattgact60gatgcaatgaatatgatggcagattatggatttacccgtaccttaattgtcactgacaat120atgttaacgaaattaggtatggcgggcgatgtgcaaaaagcactggaagaacgcaatatt180tttagcgttatttatgatggcacccaacctaaccccaccacggaaaacgtcgccgcaggt240ttgaaattacttaaagagaataattgcgatagcgtgatctccttaggcggtggttctcca300cacgactgcgcaaaaggtattgcgctggtggcagccaatggcggcgatattcgcgattac360gaaggcgttgaccgctctgcaaaaccgcagctgccgatgatcgccatcaataccacggcg420ggtacggcctctgaaatgacccgtttctgcatcatcactgacgaagcgcgtcatatcaaa480atggcgattgttgataaacatgtcactccgctgctttctgtcaatgactcctctctgatg540attggtatgccgaagtcactgaccgccgcaacgggtatggatgccttaacgcacgctatc600gaagcatatgtttctattgccgccacgccgatcactgacgcttgtgcactgaaagccgtg660accatgattgccgaaaacctgccgttagccgttgaagatggcagtaatgcgaaagcgcgt720gaagcaatggcttatgcccagttcctcgccggtatggcgttcaataatgcttctctgggt780tatgttcatgcgatggcgcaccagctgggcggtttctacaacctgccacacggtgtatgt840aacgccgttttgctgccgcacgttcaggtattcaacagcaaagtcgccgctgcacgtctg900cgtgactgtgccgctgcaatgggcgtgaacgtgacaggtaaaaacgacgcggaaggtgct960gaagcctgcattaacgccatccgtgaactggcgaagaaagtggatatcccggcaggccta1020cgcgacctgaacgtgaaagaagaagatttcgcggtattggcgactaatgccctgaaagat1080gcctgtggctttactaacccgatccaggcaactcacgaagaaattgtggcgatttatcgc1140gcagcgatgtaa1152<210>117<211>256<212>prt<213>肺炎克雷伯菌<400>117metlyslysvalalaleuvalthrglyalaglyglnglyileglylys151015alailealaleuargleuvallysaspglyphealavalalaileala202530asptyrasnaspalathralalysalavalalasergluileasngln354045alaglyglyargalametalavallysvalaspvalseraspargasp505560glnvalphealaalavalgluglnalaarglysthrleuglyglyphe65707580aspvalilevalasnasnalaglyvalalaproserthrproileglu859095serilethrprogluilevalasplysvaltyrasnileasnvallys100105110glyvaliletrpglyileglnalaalavalglualaphelyslysglu115120125glyhisglyglylysileileasnalacysserglnalaglyhisval130135140glyasnprogluleualavaltyrserserserlysphealavalarg145150155160glyleuthrglnthralaalaargaspleualaproleuglyilethr165170175valasnglytyrcysproglyilevallysthrpromettrpalaglu180185190ileaspargglnvalserglualaalaglylysproleuglytyrgly195200205thralagluphealalysargilethrleuglyargleuserglupro210215220gluaspvalalaalacysvalsertyrleualaserproaspserasp225230235240tyrmetthrglyglnserleuleuileaspglyglymetvalpheasn245250255<210>118<211>771<212>dna<213>肺炎克雷伯菌<400>118atgaaaaaagtcgcacttgttaccggcgccggccaggggattggtaaagctatcgccctt60cgtctggtgaaggatggatttgccgtggccattgccgattataacgacaccaccgccaaa120gcggtcgcctccgaaatcaaccaggccggcggccgcgccatggcggtgaaagtggatgtc180tccgaccgcgatcaggtgtttgccgccgtcgaacaggcgcgcaaaacgctgggcggcttc240gacgtcatcgtcaacaacgccggcgtggcgccgtccacgccgatcgagtccattaccccg300gagattgtcgataaagtctacaacatcaacgttaaaggggtgatctggggcattcaggcg360gcggtcgaggcctttaagaaagagggtcacggcgggaaaatcatcaacgcctgttcccag420gccggccacgtcggcaacccggagctggcggtatatagctcgagtaaattcgccgtacgc480ggcttaacccagaccgccgctcgcgacctcgcgccgctgggcatcacagtcaacggctac540tgcccggggattgtcaaaacgccaatgtgggccgaaattgaccgccaggtgtccgaagcc600gccggtaaaccgctgggttacggtaccgccgagttcgccaaacgcatcaccctcggccgc660ctgtccgagccggaagatgtcgccgcctgcgtctcctatcttgccagcccggattctgat720tatatgaccggtcagtcattgctgatcgacggcgggatggtgtttaactaa771<210>119<211>101<212>dna<213>人工的<220><223>用于缺失yqhd的寡核苷酸<400>119atgaacaactttaatctgcacaccccaacccgcattctgtttggtaaaggcgcaatcgct60ggtttacgcgaacaaattccgtgtaggctggagctgcttcg101<210>120<211>101<212>dna<213>人工的<220><223>用于缺失yqhd的寡核苷酸<400>120ttagcgggcggcttcgtatatacggcggctgacatccaacgtaatgtcatgattttcgcc60cagttgggtcatgccgtgctccatatgaatatcctccttag101<210>121<211>101<212>dna<213>人工的<220><223>用于缺失ydjg的寡核苷酸<400>121ttaacgctccagggcctctgccatttccctcatcaatgttgcgtctgcatccgataagtt60gatattcagtgccgcgacatgtgtaggctggagctgcttcg101<210>122<211>100<212>dna<213>人工的<220><223>用于缺失ydjg的寡核苷酸<400>122atgaaaaagatacctttaggcacaacggatattacgctttcgcgaatggggttggggaca60tgggccattggcggcggtcccatatgaatatcctccttag100<210>123<211>100<212>dna<213>人工的<220><223>用于缺失ptsg的寡核苷酸<400>123gtacacggcgaggctctccccccttgccacgcgtgagaacgtaaaaaaagcacccatact60caggagcactctcaattatgtgtaggctggagctgcttcg100<210>124<211>100<212>dna<213>人工的<220><223>用于缺失ptsg的寡核苷酸<400>124ataacacctgtaaaaaaggcagccatctggctgccttagtctccccaacgtcttacggat60tagtggttacggatgtactccatatgaatatcctccttag100<210>125<211>339<212>prt<213>人工的<220><223>yjgb*(i165v)<400>125metsermetilelyssertyralaalalysglualaglyglygluleu151015gluvaltyrglutyraspproglygluleuargproglnaspvalglu202530valglnvalasptyrcysglyilecyshisseraspleusermetile354045aspasnglutrpglypheserglntyrproleuvalalaglyhisglu505560valileglyargvalvalalaleuglyseralaalaglnasplysgly65707580leuglnvalglyglnargvalglyileglytrpthralaargsercys859095glyhiscysaspalacysileserglyasnglnileasncysglugln100105110glyalavalprothrilemetasnargglyglyphealaglulysleu115120125argalaasptrpglntrpvalileproleuprogluasnileaspile130135140gluseralaglyproleuleucysglyglyvalthrvalphelyspro145150155160leuleumethishisilethralathrserargvalglyvalilegly165170175ileglyglyleuglyhisilealailelysleuleuhisalametgly180185190cysgluvalthralapheserserasnproalalysgluglngluval195200205leualametglyalaasplysvalvalasnserargaspproglnala210215220leulysalaleualaglyglnpheaspleuileileasnthrvalasn225230235240valserleuasptrpglnprotyrpheglualaleuthrtyrglygly245250255asnphehisthrvalglyalavalleuthrproleuservalproala260265270phethrleuilealaglyaspargservalserglyseralathrgly275280285thrprotyrgluleuarglysleumetargphealaalaargserlys290295300valalaprothrthrgluleupheprometserlysileasnaspala305310315320ileglnhisvalargaspglylysalaargtyrargvalvalleulys325330335alaaspphe<210>126<211>1020<212>dna<213>人工的<220><223>yjgb*(i165v)<400>126atgtcaatgattaaatcgtatgcggcgaaagaagcgggtggcgaactggaagtgtatgaa60tacgacccaggcgaactgcgtccgcaggatgtggaagtccaggtggattattgcggtatt120tgccatagcgacctgtctatgattgacaacgaatggggctttagccagtatccgctggtg180gcgggtcacgaagtgattggtcgtgtggttgccctgggcagtgccgcccaggataaaggc240ttacaggtgggtcagcgtgtgggcattggctggacggcacgcagttgcggtcattgcgat300gcgtgtatcagcggcaaccaaatcaactgcgaacagggtgccgttccgacgattatgaac360cgtggcggctttgccgagaaactgcgtgccgattggcagtgggtgattccgctgcccgaa420aacattgatattgaatctgcgggtccgctgttatgtggcggtgttaccgtgttcaaaccg480ctgttgatgcaccacattaccgccaccagccgtgttggcgtgattggcatcggcggtctg540ggtcacattgccatcaaactgttacacgcaatgggctgcgaagtgacggcgtttagcagt600aacccagcgaaagaacaggaagtgctggcgatgggtgcggataaagtggtgaacagccgt660gacccgcaggcactgaaagccctggcgggtcagtttgacctgattatcaacaccgtgaat720gtcagcctggattggcagccgtattttgaagccctgacctatggcggtaacttccatacc780gtgggtgccgtgctgaccccgctgagcgtgcctgcgtttaccctgattgcgggtgaccgc840agcgtgagcggcagtgcgacgggcaccccgtatgaactgcgtaaactgatgcgttttgcc900gctcgcagcaaagtggcaccgaccaccgaactgtttccgatgagcaaaatcaacgatgcg960attcagcatgtgcgtgacggcaaagcccgctatcgtgtggtgctgaaagcggatttttaa1020<210>127<211>339<212>prt<213>人工的<220><223>yjgb*(q39r/i165v/a296v)<400>127metsermetilelyssertyralaalalysglualaglyglygluleu151015gluvaltyrglutyraspproglygluleuargproargaspvalglu202530valglnvalasptyrcysglyilecyshisseraspleusermetile354045aspasnglutrpglypheserglntyrproleuvalalaglyhisglu505560valileglyargvalvalalaleuglyseralaalaglnasplysgly65707580leuglnvalglyglnargvalglyileglytrpthralaargsercys859095glyhiscysaspalacysileserglyasnglnileasncysglugln100105110glyalavalprothrilemetasnargglyglyphealaglulysleu115120125argalaasptrpglntrpvalileproleuprogluasnileaspile130135140gluseralaglyproleuleucysglyglyvalthrvalphelyspro145150155160leuleumethishisilethralathrserargvalglyvalilegly165170175ileglyglyleuglyhisilealailelysleuleuhisalametgly180185190cysgluvalthralapheserserasnproalalysgluglngluval195200205leualametglyalaasplysvalvalasnserargaspproglnala210215220leulysalaleualaglyglnpheaspleuileileasnthrvalasn225230235240valserleuasptrpglnprotyrpheglualaleuthrtyrglygly245250255asnphehisthrvalglyalavalleuthrproleuservalproala260265270phethrleuilealaglyaspargservalserglyservalthrgly275280285thrprotyrgluleuarglysleumetargphealaalaargserlys290295300valalaprothrthrgluleupheprometserlysileasnaspala305310315320ileglnhisvalargaspglylysalaargtyrargvalvalleulys325330335alaaspphe<210>128<211>1020<212>dna<213>人工的<220><223>yjgb*(q39r/i165v/a296v)<400>128atgtcaatgattaaatcgtatgcggcgaaagaagcgggtggcgaactggaagtgtatgaa60tacgacccaggcgaactgcgtccgcgggatgtggaagtccaggtggattattgcggtatt120tgccatagcgacctgtctatgattgacaacgaatggggctttagccagtatccgctggtg180gcgggtcacgaagtgattggtcgtgtggttgccctgggcagtgccgcccaggataaaggc240ttacaggtgggtcagcgtgtgggcattggctggacggcacgcagttgcggtcattgcgat300gcgtgtatcagcggcaaccaaatcaactgcgaacagggtgccgttccgacgattatgaac360cgtggcggctttgccgagaaactgcgtgccgattggcagtgggtgattccgctgcccgaa420aacattgatattgaatctgcgggtccgctgttatgtggcggtgttaccgtgttcaaaccg480ctgttgatgcaccacattaccgccaccagccgtgttggcgtgattggcatcggcggtctg540ggtcacattgccatcaaactgttacacgcaatgggctgcgaagtgacggcgtttagcagt600aacccagcgaaagaacaggaagtgctggcgatgggtgcggataaagtggtgaacagccgt660gacccgcaggcactgaaagccctggcgggtcagtttgacctgattatcaacaccgtgaat720gtcagcctggattggcagccgtattttgaagccctgacctatggcggtaacttccatacc780gtgggtgccgtgctgaccccgctgagcgtgcctgcgtttaccctgattgcgggtgaccgc840agcgtgagcggcagtgtgacgggcaccccgtatgaactgcgtaaactgatgcgttttgcc900gctcgcagcaaagtggcaccgaccaccgaactgtttccgatgagcaaaatcaacgatgcg960attcagcatgtgcgtgacggcaaagcccgctatcgtgtggtgctgaaagcggatttttaa1020当前第1页12