用于2,4‑二羟基丁酸的优化生产的经修饰的微生物的制作方法

导言本发明涉及经遗传修饰的微生物，用于通过经由1,2,4-丁三醇中间体代谢转化木糖来生产2,4-二羟基丁酸。本发明还涉及生产2,4-二羟基丁酸的方法，其通过在发酵培养基中培养所述经遗传修饰的微生物并从所述培养基中回收2,4-dhb进行。2,4-二羟基丁酸(即2,4-dhb或dhb)，也称为2,4-二羟基丁(烷)酸或2,4-二羟基丁酸酯/盐，是一种具有高经济利益的化合物，因为它可充当用于化学合成2-羟基-4-(甲基硫代)-丁酸酯/盐(hmtb)(一种甲硫氨酸类似物，在动物营养中，尤其是家禽中广泛使用的食品添加剂)的前体。实际上，hmtb可作为甲硫氨酸的低价氨基酸替代物包括在动物饲料中，以显著增加肉产率(us2009/318715)。常规地，通过在甲硫醇和碱性或酸性催化剂存在下采用α-羟基-γ-丁内酯(2hbl)的化学合成方法生产hmtb。在工业上在三个步骤中从苹果酸(de19735575a1,au2004200948a)，或者在两个步骤中从γ-丁内酯(wo2008/022953a1)以已知的方式制备2hbl。这两条路线引起的问题在于分离2hbl的困难以及非常显著的盐的产生，所述盐然后必须被除去。此外，hmtb的石油化学合成在经济上不可行，因为它依赖于使用有害的材料和条件以及昂贵的材料和试剂。最近，已显示可通过调节适当的ph在水性介质中将2,4-dhb转化成α-羟基-γ-丁内酯(2hbl)(us2013/0204016)。因此，即使没有用于2,4-二羟基丁酸的生化生产的天然代谢途径，微生物中用于dhb产生的合成代谢途径代表一种有吸引力的替代方案，因为它减轻了许多上文提及的问题。事实上，最近开发了依赖于微生物中特定酶的重组表达的代谢工程方法：wo2012/056318、wo2013/160762、wo2014/009435和ep14306564.7特别描述了通过经由不同代谢途径在经遗传修饰的微生物中发酵葡萄糖来生产2,4-dhb。通过基于作用于空间上相似的同族底物的候选酶的结构和机械知识的合理工程化，或通过筛选天然酶并通过合理设计进一步改善来获得这些专利申请中鉴定的大多数酶。更具体地，wo2012/056318已鉴定了三种非天然存在的酶(苹果酸激酶、苹果酸半醛脱氢酶和dhb脱氢酶，均被突变)，它们可在微生物中过表达以将代谢中间体(l)-苹果酸转化成2,4-dhb；wo2013/160762要求多种酶的异源表达，其中一些经突变而改善酶活性和/或底物亲和性(苹果酰coa合成酶或琥珀酰coa:(l)-苹果酸-coa转移酶和/或苹果酰-coa裂合酶；苹果酰coa还原酶；和dhb脱氢酶)以将代谢中间体苹果酸或琥珀酰-coa或乙醇酰-coa转化成2,4-dhb；而wo2014/009435的方法依赖于将代谢中间体(l)-高丝氨酸转化成2-氧代-4-羟基丁酸(ohb)以及通过重组表达两种突变酶(高丝氨酸转氨酶和dhb还原酶)而在2,4-dhb中还原ohb。本发明提出了用于在两个单一步骤中从1,2,4-丁三醇代谢中间体以微生物生产2,4-二羟基丁酸的备选的合成途径，所述步骤需要连续地将1,2,4-丁三醇氧化成2,4-二羟基丁醛，然后将2,4-二羟基丁醛氧化成2,4-dhb。本发明人确实令人惊讶地发现，可通过在能够将碳源(更特别是木糖)转化成1,2,4-丁三醇的微生物中对上述两个反应进行遗传工程改造而大大改善2,4-dhb的总产量。因此，本发明在本文中提供了备选的微生物，其经遗传修饰用于从木糖优化生产2,4-二羟基丁酸。经遗传工程改造而将木糖转化成1,2,4-丁三醇的此微生物包含进一步的遗传修饰以：-将1,2,4-丁三醇氧化成2,4-二羟基丁醛，并且-将2,4-二羟基丁醛氧化成2,4-dhb。本发明还涉及通过发酵生产2,4-二羟基丁酸的方法，包括在含有木糖的培养基中培养本发明的微生物，并从培养基中回收产生的2,4-dhb。发明详述应该理解的是，下面的详细描述不是限制性的，并且可在不背离本发明的范围的情况下进行各种修改、替换、省略和改变。还应该理解，本文使用的术语是为了描述本发明的具体实施方案的目的，而并不意图为限制性的。本文引用的所有出版物、专利和专利申请(无论是上文还是下文)均通过提述以其整体由此并入。此外，除非另有说明，本文使用的所有技术和科学术语具有与本领域普通技术人员通常理解的相同的含义。常规的微生物学和分子生物学技术也是本领域中公知和常用的那些。此类技术对于本领域技术人员来说是公知的，并且在文献中充分地解释(参见例如sambrook等，2012)。尽管如此，就贯穿本说明书的不同术语的使用而言，下述定义更特别适用。除非上下文另有明确说明，单数形式“一种”、“一个”和“该/所述”在本文中包括复数指称。因此，例如，提及“微生物”包括多种此类微生物，并且提及“内源基因”是提及一个或多个内源基因，等等。术语“包含”、“含有”、“涉及”或“包括”或者变型在本文中以包含的意义使用，即以详明所述特征的存在，但是不排除本发明的各个实施方案中其它特征的存在或添加。如本文所用，术语“微生物”指活的微观生物体，其可为单细胞或多细胞生物，并通常可在自然界中找到。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，本发明的微生物选自肠杆菌科(enterobacteriaceae)、芽孢杆菌科(bacillaceae)、梭菌科(clostridiaceae)、链霉菌科(streptomycetaceae)、棒杆菌科(corynebacteriaceae)和酵母。甚至更优选地，本发明的微生物是埃希氏菌属(escherichia)、克雷伯氏菌属(klebsiella)、热厌氧杆菌属(thermoanaerobacterium)、梭菌属(clostridium)、棒杆菌属(corynebacterium)或酵母属的菌种。还甚至更优选地，本发明的微生物选自大肠杆菌(escherichiacoli)、肺炎克雷伯氏菌(klebsiellapneumoniae)、热解糖热厌氧杆菌(thermoanaerobacteriumthermosaccharolyticum)、丙酮丁醇梭菌(clostridiumacetobutylicum)、谷氨酸棒杆菌(corynebacteriumglutamicum)和酿酒酵母(saccharomycescerevisiae)。最优选地，本发明的微生物是大肠杆菌。如本文所用，术语“重组微生物”、“经遗传修饰的微生物”或“遗传工程化微生物”是指如上文定义的微生物，所述微生物在自然界中找不到，因此在遗传上与其天然对应物不同。换言之，它是指通过导入和/或通过缺失和/或通过修饰其遗传元件而经修饰的微生物。此类修饰可通过基因工程、通过在特定选择压力下培养微生物而迫使新代谢途径的形成和进化、或通过组合这两种方法进行(参见例如wo2005/073364或wo2008/116852)。因此，根据本发明的经遗传修饰用于产生2,4-dhb的微生物意指所述微生物是能够产生2,4-dhb的如上文定义的重组微生物。换言之，所述微生物已经经遗传修饰而允许产生2,4-dhb。在本发明的上下文中，可通过调节一个或多个内源基因的表达水平来遗传修饰微生物。“调节”在本文中指所述基因的表达水平与其天然表达水平相比被上调、下调或甚至完全消除。因此，此类调节可导致基因产物活性的增强，或者可替换地导致内源基因产物的较低活性或无活性。术语“内源基因”在本文中是指天然存在于微生物中的基因。可通过引入除内源调控元件外有利于上调的异源序列或通过用此类异源序列替换那些内源调控元件，或者通过将内源基因的一个或多个补充拷贝引入微生物内的染色体或质粒来过表达内源基因。也可通过将突变引入其编码序列中以修饰基因产物来修饰内源基因活性和/或表达水平。还可进行内源基因的缺失以完全抑制其在微生物内的表达。调节内源基因表达的另一种方式是用更强或更弱的启动子交换其启动子(即野生型启动子)以上调或下调此基因的表达水平。适于此类目的的启动子可为同源的或异源的并且是本领域中公知的。选择适当的启动子来调节内源基因的表达在本领域技术人员的能力范围内。另外/或者，微生物可经遗传修饰而表达一个或多个外源基因，条件是将所述基因与对于其在宿主微生物中表达必需的所有调节元件一起导入微生物中。用外源dna修饰或“转化”微生物是本领域技术人员的常规任务。在本发明的上下文中，术语“过表达”在本文中也就外源基因在微生物中的表达而言使用。“外源基因”或“异源基因”在本文中指所述基因在微生物中不是天然存在的。为了在微生物中表达外源基因，可将此类基因直接整合入微生物染色体中，或者通过微生物内的质粒或载体在染色体外表达。多种质粒(在其复制起点和其在细胞中的拷贝数方面不同)在本领域中是公知的，并可由熟练技术人员容易地选择用于此目的。根据本发明的外源基因有利地为同源基因。在本发明的上下文中，术语“同源基因”或“同源物”不仅指通过理论上共同的遗传祖先由两个物种(即微生物物种)遗传的基因，而且包括可为遗传上无关但已进化以编码进行相似功能和/或具有相似结构的蛋白质的基因(即功能同系物)。因此，术语“功能同源物”在本文中指编码功能同源蛋白质的基因。使用数据库，如uniprot(用于蛋白质)、genbank(用于基因)或ncbi(用于蛋白质或基因)中可用的信息，本领域技术人员可容易地确定微生物的特定蛋白质和/或基因的序列，并基于此序列鉴定另一种微生物中的等同基因或同源物之一。此常规工作可通过一种微生物的特定基因序列与其它微生物的基因序列或基因组(其可在上文提及的数据库中找到)的序列比对来进行。有利地，可使用altschul等(1990)开发的blast算法进行此类序列比对。一旦在那些序列之间建立了序列同源性，可得出共有序列并用于设计简并探针以克隆相关微生物的相应同源物基因。分子生物学的这些常规方法是本领域技术人员公知的。应当进一步理解的是，在本发明的上下文中，如果在特定的微生物中表达编码感兴趣的蛋白质的外源基因，则优选通过将非优选的密码子或不太优选的密码子替换为编码相同氨基酸的所述微生物的优选密码子来构建此基因的合成形式。本领域确实公知密码子选择在微生物物种之间变化，这可影响感兴趣的蛋白质的重组表达水平。为了克服此问题，已开发了密码子优化方法，并由graf等(2000),deml等(2001)和davis&olsen(2011)广泛描述。值得注意地，已为了密码子优化测定而开发出几个软件，如软件(lifetechnologies)或(genscript)的optimumgenetm软件。换言之，编码感兴趣蛋白质的外源基因优选进行密码子优化用于在特定微生物中表达。根据本发明的微生物也可经遗传修饰而增加或降低一个或多个蛋白质的活性。可通过改善蛋白质催化效率、通过降低蛋白质周转率、通过降低信使rna(mrna)周转率、通过增加基因的转录、或通过增加mrna的翻译获得增加此类活性。改善蛋白质催化效率意指对于给定底物和/或给定辅因子，增加kcat和/或降低km，和/或对于给定抑制剂，增加ki。kcat、km和ki是本领域技术人员能够确定的michaelis-menten常数(segel，1993)。减少蛋白质周转率意指稳定蛋白质。改善蛋白质催化效率和/或降低蛋白质周转率的方法是本领域技术人员公知的。那些包括合理的工程化改造及序列和/或结构分析和定向诱变，以及随机诱变和筛选。可通过常规方法如聚合酶链式反应(pcr)进行定点诱变，通过随机诱变技术例如经由诱变剂(紫外线或化学试剂如亚硝基胍(ntg)或乙基甲磺酸(ems))或dna改组或易错pcr引入突变。稳定蛋白质也可通过在蛋白质的n-末端或c-末端添加“标签”肽序列来实现。此类标签在本领域中是公知的，并且包括谷胱甘肽-s-转移酶(gst)等等。可通过修饰5’-非翻译区(5’-utr)和/或编码区和/或3’-utr的基因序列减少mrna周转率(carrier和keasling，1999)。增加基因的转录(无论是内源的还是外源的)可通过增加微生物内其拷贝的数目和/或通过使用与野生型启动子相比导致更高的基因表达水平的启动子来实现。在本发明的上下文中，术语“过表达”也用于指微生物中基因转录的增加。如上文指示，为了增加微生物中基因的拷贝数，可在染色体上或在染色体外编码所述基因。当要在染色体上编码感兴趣的基因时，可通过本领域公知的遗传重组方法(例如基因置换)在染色体上引入基因的几个拷贝。当要在微生物中在染色体外编码基因时，它可由不同类型的质粒携带，所述质粒就其复制起点而言取决于它们可在其中复制的微生物以及它们在细胞中的拷贝数而不同。根据质粒的性质，由所述质粒转化的微生物可含有质粒的1至5个拷贝，或其约20个拷贝，或甚至其多达500个拷贝。可在大肠杆菌中复制的低拷贝数质粒的实例包括但不限于psc101质粒(紧密复制)、rk2质粒(紧密复制)以及pacyc和prsf1010质粒，而可在大肠杆菌中复制的高拷贝数质粒的例子是pskbluescriptii。可增加基因表达水平的启动子也是本领域技术人员公知的，并且可为同源的(起源于相同的物种)或异源的(起源于不同的物种)。此类启动子的实例包括但不限于启动子ptrc、ptac、plac和λ启动子ci。这些启动子也可由特定的化合物或由特定的外部条件如温度或光诱导(“诱导型启动子”)。可通过修饰核糖体结合位点(rbs)增加mrna的翻译。rbs是启动蛋白质翻译时与核糖体结合的mrna上的序列。它可为真核生物中mrna的5’帽、原核生物中起始密码子aug上游6-7个核苷酸区(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，可改变蛋白质翻译起始速率，按比例改变其生产速率，和控制其在细胞内的活性。也可通过使用软件rbscalculator(salis，2011)来优化rbs序列的强度以达到靶向的翻译起始速率。基于mrna的性质选择rbs序列在本领域技术人员的能力范围内。降低蛋白质的活性可意指通过突变编码所述蛋白质的基因来降低其特异的催化活性，从而通过突变核苷酸序列或通过缺失所述基因的编码区来改变相应的氨基酸序列和/或降低细胞中蛋白质的浓度。如本文所用，术语“氧化还原酶”指能够根据操作条件可逆地或不可逆地还原或氧化其底物的酶。更具体地说，此类酶通过催化电子从其底物(电子供体)转移到称为氧化剂(电子受体)的另一种分子而起作用。在本发明的上下文中，选择的氧化还原酶氧化它们各自的底物(1,2,4-丁三醇氧化成2,4-二羟基丁醛，2,4-二羟基丁醛氧化成2,4-dhb)，更优选以不可逆的方式进行。适应操作条件以使本发明的氧化还原酶氧化它们的底物在本领域技术人员的能力范围内。通常将氧化还原酶分类为酶的酶委员会(ec)编号分类中的ec1，其分组超过20种酶的亚类(如ec1.1，包括作用于供体的ch-oh基团的氧化还原酶，也称为醇氧化还原酶；ec1.2，包括作用于供体的醛基或氧代基团的氧化还原酶；ec1.3，包括作用于供体的ch-ch基团的氧化还原酶，也称为ch-ch氧化还原酶；ec1.4，包括作用于供体的ch-nh2基团的氧化还原酶供体，也称为氨基酸氧化还原酶或单胺氧化酶；ec1.5，包括作用于供体的ch-nh基团的氧化还原酶；ec1.6，包括作用于nadh或nadph的氧化还原酶；ec1.7，包括作用于作为供体的其它含氮化合物的氧化还原酶；ec1.8，包括作用于供体的硫基团的氧化还原酶；ec1.9，包括作用于供体的血红素基团的氧化还原酶；ec1.10，包括作用于作为供体的二酚和相关物质的氧化还原酶；ec1.11，包括作用于作为受体的过氧化物的氧化还原酶，也称为过氧化物酶；ec1.12，包括作用于作为供体的氢的氧化还原酶；ec1.13，包括作用于单一供体及并入分子氧的氧化还原酶，也称为加氧酶；ec1.14，包括作用于配对的供体及并入分子氧的氧化还原酶；ec1.15，包括作用于作为受体的超氧化物自由基的氧化还原酶；ec1.16，包括氧化金属离子的氧化还原酶；ec1.17，包括作用于ch或ch2基团的氧化还原酶；ec1.18，包括作用于作为供体的铁硫蛋白的氧化还原酶；ec1.19，包括作用于作为供体的还原性黄素氧还蛋白的氧化还原酶；ec1.20，包括作用于供体中的磷或砷的氧化还原酶；ec1.21，包括作用于x-h和y-h以形成x-y键的氧化还原酶；和包括其它氧化还原酶的ec1.97)。这些氧化还原酶中的一些能够使用nadp或nad+作为辅因子，并且因此称为nad+/nadp+氧化还原酶。根据本发明的术语“碳的来源”、“碳源”或“碳底物”指能够被微生物代谢且含有至少一个碳原子的任何碳源。碳源可源自可再生原料。可再生原料定义为某些工业过程所需要的原料，其可在短暂的延迟内再生并且处于足够的量以允许其转化成期望的产物。植物生物量(经处理或未处理)是一种令人感兴趣的可再生碳源。在本发明的上下文中，木糖是碳源，并且除木糖外的碳源有利地是碳水化合物。术语“碳水化合物”在本文中是指能够被微生物代谢且含有至少一个碳原子、两个氢原子和一个氧原子的任何碳源。本发明的碳水化合物优选选自下组：单糖如葡萄糖、果糖、甘露糖、半乳糖等，二糖如蔗糖、纤维二糖、麦芽糖、乳糖等，寡糖如棉子糖、水苏糖、麦芽糖糊精等，和多糖诸如纤维素、半纤维素、淀粉等、甲醇、甲醛和甘油。根据本发明的优选实施方案，除木糖外的碳源有利地是包含3、6或12个碳原子的碳水化合物，或其任何组合。在本发明的一个更优选的实施方案中，除木糖外的碳源选自甘油、葡萄糖、半乳糖、果糖、乳糖、麦芽糖、蔗糖及其任何组合。更优选地，除木糖外的碳源选自甘油、葡萄糖、蔗糖及其任何组合，并且最优选是葡萄糖。在本发明的第一方面，本发明涉及微生物，所述微生物经遗传修饰而通过将木糖转化成1,2,4-丁三醇来产生2,4-二羟基丁酸，其中所述微生物进一步经遗传修饰用于：i)将1,2,4-丁三醇氧化成2,4-二羟基丁醛；和ii)将2,4-二羟基丁醛氧化成2,4-二羟基丁酸。更优选地，氧化步骤i)和ii)分别涉及排他性地将1,2,4-丁三醇转化成2,4-二羟基丁醛和将2,4-二羟基丁醛转化成2,4-二羟基丁酸。换言之，特别优选的是，在将1,2,4-丁三醇氧化成2,4-二羟基丁醛和将2,4-二羟基丁醛氧化成2,4-dhb的过程中，微生物不产生代谢中间体以优化2,4-dhb产生。此类排他性转化可通过使用下面进一步描述的酶来实现。根据优选的实施方案，遗传修饰i)是编码作用于供体的ch-oh基团的氧化还原酶(ec1.1酶)的至少一个基因的过表达，和/或遗传修饰ii)是编码作用于供体的醛基或氧代基团的氧化还原酶(ec1.2酶)的至少一个基因的过表达。作用于供体的ch-oh基团的氧化还原酶的实例包括但不限于ec1.1.1酶(用nad+或nadp+作为受体的氧化还原酶，也称为nad+/nadp+氧化还原酶)、ec1.1.2酶(用细胞色素作为受体的氧化还原酶)、ec1.1.3酶(用氧作为受体的氧化还原酶)、ec1.1.4酶(用二硫化物作为受体的氧化还原酶)、ec1.1.5酶(用醌或类似化合物作为受体的氧化还原酶)、ec1.1.98酶(用其它已知受体的氧化还原酶)和ec1.1.99酶(用其它受体的氧化还原酶)。作用于供体的醛基或氧代基团的氧化还原酶的实例包括但不限于ec1.2.1酶(用nad+或nadp+作为受体的氧化还原酶，也称为nad+/nadp+氧化还原酶)、ec1.2.2酶(用细胞色素作为受体的氧化还原酶)、ec1.2.3酶(用氧作为受体的氧化还原酶)、ec1.2.4酶(用二硫化物作为受体的氧化还原酶)、ec1.2.5酶(用醌或类似化合物作为受体的氧化还原酶)、ec1.2.7(用铁硫蛋白作为受体的氧化还原酶)和ec1.2.99酶(用其它受体的氧化还原酶)。显示上述活性的酶在本领域中是公知的，并且可由本领域技术人员容易地鉴定，例如从公众可得的数据库如brenda鉴定。为了将1,2,4-丁三醇氧化成2,4-二羟基丁醛，作用于供体的ch-oh基团的氧化还原酶(ec1.1酶)优选选自下组：醇脱氢酶(即醛还原酶)、醛脱氢酶、乳醛还原酶、乙醛酸还原酶、二脱氢葡糖酸还原酶及其任何组合。更优选地，所述ec1.1氧化还原酶是醇脱氢酶(即醛还原酶)或乳醛还原酶。还更优选地，所述ec1.1氧化还原酶是作用于供体的ch-oh基团的nad+/nadp+氧化还原酶(即ec1.1.1酶)，或以其它受体作用于供体的ch-oh基团的氧化还原酶(即ec1.1.99酶)。最优选地，所述ec1.1酶是ec1.1.1酶。选择适于本发明目的的ec1.1酶并且鉴定其相应的基因序列在本领域技术人员的能力范围内。作用于供体的ch-oh基团的特别优选的氧化还原酶(ec1.1酶)根据其在数据库中的序列标识和登录号在下面的表1中描述：醇脱氢酶包括但不限于序列seqidno:1至seqidno:40；乳醛还原酶包括但不限于序列seqidno:41的酶；乙醛酸还原酶包括但不限于序列seqidno:42和seqidno:43的酶，并且二脱氢葡糖酸还原酶包括但不限于序列seqidno:44和seqidno:45的酶。在本发明的特别优选的实施方案中，根据如图1中所述的1,2,4-丁三醇转化成2,4-二羟基丁醛的转化代谢途径，作用于供体的ch-oh基团的氧化还原酶(ec1.1酶)是序列seqidno:36或seqidno:41的酶。编码上文提及的氧化还原酶的基因在本领域中也是公知的，并在下面的表1中描述：-编码醇脱氢酶的基因包括但不限于大肠杆菌基因frma(也称为adhc)、adhp(也称为yddn)、yiay、eutg(也称为yffv)、yqhd、yeae、ydhf、yhdn、ybdr、ybdh、ybjj、ydjl、ahr、yahk、tas(也称为ygds)和glda；丙酮丁醇梭菌基因bdhb、bdha和ca_c3392，酿酒酵母基因adh1、adh2、adh3、adh4和bdh1；枯草芽孢杆菌基因yhdn和bdha；氧化葡糖杆菌(gluconobacteroxydans)基因gox1615；拟南芥基因akr4c9；杜氏利什曼原虫(leishmaniadonovani)基因a4utp6；产乙醇热厌氧杆菌(thermoanaerobacterethanolicus)基因adha；谷氨酸棒杆菌(corynebacteriumglutamicum)基因buta和budc；肺炎克雷伯氏菌(klebsiellapneumoniae)基因budc；粘质沙雷氏菌(serratiamarcescens)基因slac；红褐肉座菌(hypocreajecorina)基因gld2；丁酸梭菌(clostridiumbutyricum)基因dhat；弗氏柠檬酸杆菌(citrobacterfreundii)基因dhat；肺炎克雷伯氏菌(klebsiellapneumoniae)基因dhat；和编码nmda依赖性醇脱氢酶的红串红球菌(rhodoccuserythropolis)基因；和编码nmda依赖性醇脱氢酶的甲醇拟无枝酸菌(amycolatopsismethanolica)基因；-编码乳醛还原酶的基因包括但不限于大肠杆菌基因fuco；-编码乙醛酸还原酶的基因包括但不限于大肠杆菌基因ghra(也称为ycdw)和ghrb(也称为yiae)；和-编码二脱氢葡糖酸还原酶的基因包括但不限于大肠杆菌基因dkga(也称为yqhe)和dkgb(也称为yafb)。在本发明的一个特别优选的实施方案中，作用于供体的ch-oh基团的氧化还原酶(ec1.1酶)由来自大肠杆菌的fuco基因和/或来自丁酸梭菌、弗氏柠檬酸杆菌或肺炎克雷伯氏菌的dhat基因编码，即如图1所示根据1,2,4-丁三醇变为2,4-二羟基丁醛的转化代谢途径。如上文指示，本发明的微生物也经遗传修饰用于ii)将2,4-二羟基丁醛氧化成2,4-二羟基丁酸。为此，可在微生物中过表达编码作用于供体的醛基或氧代基团的氧化还原酶(ec1.2酶)的至少一个基因。因此，作用于供体的醛或氧代基团的氧化还原酶(ec1.2酶)优选选自下组：醛脱氢酶、醛氧化酶及其任何组合。更优选地，所述ec1.2氧化还原酶是醛脱氢酶。还更优选地，所述ec1.2氧化还原酶是作用于供体的醛基或氧代基团的nad+/nadp+氧化还原酶(即ec1.2.1酶)，或用氧作为受体作用于供体的醛基或氧代基团的氧化还原酶(即ec1.2.3酶)。最优选地，所述ec1.2酶是ec1.2.1酶。选择适于本发明目的的ec1.2酶并鉴定其相应的基因序列在本领域技术人员的能力范围内。作用于供体的醛基或氧代基团的特别优选的氧化还原酶(ec1.2酶)根据其在数据库中的序列标识和登录号在下面的表1中描述：醛脱氢酶包括但不限于序列seqidno:46至seqidno:60的酶；醛氧化酶包括但不限于序列seqidno:61的酶。在本发明的特别优选的实施方案中，根据如图1中所述将2,4-二羟基丁醛变为2,4-二羟基丁酸的转化代谢途径，作用于供体醛基或氧代基团的氧化还原酶(ec1.2酶)是序列seqidno:46、seqidno:54或seqidno:55的酶。编码上文提及的氧化还原酶的基因在本领域中也是公知的，并在下面的表1中描述：-编码醛脱氢酶的基因包括但不限于大肠杆菌基因puuc、sad、gabd、prr(也称为ydcw)、alda、aldb(也称为yiax)、feab和astd；肺炎克雷伯氏菌kpn_01018；酿酒酵母基因ald4和ald5；恶臭假单胞菌(pseudomonasputida)基因davd；假单胞菌属菌种基因amnc；和詹氏甲烷球菌(methanocaldococcusjannaschii)基因mj1411；-编码二氧戊酸脱氢酶的基因包括但不限于地衣芽孢杆菌基因ycbd；和-编码醛氧化酶的基因包括但不限于玉蜀黍基因ao1。在本发明的一个特别优选的实施方案中，作用于供体的醛基或氧代基团的氧化还原酶(ec1.2酶)由来自大肠杆菌的alda、aldb或puuc基因编码，即根据如图1中所述将2,4-二羟基丁醛变为2,4-二羟基丁酸的转化代谢途径。必须进一步注意到，当编码上述活性的内源基因已经存在于感兴趣的微生物中时，它们有利地在所述微生物中过表达。相反，当微生物不天然地包含编码此类活性的基因时，有利地用一种或多种编码所述酶的外源基因转化微生物：认为也过表达所述外源基因。如上文解释，外源基因优选为进行了密码子优化用于其在感兴趣的微生物中表达的合成基因。在本发明的上下文中，微生物能够将木糖转化成1,2,4-丁三醇。经遗传修饰用于将木糖转化成1,2,4-丁三醇的微生物，特别是用于排他性的将木糖转化成1,2,4-丁三醇的微生物是本领域公知的，并且已经特别通过专利申请wo2008/091288和us2013/0203141描述，其通过提述并入本文。因此，在一个优选的实施方案中，用于将木糖转化成1,2,4-丁三醇的遗传修饰是至少一个以下基因的过表达：-编码木糖脱氢酶的基因，-编码木糖酸内酯酶的基因，-编码木糖酸脱水酶的基因，-编码3-脱氧-d-甘油-戊酮糖酸(dgp)脱羧酶的基因，-编码1,2,4-丁三醇脱氢酶的基因，和-其任何组合。编码上文所述酶的基因在本领域中是公知的：-编码木糖脱氢酶的基因公开于us2013/0203141和wo2008/091288中，通过提述并入本文，并且包括但不限于来自新月柄杆菌(caulobactercrescentus)、死海盐盒菌(haloarculamarismortui)、真菌伯克霍尔德氏菌(burkholderiafugorum)lb400和沃氏富盐菌(haloferaxvolcanii)ds2的xdh编码基因，-编码木糖酸内酯酶的基因包括但不限于来自新月柄杆菌的xylc基因，-编码d-木糖酸脱水酶的基因在us2013/0203141和wo2008/091288中公开，其通过提述并入本文，并包括但不限于来自大肠杆菌的yjhg和yagf基因，-编码3-脱氧-d-甘油-戊酮糖酸脱羧酶的基因公开于us2013/0203141和wo2008/091288中，其通过提述并入本文，并且包括但不限于来自恶臭假单胞菌的mdlc基因，和-编码nadph依赖性1,2,4-丁三醇脱氢酶的基因公开于us2013/0203141和wo2008/091288中，通过提述并入本文，并且包括但不限于来自大肠杆菌的adhp和yqhd基因。在一个优选的实施方案中，特别是当经遗传修饰的微生物是大肠杆菌时：α)编码木糖脱氢酶、木糖酸内酯酶和3-脱氧-d-甘油-戊酮糖酸脱羧酶的基因是导入微生物中的异源基因，和β)编码木糖酸脱水酶和1,2,4-丁三醇脱氢酶的基因是内源性基因，因此有利地是过表达的。在本发明的一个特别优选的实施方案中，木糖脱氢酶由新月柄杆菌的xdh基因编码，木糖酸内酯酶由新月柄杆菌的xylc基因编码，木糖酸脱水酶由来自大肠杆菌的yjhg和/或yagf基因编码，3-脱氧-d-甘油-戊酮糖酸脱羧酶由来自恶臭假单胞菌的mdlc基因编码，并且1,2,4-丁三醇脱氢酶由来自大肠杆菌的adhp和/或yqhd基因编码，即根据如图1中所述将木糖变为1,2,4-丁三醇的转化代谢途径。上文提及基因的核苷酸或由所述基因编码的氨基酸序列根据它们在数据库中的登录号和版本和/或根据它们的序列标识在下面的表1中描述。当编码上述活性的内源基因已经存在于感兴趣的微生物中时，它们有利地在所述微生物中过表达。相反，当微生物不天然地包含编码此类活性的基因时，有利地用编码所述酶的一个或多个外源基因转化微生物：认为也过表达所述外源基因。如上文解释，外源基因优选为进行了密码子优化用于在感兴趣的微生物中表达的合成基因。根据一个优选的实施方案，本发明的微生物进一步经遗传修饰用于：iii)从除木糖外的碳源提供用于2,4-二羟基丁酸生产和微生物生长的还原能力和/或能量，和/或iv)至少部分地，优选完全地抑制碳分解代谢物阻抑。如上文所述的遗传修饰iii)是特别有利的，因为其优化了2,4-dhb生产，通过使用备选碳源而非木糖来提供还原能力和/或能量，从而可将木糖排他性地转化成2,4-二羟基丁酸。为了从除木糖外的碳源提供1,2,4-丁三醇生产和微生物生长的还原能力和/或能量，优选通过缺失和/或弱化编码使用木糖或其它代谢物的酶的基因来遗传修饰根据本发明的微生物，所述代谢物产生与木糖变为2,4-二羟基丁酸的转化相竞争的流量(flux)。因此，根据本发明的一个优选实施方案，遗传修饰iii)是以下基因中的至少一个的弱化和/或缺失：-编码木糖异构酶的基因(例如来自大肠杆菌的xyla基因)，-编码木酮糖激酶的基因(例如来自大肠杆菌的xylb基因)，-编码3-脱氧-d-甘油-戊酮糖酸醛缩酶的基因(例如来自大肠杆菌的yjhh和/或yage基因)，-编码酮酸脱氢酶的基因(例如来自大肠杆菌的yiae和/或ycdw基因)，和-其任何组合。这些基因以及用于缺失或弱化它们的方法在us2013/0203141中充分公开，通过提述并入本文。上文提及的基因的核苷酸或由所述基因编码的氨基酸序列也根据它们在数据库中的登录号和版本和/或根据它们的序列标识在下面的表1中描述。如果本发明的微生物如上文所述进行遗传修饰，则相应优选的是微生物使用除木糖外的碳源来提供2,4-dhb生产和微生物生长必需的还原能力和/或能量。在这方面，在本领域中公知的是，一些微生物相对于其它碳源优选某些特定的碳源。值得注意的是，大多数天然存在的微生物，特别是大肠杆菌相对于其它糖优选使用葡萄糖，即使它们能够代谢一系列单糖(kim等，2010)。然而，一些微生物不能以有效的方式共利用葡萄糖和木糖。实际上，当两种糖存在于培养基中时，微生物中可发生二期(dizuxic)发酵模式：此调节机制在本领域中公知为分解代谢物阻抑，并且在20世纪40年代已由monod对葡萄糖和乳糖报道。已经在肠杆菌科中广泛研究此类机制。在大肠杆菌中，已经明显认识到分解代谢物阻抑如下发生：葡萄糖借助葡萄糖特异性通透酶eiicbglc(由ptsg编码)进入磷酸烯醇丙酮酸(pep):碳水化合物磷酸转移酶系统(pts)；在葡萄糖转运期间，通过去磷酸化的eiiaglc(由crr编码)降低了环amp(camp)的水平，这又限制了分解代谢物阻遏蛋白(crp)和camp复合物(camp-crp)的可用性；随后，涉及除葡萄糖外的糖的分解代谢并通常由camp-crp调节的所有基因都受到阻抑。不被pts系统转运的碳水化合物的分解代谢物阻抑也在文献中报道，但尚未得到充分理解(hogema等，1997)。因此，在一个优选的实施方案中，允许抑制碳分解代谢物阻抑的遗传修饰iv)选自以下的至少一种：-缺失编码磷酸转移酶系统的葡萄糖通透酶的基因，-缺失编码磷酸载体hpr蛋白的基因，-优选从不受camp-crp调节的组成性或诱导型启动子表达糖输入蛋白系统中所涉及的基因和/或操纵子，其中所述糖是除木糖外的碳源，-表达编码木糖转运蛋白，如同向转运蛋白或abc转运蛋白的基因，优选从不受camp-crp调节的组成性或诱导型启动子表达，-过表达编码葡萄糖同向转运蛋白的基因，-过表达编码葡萄糖易化蛋白的基因，-过表达编码葡糖激酶的基因，-调节牵涉camp水平的基因，优选编码腺苷酸环化酶的基因的表达，-调节编码crp和/或crp样蛋白质的基因的表达，-表达编码不依赖camp的crp蛋白质的基因，优选从不受camp-crp调节的组成性或诱导型启动子表达，以及-其任何组合。更优选地，为了共利用木糖和葡萄糖，缺失编码磷酸转移酶和/或磷酸载体hpr蛋白的基因有利地与过表达编码葡萄糖通透酶或葡萄糖易化蛋白的基因连同过表达编码葡糖激酶的基因组合。编码上文描述的蛋白质的基因在本领域是公知的：-编码磷酸转移酶系统的葡萄糖通透酶的基因包括但不限于来自大肠杆菌的pstg基因，-编码磷酸载体hpr蛋白质(其为磷酸烯醇丙酮酸：糖磷酸转移酶系统(pts糖)的两种糖非特异性蛋白质组分之一)的基因包括但不限于来自大肠杆菌的ptsh基因，-涉及糖输入蛋白系统(pts或其它系统)的基因/操纵子包括但不限于来自大肠杆菌的用于同化乳糖的lacy基因、来自大肠杆菌的用于同化麦芽糖的malfg基因、以及分别来自鼠伤寒沙门氏菌和大肠杆菌的用于同化蔗糖的scrkyabr或cscbkar操纵子，-编码木糖转运蛋白的基因包括但不限于来自大肠杆菌的xylfgh操纵子，来自大肠杆菌的xyle和arae基因，-编码葡萄糖同向转运蛋白的基因包括但不限于来自大肠杆菌的galp基因，-编码葡萄糖易化蛋白的基因包括但不限于运动发酵单胞菌(zymomonasmobilis)的glf基因，-编码葡糖激酶的基因包括但不限于来自大肠杆菌的glk基因，-编码腺苷酸环化酶的基因包括但不限于来自大肠杆菌的cyaa基因，-编码crp和/或crp样蛋白质的基因包括但不限于来自大肠杆菌的crp基因和来自枯草芽孢杆菌和其它硬壁菌门(firmicutes)的ccpa基因，-hogema等(1997)已经描述了编码不依赖camp的crp蛋白质的基因，包括但不限于编码在两个区中突变的crp蛋白的基因：对应于crp蛋白的camp结合域的残基53至62和对应于dna结合域(螺旋-转角-螺旋)的近端边界的残基141至148。此类蛋白质突变体的实例包括crp突变体asp至his残基53、ser至phe残基62、gly至asp残基141、arg至asp残基142、leu至arg残基148及其任何组合。更优选地，为了在大肠杆菌中共利用木糖和葡萄糖，ptsg或ptsh基因缺失可与galp或glf基因的过表达连同glk基因的过表达组合。上文提及基因的核苷酸序列或由所述基因编码的氨基酸序列根据它们在数据库中的登录号和版本和/或根据它们的序列标识在下面的表1中描述。如上文解释，当编码上述活性的内源基因已经存在于感兴趣的微生物中时，它们有利地在所述微生物中过表达。然而，如果微生物不天然地包含编码此类活性的基因，则有利地用一个或多个编码所述酶的外源基因转化微生物：认为也过表达所述外源基因。如上文解释，外源基因优选为进行了密码子优化用于在感兴趣的微生物中表达的合成基因。相反，可根据上文描述的任一种方法缺失内源基因或弱化其表达。根据本发明的除木糖外的碳源如上文定义，并且包括但不限于包含3、6或12个碳原子的碳水化合物，或其任何组合。优选地，除木糖外的碳源选自下组：甘油、葡萄糖、半乳糖、果糖、乳糖、麦芽糖、蔗糖及其任何组合。最优选地，本发明中使用的除木糖外的碳源是葡萄糖。或者，除木糖外的碳源可为蔗糖。因此，在本发明的优选实施方案中，微生物包含进一步的遗传修饰以使用蔗糖作为除木糖外的碳源，如wo2012/004247中所述，其通过提述并入本文。为此，微生物优选包含编码pts蔗糖利用系统和/或非pts蔗糖利用系统的功能基因。pts蔗糖利用系统是基于通过磷酸烯醇丙酮酸(pep)-依赖性蔗糖磷酸转移酶系统(蔗糖-pts)转运蔗糖的蔗糖利用系统。磷酸转移酶系统将糖(例如蔗糖或葡萄糖)的转运与使用pep作为磷酸供体的糖的磷酸化偶联。在转运入细胞后，蔗糖磷酸被转化酶切割成葡萄糖-6-磷酸和果糖。果糖然后被果糖激酶磷酸化成果糖-6-磷酸。编码此pts蔗糖利用系统的基因可由调节蛋白控制。非pts蔗糖利用系统是基于通过独立于磷酸烯醇丙酮酸的系统转运蔗糖的蔗糖利用系统。在转运入细胞后，蔗糖被转化酶切割成葡萄糖和果糖。果糖然后被果糖激酶磷酸化成果糖-6-磷酸，且葡萄糖被葡糖激酶磷酸化成葡萄糖-6-磷酸。编码此非pts蔗糖利用系统的基因可由调节蛋白控制。在本发明的一个特别优选的实施方案中，微生物天然表达或已经经遗传修饰以表达来自沙门氏菌属的操纵子scrkyabr的基因，即编码果糖激酶的scrk基因、编码孔蛋白的scry基因、编码iibc蛋白的scra基因、编码蔗糖-6-p转化酶的scrb基因和编码阻遏物的scrr基因，以及它们的任何组合。可使用携带scrkyabr的接合质粒pur400转化微生物。这些基因可单独、以包含这些基因的至少两个的任何组合、或全部组合在一起在微生物中表达。可优选省略基因scrr。在本发明的特别优选的实施方案中，微生物天然表达或已经经遗传修饰以表达来自大肠杆菌菌株ec3132的基因，即编码蔗糖:质子同向运输转运系统(cscb基因)、果糖激酶(csck)、转化酶(csca基因)和蔗糖特异性阻遏物(cscr基因)的操纵子cscbkar基因。这些基因可单独、以包含这些基因的至少两个的任何组合、或全部组合在一起在微生物中表达。可优选省略基因cscr。也可使用来自其它生物的同源基因。然而，在一个优选的实施方案中，本发明的微生物包含牵涉产生nadph作为还原能力来源的至少一个基因的进一步遗传修饰。事实上，还原酶如脱氢酶需要微生物中可用的还原能力，特别是以nadph的形式。增加细胞中nadph可用性的策略在本领域中是公知的，并且已经特别由lee等(2013)综述，并且还由us8088620、wo2012/055798和ep14305691.9描述，上述文件通过提述并入本文。根据本发明，用于改善nadph产生并因此改善其在微生物中的可用性的遗传修饰优选选自：-过表达编码膜结合转氢酶的基因或操纵子，-缺失或弱化编码可溶性转氢酶的基因，-过表达编码产生nadph的甘油醛-3-磷酸脱氢酶的基因，-缺失或弱化编码磷酸葡糖异构酶的基因，-缺失或弱化编码磷酸果糖激酶的基因，-过表达编码葡萄糖-6-磷酸脱氢酶的基因，-过表达编码能够产生nadph的硫辛酰胺脱氢酶的突变体基因，-过表达编码双功能nad(p)h水合物修复酶的基因，和-其任何组合。缺失或弱化编码磷酸果糖激酶的基因更优选与过表达编码葡萄糖-6-磷酸脱氢酶的基因组合，以增加通过戊糖磷酸途径的nadph流量。更优选地，用于改善nadph产生的遗传修饰选自：-过表达编码膜结合转氢酶的基因，-缺失或弱化编码磷酸葡糖异构酶和/或可溶性转氢酶的基因，和-过表达编码产生nadph的甘油醛-3-磷酸脱氢酶的基因。编码上文描述的蛋白质的基因在本领域中是公知的：-编码膜结合转氢酶的基因或操纵子包括但不限于来自大肠杆菌的pntab操纵子，特别由wo2012/055798a1所述，-编码可溶性转氢酶的基因包括但不限于来自大肠杆菌的udha基因，-编码产生nadph的甘油醛-3-磷酸脱氢酶的基因包括但不限于来自变异链球菌的gapn(如centeno-leija等，2013所述)，其可用于例如取代来自大肠杆菌的内源gapa基因，-编码磷酸葡糖异构酶的基因包括但不限于来自大肠杆菌的pgi基因，-编码磷酸果糖激酶的基因包括但不限于来自大肠杆菌的pfka基因，如特别由wo2005/047498描述，-编码葡萄糖-6-磷酸脱氢酶的基因包括但不限于来自大肠杆菌的zwf基因，如特别由lim等(2002)描述，-编码能够产生nadph的硫辛酰胺脱氢酶的突变体基因包括但不限于来自大肠杆菌的突变lpd基因(lpd*)，如特别由bocanegra等(1993)描述，和-编码双功能nad(p)h水合物修复酶的基因包括但不限于来自大肠杆菌的yjef基因，如特别由marbaix等(2011)描述。在大肠杆菌中，如果缺失或弱化pfka基因，则所述遗传修饰优选与过表达zwf基因组合。更优选地，用于改善nadph产生的遗传修饰选自：-过表达来自大肠杆菌的pntab操纵子，-缺失来自大肠杆菌的pfka基因和/或udha基因，-用来自变异链球菌的gapn基因取代来自大肠杆菌的gapa。上文提及的基因的核苷酸序列或由所述基因编码的氨基酸序列根据它们在数据库中的登录号和版本和/或根据它们的序列标识在下面的表1中描述。如上文解释，当编码上述活性的内源基因已经存在于感兴趣的微生物中时，它们有利地在所述微生物中过表达。然而，如果微生物不天然地包含编码此类活性的基因，则有利地用一个或多个编码所述酶的外源基因转化微生物：认为也过表达所述外源基因。如上文解释，外源基因优选为进行了密码子优化用于在感兴趣的微生物中表达的合成基因。相反，可根据上文描述的任一种方法缺失内源基因或弱化其表达。表1：本发明的基因和蛋白质(nd＝未公开)另一方面，本发明涉及生产2,4-二羟基丁酸的方法，其包括：a)在包含木糖的培养基中，在允许将木糖转化成2,4-二羟基丁酸的发酵条件下培养如上文所述的本发明的经遗传修饰的微生物，以及b)从所述培养基中回收2,4-二羟基丁酸。发酵培养基和碳源是本领域中公知的。根据本发明，术语“发酵过程”、“发酵”或“培养”可互换使用，指允许给定微生物生长的实验条件。微生物的生长通常在具有适于所用微生物的适当生长培养基的发酵罐中进行。“适当的培养基”在本文中指培养基(例如无菌液体培养基)，其包含对于微生物的维持和/或生长必需的或有益的营养物如碳源或碳底物；氮源，例如蛋白胨、酵母提取物、肉提取物、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；以及生长因子如氨基酸和维生素。根据一个优选的实施方案，步骤a)的培养基进一步包含除木糖外的碳源，其优选为碳水化合物。所述碳水化合物可选自下组：单糖如葡萄糖、果糖、甘露糖、阿拉伯糖、半乳糖等，二糖如蔗糖、纤维二糖、麦芽糖、乳糖等，寡糖如棉子糖、水苏糖(stacchyose)、麦芽糖糊精等，多糖如纤维素、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包含3、6或12个碳原子，更优选甘油、葡萄糖、果糖、半乳糖、乳糖、麦芽糖、蔗糖及其任何组合。最优选地，碳水化合物是葡萄糖。或者，所述碳水化合物可为蔗糖。在本发明的一个优选实施方案中，碳源(优选碳水化合物)源自可再生原料，如植物生物质。本领域技术人员可容易地确定用于培养根据本发明的微生物必需的培养条件。特别地，公知的是可在20℃-55℃，优选在25℃-40℃的温度使细菌发酵。更特别地，可在约30℃至约37℃的温度培养大肠杆菌。本发明的方法可以分批过程、补料分批过程或连续过程并在需氧、微需氧或厌氧条件下进行。“在需氧条件下”的发酵意指通过将气体溶解到培养物的液相中来将氧气提供给培养物。这可通过如下实现：(1)将含氧气体(例如空气)喷入液相中，或者(2)摇动含有培养基的容器以将顶部空间中所含的氧气转移到液相中。在需氧条件下发酵的主要优点是氧作为电子受体的存在改善菌株产生更多atp形式的能量用于细胞过程的能力，由此改善菌株的一般代谢。微需氧条件可在本文中使用并且定义为将低百分比的氧气(例如，使用含有0.1-10％的氧气，用氮气填充至100％的气体混合物)溶解到液相中的培养条件。相反，“厌氧条件”定义为不向培养基提供氧气的培养条件。严格厌氧条件可通过向培养基中喷入惰性气体(如氮气)来除去痕量的其它气体来获得。硝酸盐可用作电子受体以改善菌株的atp产生并改善其代谢。根据优选的实施方案，本发明的方法还包括纯化步骤b)的2,4-dhb的步骤c)。用于纯化羧酸和具体地羟基酸的方法在本领域中是公知的，并且特别在wo2002/090312、wo2002/022544和wo2012/153042中描述，其通过提述并入本文。优选地，通过本领域技术人员公知的手段，且特别是离子交换层析技术如离子交换树脂或固定或模拟移动床离子交换树脂，将2,4-dhb在其于培养基中回收后纯化。附图简述图1表示将d-木糖转化成2,4-dhb的代谢途径(*：任选缺失或弱化的基因)。实施例在以下实施例中进一步限定了本发明。应该理解的是，这些实施例虽然指示了本发明的优选实施方案，但仅以说明的方式给出。从上面的公开和这些实例中，本领域的技术人员可对本发明进行各种变化以使其适于各种用途和条件，而不需要修改本发明的基本手段。描述了构建具有这些能力的微生物所需要的示例性基因和酶以及用于克隆和转化、监测产物形成和使用工程化微生物进行生产的方法。具体地，实施例显示了经修饰的大肠杆菌菌株，但是这些修饰可容易地在相同科的其它微生物中进行。大肠杆菌属于肠杆菌科，其包含革兰氏阴性、杆状、非孢子形成，并且长度通常为1-5μm的成员。大多数成员具有用于活动的鞭毛，但一些属是不能运动的。此科的许多成员是在人类和其它动物的肠中发现的肠道菌群的正常部分，而其它在水中或土壤中发现，或者是多种不同动物和植物上的寄生物。大肠杆菌是最重要的模式生物体之一，但肠杆菌科的其它重要成员包括克雷伯氏菌属(特别是土生克雷伯氏菌、植物克雷伯氏菌或产酸克雷伯氏菌)，泛菌属和沙门氏菌属。方案以下实施例中描述了用于构建2,4-二羟基丁酸生产菌株的几种方案。在本发明中使用的方案1(通过同源重组的染色体修饰、重组体的选择和抗生素盒切除)和方案2(噬菌体p1的转导)已经在专利申请wo2013/001055中充分描述。方案3：构建重组质粒本领域技术人员已知充分描述和知道重组dna技术。简言之，使用寡核苷酸(本领域技术人员能够设计)和mg1655大肠杆菌k-12或其它微生物基因组dna作为基质(根据待扩增的靶向基因)对dna片段进行pcr扩增。用相容的限制酶消化dna片段和选择的质粒，连接，然后转化到感受态细胞中。分析转化体并通过dna测序验证感兴趣的重组质粒。实施例1：通过在mg1655大肠杆菌中过量产生来自新途径的酶从木糖生产2,4-dhb-构建菌株1至11。修饰大肠杆菌菌株mg1655以使用图1所示的途径从d-木糖生产2,4-二羟基丁酸(2,4-dhb)。该工作寻求使朝向2,4-dhb生产的碳流量最大化，从而除去所有涉及其它木糖消耗途径或涉及2,4-dhb-中间体化合物的转化(其代表了产物的损失)的酶。除由大肠杆菌天然表达的基因(序列seqidno:64的yjhg基因和序列seqidno:65的yagf基因，其编码木糖酸脱水酶；以及序列seqidno:66的adhp基因和序列seqidno:67的yqhd基因，其编码醇脱氢酶-nad(p)h依赖性1,2,4-丁三醇脱氢酶)外，使用pcl1920质粒(lerner&inouye,1990)，用ptrc人工启动子(专利wo2007/0770441中给出的序列)及其自身的核糖体结合位点分别表达编码以下酶的基因：新月柄杆菌的木糖脱氢酶和木糖酸内酯酶(分别为xdh[caulocyc上的cc0821，seqidno:62]和xylc[caulocyc上的cc0820，seqidno:63])，恶臭假单胞菌的3-脱氧-d-甘油-戊酮糖酸脱羧酶(序列seqidno:68的mdlc基因)，大肠杆菌或丁酸梭菌的醇脱氢酶-nad(p)+依赖性2,4-二羟基丁醛脱氢酶序列(分别为编码序列seqidno:41的酶的来自大肠杆菌的fuco基因或编码序列seqidno:36的酶的来自丁酸梭菌的dhat基因)和大肠杆菌的醛脱氢酶(编码序列seqidno:46的酶的来自大肠杆菌的puuc基因或编码序列seqidno:55的酶的来自大肠杆菌的aldb基因)。实际上，首先在pcl1920质粒上克隆基因xdh、xylc、mdlc，给出质粒pdhb0001，然后在质粒pdhb0001上序贯克隆fuco或dhat和puuc或aldb，产生4种组合，得到质粒pdhb0002至pdhb0005，如下文表中描述。此外，为了阻断天然木糖分解代谢途径，使用由datsenko&wanner，2000所述的同源重组策略并且根据方案1，从大肠杆菌mg1655染色体中缺失编码d-木糖异构酶(序列seqidno:69的xyla基因)和d-木酮糖激酶(序列seqidno:70的xylb基因)的基因。更准确地，为了缺失xylab操纵子，用seqidno:104和seqidno:105的引物产生携带抗生素抗性基因连同frt位点的被与xylab操纵子的上游和下游区同源的序列围绕的pcr产物，并将其导入先前已经转化有pkd46载体的大肠杆菌mg1655中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株1。为了避免3-脱氧-d-葡糖戊酮糖酸(dgp)的降解，也使用相同的同源重组策略缺失了编码酮酸脱氢酶(序列seqidno:73的yiae基因；和序列seqidno:74的ycdw基因)和dgp醛缩酶(序列seqidno:71的yjhh基因；序列seqidno:72的yage基因)的基因。更准确地，为了缺失yjhh基因(seqidno:71)，用seqidno:106和seqidno:107的引物产生携带抗生素抗性基因连同frt位点的被与yjhh基因的上游和下游区同源的序列围绕的pcr产物，并将其导入先前转化有pkd46载体的菌株1中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株2。然后，为了缺失yage基因(seqidno:72)，产生携带抗生素抗性基因连同frt位点的被与yage基因(seqidno:108和seqidno:109)的上游和下游区同源的序列围绕的pcr产物，并将其导入先前转化有pkd46载体的菌株2中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株3。然后，为了缺失yiae基因(seqidno:73)，用seqidno:110和seqidno:111的引物产生携带抗生素抗性基因连同frt位点的被与yiae基因的上游和下游区同源的序列围绕的pcr产物，并将其导入预先转化有pkd46载体的菌株3中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株4。最后，为了缺失ycdw基因(seqidno:74)，用seqidno:112和seqidno:113的引物产生携带抗生素抗性基因连同frt位点的被与ycdw基因的上游和下游区同源的序列围绕的pcr产物，并将其导入预先转化有pkd46载体的菌株4中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株5。由于yjhh基因与yjhg(seqidno:64)和yjhi一起属于yjhihg操纵子，必须选择与yjhh的上游和下游区同源的序列，从而不改变周围基因的表达。对于属于yagef操纵子的yage基因(sedidno:72)是相同的。最后，为了抑制分解代谢物阻抑，通过使用由datsenko&wanner，2000描述的同源重组策略(根据方案1)，如专利申请ep14305691.9中，特别是在所述文献的实施例2中所述，使用seqidno:114和seqidno:115的引物缺失由ptsg基因(seqidno:75)编码的葡萄糖磷酸转移酶iibc(glc)。将适当的pcr产物导入先前转化有pkd46载体的菌株5中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株6。每次使用卡那霉素、氯霉素、庆大霉素、四环素、杀稻瘟素或壮观霉素中不同的抗生素抗性基因。在使用菌株6之前，如由datsenko&wanner，2000所述(根据方案1)，使用flp重组酶从δxylab,δyjhh,δyage,δyiae,δycdw修饰中除去抗生素盒，产生菌株7。然后，将上文所述的每个质粒pdhb0002、pdhb0003、pdhb0004或pdhb0005导入菌株7中，产生菌株8至11，如下表所述。实施例2：通过增加生产菌株的nadph可用性来改善2,4-二羟基-丁酸产量-构建菌株12至20。由adhp和yqhd编码的1,2,4-丁三醇脱氢酶需要生物体中可用的还原力，特别是nadph形式，因此过表达参与nadph产生的基因。通过将大肠杆菌mg1655的pnta基因的内源启动子和核糖体结合位点替换为诱导型ptrc启动子(来自质粒ptrc99a，amershampharmacia)和确定的核糖体结合位点rbs120(来自rbscalculator软件)，如专利申请ep14305691.9中所述，特别是所述文献的实施例4中所述(本文称为seqidno:116)过量产生由pntab操纵子(seqidno:94)编码的膜结合质子易位吡啶核苷酸转氢酶。将专利申请ep14305691.9中描述的适当的pcr产物导入先前转化有pkd46载体的菌株7中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株12。使用seqidno:117和seqidno:118的引物，通过使用由datsenko&wanner，2000描述的同源重组策略(根据方案1)并如专利申请wo2012/055798中特别是在所述申请的实施例2中所述，缺失由stha基因(先前称为udha，序列seqidno:95)编码的可溶性吡啶核苷酸转氢酶。将适当的pcr产物导入先前转化有pkd46载体的菌株12中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株13。使用seqidno:119和seqidno:120的引物，通过使用由datsenko&wanner，2000描述的同源重组策略(根据方案1)并如专利申请ep14305691.9中特别是在所述申请的实施例5中所述，缺失由pfka基因(seqidno:99)编码的果糖磷酸激酶。将适当的pcr产物导入预先转化了pkd46载体的菌株13中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株14。通过使用datsenko&wanner，2000描述的同源重组策略(根据方案1)将由来自大肠杆菌的gapa基因(seqidno:97)编码的nad依赖性磷酸甘油醛脱氢酶替换为编码nadp依赖性甘油醛-3-磷酸脱氢酶的来自变异链球菌的gapn基因(seqidno:96)(centeno-leija等，2013)。通过如下完成gapn基因对gapa基因的取代：将gapa启动子和核糖体结合位点同时取代为热诱导型pr01启动子(seqidno:121)及其关联的不耐热性阻抑物ci857(seqidno122)(如专利申请ep2532751的实施例1中所述)和确定的核糖体结合位点rbs150(来自rbscalculator软件，seqidno:123)。更准确地，使用seqidno:124和seqidno:125的引物产生携带ci857基因、pr01启动子、rbs150核糖体结合位点、来自变异链球菌的gapn基因和抗生素抗性基因连同frt位点的被与gapa基因的上游和下游区同源的序列围绕的pcr产物，并将其导入先前转化为pkd46载体的菌株14中。用适当的寡核苷酸验证抗生素抗性转化体，并将保留的菌株命名为菌株15。在使用菌株15之前，根据方案1使用flp重组酶从pntab、udha、pfka和gapa基因座除去抗生素盒，产生菌株16。最后，将上文所述的每个质粒pdhb0002、pdhb0003、pdhb0004或pdhb0005导入菌株16中，产生菌株17至20，如下表中所述。实施例3：在产生2,4-二羟基-丁酸的大肠杆菌重组菌株中过量生产涉及蔗糖代谢的酶-构建菌株21至26。为了避免当在蔗糖和木糖的混合物上培养菌株时的分解代谢物阻遏，通过使用由datsenko&wanner，2000描述的同源重组策略(根据方案1)将编码木糖的输入蛋白的操纵子xylfgh(xylf周质蛋白，xylgatp结合亚基，xylh膜亚基)的天然启动子替换为人工启动子。更准确地，使用seqidno:127和seqidno:128的引物产生携带ptrc人工启动子(seqidno:126)和卡那霉素抗性基因连同frt位点的被与xylf基因且与染色体上xylf基因的上游区同源的序列围绕的pcr产物，并将其导入先前转化有pkd46载体的菌株16中。用适当的寡核苷酸验证卡那霉素抗性转化体，并将保留的菌株命名为菌株21。在使用菌株21之前，根据方案1使用flp重组酶从xylfgh操纵子除去抗生素盒，产生菌株22。为了允许大肠杆菌在蔗糖上生长，将来自鼠伤寒沙门氏菌的质粒pur400(schmidt等,1982,seqidno:81)的编码果糖激酶的基因scrk、孔蛋白(porine)的基因scry、蛋白质iibc的基因scra、蔗糖-6-磷酸转化酶的基因scrb和阻抑物的基因scrr克隆于pbbr1mcs质粒(kovach等，1995)上的天然启动子下，得到质粒pbdo0003。最后，如下表所述，将质粒pbdo0003与质粒pdhb0002、pdhb0003、pdhb0004或pdhb0005之一组合引入菌株22，产生菌株23至菌株26。实施例4：2,4-二羟基丁酸的微生物生产。使用改良的m9培养基(anderson,1946)在500ml带挡板的锥形瓶中评价生产菌株，所述改良的m9培养基补充有30g/lmops、20g/ld-木糖和10g/l葡萄糖，并且调节为ph6,8。当在预培养和培养物中有必要时添加浓度50mg.l-1的壮观霉素和5mg.l-1的四环素。在37℃在lb培养基(sigma)中培养预培养物。在培养24小时后，使用它于30℃和以200rpm接种改良的m9培养基的50ml培养物至od600约0.2。当培养基中的糖耗尽时，将培养物离心，并且通过lc-ms/ms对培养液分析2,4-二羟基-丁酸。2,4-二羟基丁酸滴度/效价(titer)表示如下：表2：锥形瓶中评价的每种菌株的2,4-二羟基丁酸(dhb)滴度。符号(-)是指不及5mg/l的2,4-dhb产生，而符号(+)对应于5-15mg/l的2,4-dhb产生，并且符号(++)对应于高于15mg/l的2,4-dhb产生。菌株dhb滴度mg1655-菌株8至11+菌株17至20++对所有重组菌株观察2,4-二羟基丁酸产生。通过为增加细胞中nadph汇集物而进行的遗传修饰来增强产量。实施例5中在与上文所述相同的条件下，在木糖和蔗糖的混合物上测试菌株23至26(未在表2中呈现)，并且其2,4-dhb产生与菌株8至11相似。参考文献-altschuls,gishw,millerw,myerse,lipmandj,j.mol.biol,1990,215(3):403-410-anderson,proc.natl.acad.sci.usa.,1946,32:120-128.-bocanegraj,scruttonn,perhamr,biochemistry,1993,32(11):2737-2740.-carriert&keaslingj,biotechnolprog.,1999,15(1):58-64.-centeno-leijas,utrillaj,floresn,rodrigueza,gossetg,martineza,antonievanleeuwenhoek.,2013,104(6),913-924.-datsenkoka&wannerbl,procnatlacadsciusa.,2000,97:6640-6645.-davisjj&olsengj.,mol.biol.evol.,2011,28(1):211-221.-demll,bojaka,stecks,grafm,wildj,schirmbeckr,wolfh,wagnerr.,2011,j.virol.,75(22):10991-11001.-grafm,bojaka,demll,bielerk,wolfh,wagnerr.,2000,j.virol.,74(22):10/22-10826-hogema等,molecularmicrobiology,1997,24-857-867.-kim,j.h.等,appl.microbiol.biotechnol.,2010,88,1077-1085.-kovachme,elzerph,hillds,robertsongt,farrisma,rooprm,petersonkm.,gene,1995,166(1):175-6.-lees,mccormickm,lippards,chou,nature,2013,494:380-384.-lernerc.g.和inouyem.,nucleicacidsresearch,1990,18(15):4631.-lims,jungy,shinh,leey,jbioscibioeng.,2002,93(6):543-549.-marbaixa,noelg,detrouxa,vertommend,schaftingene,linsterc,jbiolchem.,2011,286(48):,41246-41252.-salish,methodsenzymol.,2011,498:19-42.-sambrook等,molecularcloning:alaboratorymanual4thedition,coldspringharborlaboratorypress-coldspringharbor,ny,usa,2012.-schmidtk,schupfnerm,schmittr,j.bacteriol.,1982,151:68-76.-segelih,enzymekinetics,(1993),johnwiley&sons,pp.44-54和100-112.序列表<110>代谢探索者公司(metabolicexplorersa)<120>用于2,4-二羟基丁酸的优化生产的经修饰的微生物<130>d34775<140>pctib2015/000618<141>2015-04-07<160>128<170>patentin3.5版<210>1<211>369<212>prt<213>大肠杆菌(escherichiacoli)<400>1metlysserargalaalavalalaphealaproglylysproleuglu151015ilevalgluileaspvalalaproprolyslysglygluvalleuile202530lysvalthrhisthrglyvalcyshisthraspalaphethrleuser354045glyaspaspprogluglyvalpheprovalvalleuglyhisglugly505560alaglyvalvalvalgluvalglygluglyvalthrservallyspro65707580glyasphisvalileproleutyrthralaglucysglyglucysglu859095phecysargserglylysthrasnleucysvalalavalarggluthr100105110glnglylysglyleumetproaspglythrthrargphesertyrasn115120125glyglnproleutyrhistyrmetglycysserthrpheserglutyr130135140thrvalvalalagluvalserleualalysileasnproglualaasn145150155160hisgluhisvalcysleuleuglycysglyvalthrthrglyilegly165170175alavalhisasnthralalysvalglnproglyaspservalalaval180185190pheglyleuglyalaileglyleualavalvalglnglyalaarggln195200205alalysalaglyargileilealaileaspthrasnprolyslysphe210215220aspleualaargargpheglyalathraspcysileasnproasnasp225230235240tyrasplysproilelysaspvalleuleuaspileasnlystrpgly245250255ileasphisthrpheglucysileglyasnvalasnvalmetargala260265270alaleugluseralahisargglytrpglyglnservalileilegly275280285valalavalalaglyglngluileserthrargpropheglnleuval290295300thrglyargvaltrplysglyseralapheglyglyvallysglyarg305310315320serglnleuproglymetvalgluaspalametlysglyaspileasp325330335leugluprophevalthrhisthrmetserleuaspgluileasnasp340345350alapheaspleumethisgluglylysserileargthrvalilearg355360365tyr<210>2<211>336<212>prt<213>大肠杆菌(escherichiacoli)<400>2metlysalaalavalvalthrlysasphishisvalaspvalthrtyr151015lysthrleuargserleulyshisglyglualaleuleulysmetglu202530cyscysglyvalcyshisthraspleuhisvallysasnglyaspphe354045glyasplysthrglyvalileleuglyhisgluglyileglyvalval505560alagluvalglyproglyvalthrserleulysproglyaspargala65707580servalalatrpphetyrgluglycysglyhiscysglutyrcysasn859095serglyasngluthrleucysargservallysasnalaglytyrser100105110valaspglyglymetalagluglucysilevalvalalaasptyrala115120125vallysvalproaspglyleuaspseralaalaalaserserilethr130135140cysalaglyvalthrthrtyrlysalavallysleuserlysilearg145150155160proglyglntrpilealailetyrglyleuglyglyleuglyasnleu165170175alaleuglntyralalysasnvalpheasnalalysvalilealaile180185190aspvalasnaspgluglnleulysleualathrglumetglyalaasp195200205leualaileasnserhisthrgluaspalaalalysilevalglnglu210215220lysthrglyglyalahisalaalavalvalthralavalalalysala225230235240alapheasnseralavalaspalavalargalaglyglyargvalval245250255alavalglyleuproproglusermetserleuaspileproargleu260265270valleuaspglyilegluvalvalglyserleuvalglythrarggln275280285aspleuthrglualapheglnphealaalagluglylysvalvalpro290295300lysvalalaleuargproleualaaspileasnthrilephethrglu305310315320metglugluglylysileargglyargmetvalileaspphearghis325330335<210>3<211>383<212>prt<213>大肠杆菌(escherichiacoli)<400>3metalaalaserthrphepheileproservalasnvalileglyala151015aspserleuthraspalametasnmetmetalaasptyrglyphethr202530argthrleuilevalthraspasnmetleuthrlysleuglymetala354045glyaspvalglnlysalaleuglugluargasnilepheservalile505560tyraspglythrglnproasnprothrthrgluasnvalalaalagly65707580leulysleuleulysgluasnasncysaspservalileserleugly859095glyglyserprohisaspcysalalysglyilealaleuvalalaala100105110asnglyglyaspileargasptyrgluglyvalaspargseralalys115120125proglnleuprometilealaileasnthrthralaglythralaser130135140glumetthrargphecysileilethraspglualaarghisilelys145150155160metalailevalasplyshisvalthrproleuleuservalasnasp165170175serserleumetileglymetprolysserleuthralaalathrgly180185190metaspalaleuthrhisalaileglualatyrvalserilealaala195200205thrproilethraspalacysalaleulysalavalthrmetileala210215220gluasnleuproleualavalgluaspglyserasnalalysalaarg225230235240glualametalatyralaglnpheleualaglymetalapheasnasn245250255alaserleuglytyrvalhisalametalahisglnleuglyglyphe260265270tyrasnleuprohisglyvalcysasnalavall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cgcgaccggctttgtcgtcggtctgaagaccgggattcaccgcttccacccg240gccacgggcttcagcctgctgctcgaggtcgaggacgcggcgctgaacaaccgccccaac300gacgccacggtcgacgcgcaaggccgtctgtggttcggcaccatgcacgacggggaagag360aacaatagcggctcgctctatcggatggacctcaccggcgtcgcccggatggaccgcgac420atctgcatcaccaacggcccgtgcgtctcgcccgacggcaagaccttctaccacaccgac480accctggaaaagacgatctacgccttcgacctggccgaggacggcctgctgtcgaacaag540cgcgtcttcgtgcagttcgccctgggcgacgatgtctatccggacggttcggtcgtcgat600tccgaaggctatctgtggaccgccctgtggggcggtttcggcgcggtccgcttctcgccg660caaggcgacgccgtgacgcgcatcgaactgcccgcccccaacgtcaccaagccctgcttc720ggcgggcctgacctgaagaccctctatttcaccaccgcccgcaagggcctgagcgacgag780accctggcccagtacccgctggccggcggtgtgttcgccgttccggtcgatgtggccggc840caaccccagcatgaggtccgccttgtctaa870<210>64<211>1968<212>dna<213>大肠杆菌(escherichiacoli)<400>64atgtctgttcgcaatatttttgctgacgagagccacgatatttacaccgtcagaacgcac60gccgatggcccggacggcgaactcccattaaccgcagagatgcttatcaaccgcccgagc120ggggatctgttcggtatgaccatgaatgccggaatgggttggtctccggacgagctggat180cgggacggtattttactgctcagtacactcggtggcttacgcggcgcagacggtaaaccc240gtggcgctggcgttgcaccaggggcattacgaactggacatccagatgaaagcggcggcc300gaggttattaaagccaaccatgccctgccctatgccgtgtacgtctccgatccttgtgac360gggcgtactcagggtacaacggggatgtttgattcgctaccataccgaaatgacgcatcg420atggtaatgcgccgccttattcgctctctgcccgacgcgaaagcagttattggtgtggcg480agttgcgataaggggcttccggccaccatgatggcactcgccgcgcagcacaacatcgca540accgtgctggtccccggcggcgcgacgctgcccgcaaaggatggagaagacaacggcaag600gtgcaaaccattggcgcacgcttcgccaatggcgaattatctctacaggacgcacgccgt660gcgggctgtaaagcctgtgcctcttccggcggcggctgtcaatttttgggcactgccggg720acatctcaggtggtggccgaaggattgggactggcaatcccacattcagccctggcccct780tccggtgagcctgtgtggcgggagatcgccagagcttccgcgcgagctgcgctgaacctg840agtcaaaaaggcatcaccacccgggaaattctcaccgataaagcgatagagaatgcgatg900acggtccatgccgcgttcggtggttcaacaaacctgctgttacacatcccggcaattgct960caccaggcaggttgccatatcccgaccgttgatgactggatccgcatcaacaagcgcgtg1020ccccgactggtgagcgtactgcctaatggcccggtttatcatccaacggtcaatgccttt1080atggcaggtggtgtgccggaagtcatgttgcatctgcgcagcctcggattgttgcatgaa1140gacgttatgacggttaccggcagcacgctgaaagaaaacctcgactggtgggagcactcc1200gaacggcgtcagcggttcaagcaactcctgctcgatcaggaacaaatcaacgctgacgaa1260gtgatcatgtctccgcagcaagcaaaagcgcgcggattaacctcaactatcaccttcccg1320gtgggcaatattgcgccagaaggttcggtgatcaaatccaccgccattgacccctcgatg1380attgatgagcaaggtatctattaccataaaggtgtggcgaaggtttatctgtccgagaaa1440agtgcgatttacgatatcaaacatgacaagatcaaggcgggcgatattctggtcattatt1500ggcgttggaccttcaggtacagggatggaagaaacctaccaggttaccagtgccctgaag1560catctgtcatacggtaagcatgtttcgttaatcaccgatgcacgtttctcgggcgtttct1620actggcgcgtgcatcggccatgtggggccagaagcgctggccggaggccccatcggtaaa1680ttacgcaccggggatttaattgaaattaaaattgattgtcgcgagcttcacggcgaagtc1740aatttcctcggaacccgtagcgatgaacaattaccttcacaggaggaggcaactgcaata1800ttaaatgccagacccagccatcaggatttacttcccgatcctgaattgccagatgatacc1860cggctatgggcaatgcttcaggccgtgagtggtgggacatggaccggttgtatttatgat1920gtaaacaaaattggcgcggctttgcgcgattttatgaataaaaactga1968<210>65<211>1383<212>dna<213>大肠杆菌(escherichiacoli)<400>65atgacgcaattaaccatgaaagacaaaattggctacgggctgggagacaccgcctgcggc60ttcgtctggcaggccacgatgttcctgctggcctatttctacaccgacgtcttcggcctg120tcggcggggattatgggcacgctgtttttggtctcccgcgtgctcgacgccgtcaccgac180ccgctgatggggctgctggtagaccgcacccgcacgcggcacggccagttccgcccgttc240ctgctgtggggggccatcccgttcggcatcgtctgcgtgctgaccttctacacgccggac300ttctccgcacagggcaagatcatctacgcctgcgtgacctacattctcctgaccctggtc360tacaccttcgttaacgtgccgtactgcgccatgccgggcgtcatcaccgccgacccgaaa420gagcgtcacgccctgcagtcctggcgcttcttcctggcggcggcgggctcgctcgctatc480agcggcatcgcgctgccgctggtgagcatcatcggcaaaggggacgagcaggtgggctac540ttcggcgccatgtgcgtgctggggctgagcggcgtggtgctgctctacgtctgcttcttc600acgaccaaagagcgctacacctttgaggtgcagccgggctcgtcggtggcgaaagacctt660aagctgctgctgggcaacagccagtggcgcatcatgtgcgcgttcaagatgatggcgacc720tgctccaacgtggtgcgcggcggggcgacgctctacttcgtgaaatacgtgatggatcac780ccggagttggcgacccagtttttactttacggcagcctcgccaccatgttcggctcgctt840tgctcctcacgcctgctgggccgcttcgaccgcgtcaccgccttcaagtggatcatcgtc900gcctactcgctgatcagcctgctgattttcgtcaccccggcggagcacatcgcgctcatt960tttgccctcaacatcctgttcctgttcgtctttaataccaccacgccgctgcagtggctg1020atggcttctgacgtggtggactacgaggagagccgcagcggtcgccgcctcgacgggctg1080gtgttctccacctacctgttcagcctgaagattggcctggcgattggcggggcggtggtg1140ggctggatcctggcgtacgtcaactattccgccagcagcagcgtgcagccggttgaggtg1200ctcaccaccatcaaaattctgttctgcgtggtgccggtggtgctctacgcgggcatgttc1260atcatgctgtcgctctacaagctcaccgatgcccgcgtggaggccatcagccggcagctg1320attaagcaccgcgcggcgcagggcgaggccgttcccgacgccgcgacagccgcatcccat1380taa1383<210>66<211>1041<212>dna<213>大肠杆菌(escherichiacoli)<400>66atgcagaacatcatccgaaaaggaggaactatgaaggctgcagttgttacgaaggatcat60catgttgacgttacgtataaaacactgcgctcactgaaacatggcgaagccctgctgaaa120atggagtgttgtggtgtatgtcataccgatcttcatgttaagaatggcgattttggtgac180aaaaccggcgtaattctgggccatgaaggcatcggtgtggtggcagaagtgggtccaggt240gtcacctcattaaaaccaggcgatcgtgccagcgtggcgtggttctacgaaggatgcggt300cattgcgaatactgtaacagtggtaacgaaacgctctgccgttcagttaaaaatgccgga360tacagcgttgatggcgggatggcggaagagtgcatcgtggtcgccgattacgcggtaaaa420gtgccagatggtctggactcggcggcggccagcagcattacctgtgcgggagtcaccacc480tacaaagccgttaagctgtcaaaaattcgtccagggcagtggattgctatctacggtctt540ggcggtctgggtaacctcgccctgcaatacgcgaagaatgtctttaacgccaaagtgatc600gccattgatgtcaatgatgagcagttaaaactggcaaccgaaatgggcgcagatttagcg660attaactcacacaccgaagacgccgccaaaattgtgcaggagaaaactggtggcgctcac720gctgcggtggtaacagcggtagctaaagctgcgtttaactcggcagttgatgctgtccgt780gcaggcggtcgtgttgtggctgtcggtctaccgccggagtctatgagcctggatatccca840cgtcttgtgctggatggtattgaagtggtcggttcgctggtcggcacgcgccaggattta900actgaagccttccagtttgccgccgaaggtaaagtggtgccgaaagtcgccctgcgtccg960ttagcggacatcaacaccatctttactgagatggaagaaggcaaaatccgtggccgcatg1020gtgattgatttccgtcactaa1041<210>67<211>1164<212>dna<213>大肠杆菌(escherichiacoli)<400>67atgaacaactttaatctgcacaccccaacccgcattctgtttggtaaaggcgcaatcgct60ggtttacgcgaacaaattcctcacgatgctcgcgtattgattacctacggcggcggcagc120gtgaaaaaaaccggcgttctcgatcaagttctggatgccctgaaaggcatggacgtgctg180gaatttggcggtattgagccaaacccggcttatgaaacgctgatgaacgccgtgaaactg240gttcgcgaacagaaagtgactttcctgctggcggttggcggcggttctgtactggacggc300accaaatttatcgccgcagcggctaactatccggaaaatatcgatccgtggcacattctg360caaacgggcggtaaagagattaaaagcgccatcccgatgggctgtgtgctgacgctgcca420gcaaccggttcagaatccaacgcaggcgcggtgatctcccgtaaaaccacaggcgacaag480caggcgttccattctgcccatgttcagccggtatttgccgtgctcgatccggtttatacc540tacaccctgccgccgcgtcaggtggctaacggcgtagtggacgcctttgtacacaccgtg600gaacagtatgttaccaaaccggttgatgccaaaattcaggaccgtttcgcagaaggcatt660ttgctgacgctaatcgaagatggtccgaaagccctgaaagagccagaaaactacgatgtg720cgcgccaacgtcatgtgggcggcgactcaggcgctgaacggtttgattggcgctggcgta780ccgcaggactgggcaacgcatatgctgggccacgaactgactgcgatgcacggtctggat840cacgcgcaaacactggctatcgtcctgcctgcactgtggaatgaaaaacgcgataccaag900cgcgctaagctgctgcaatatgctgaacgcgtctggaacatcactgaaggttccgatgat960gagcgtattgacgccgcgattgccgcaacccgcaatttctttgagcaattaggcgtgccg1020acccacctctccgactacggtctggacggcagctccatcccggctttgctgaaaaaactg1080gaagagcacggcatgacccaactgggcgaaaatcatgacattacgttggatgtcagccgc1140cgtatatacgaagccgcccgctaa1164<210>68<211>1587<212>dna<213>恶臭假单胞菌(pseudomonasputida)<400>68atggcttcggtacacggcaccacatacgaactcttgcgacgtcaaggcatcgatacggtc60ttcggcaatcctggctcgaacgagctcccgtttttgaaggactttccagaggactttcga120tacatcctggctttgcaggaagcgtgtgtggtgggcattgcagacggctatgcgcaagcc180agtcggaagccggctttcattaacctgcattctgctgctggtaccggcaatgctatgggt240gcactcagtaacgcctggaactcacattccccgctgatcgtcactgccggccagcagacc300agggcgatgattggcgttgaagctctgctgaccaacgtcgatgccgccaacctgccacga360ccacttgtcaaatggagctacgagcccgcaagcgcagcagaagtccctcatgcgatgagc420agggctatccatatggcaagcatggcgccacaaggccctgtctatctttcggtgccatat480gacgattgggataaggatgctgatcctcagtcccaccacctttttgatcgccatgtcagt540tcatcagtacgcctgaacgaccaggatctcgatattctggtgaaagctctcaacagcgca600tccaacccggcgatcgtcctgggcccggacgtcgacgcagcaaatgcgaacgcagactgc660gtcatgttggccgaacgcctcaaagctccggtttgggttgcgccatccgctccacgctgc720ccattccctacccgtcatccttgcttccgtggattgatgccagctggcatcgcagcgatt780tctcagctgctcgaaggtcacgatgtggttttggtaatcggcgctccagtgttccgttac840caccaatacgacccaggtcaatatctcaaacctggcacgcgattgatttcggtgacctgc900gacccgctcgaagctgcacgcgcgccaatgggcgatgcgatcgtggcagacattggtgcg960atggctagcgctcttgccaacttggttgaagagagcagccgccagctcccaactgcagct1020ccggaacccgcgaaggttgaccaagacgctggccgacttcacccagagacagtgttcgac1080acactgaacgacatggccccggagaatgcgatttacctgaacgagtcgacttcaacgacc1140gcccaaatgtggcagcgcctgaacatgcgcaaccctggtagctactacttctgtgcagct1200ggcggactgggcttcgccctgcctgcagcaattggcgttcaactcgcagaacccgagcga1260caagtcatcgccgtcattggcgacggatcggcgaactacagcattagtgcgttgtggact1320gcagctcagtacaacatccccactatcttcgtgatcatgaacaacggcacctacggtgcg1380ttgcgatggtttgccggcgttctcgaagcagaaaacgttcctgggctggatgtgccaggg1440atcgacttccgcgcactcgccaagggctatggtgtccaagcgctgaaagccgacaacctt1500gagcagctcaagggttcgctacaagaagcgctttctgccaaaggcccggtacttatcgaa1560gtaagcaccgtaagcccggtgaagtga1587<210>69<211>1323<212>dna<213>大肠杆菌(escherichiacoli)<400>69atgcaagcctattttgaccagctcgatcgcgttcgttatgaaggctcaaaatcctcaaac60ccgttagcattccgtcactacaatcccgacgaactggtgttgggtaagcgtatggaagag120cacttgcgttttgccgcctgctactggcacaccttctgctggaacggggcggatatgttt180ggtgtgggggcgtttaatcgtccgtggcagcagcctggtgaggcactggcgttggcgaag240cgtaaagcagatgtcgcatttgagtttttccacaagttacatgtgccattttattgcttc300cacgatgtggatgtttcccctgagggcgcgtcgttaaaagagtacatcaataattttgcg360caaatggttgatgtcctggcaggcaagcaagaagagagcggcgtgaagctgctgtgggga420acggccaactgctttacaaaccctcgctacggcgcgggtgcggcgacgaacccagatcct480gaagtcttcagctgggcggcaacgcaagttgttacagcgatggaagcaacccataaattg540ggcggtgaaaactatgtcctgtggggcggtcgtgaaggttacgaaacgctgttaaatacc600gacttgcgtcaggagcgtgaacaactgggccgctttatgcagatggtggttgagcataaa660cataaaatcggtttccagggcacgttgcttatcgaaccgaaaccgcaagaaccgaccaaa720catcaatatgattacgatgccgcgacggtctatggcttcctgaaacagtttggtctggaa780aaagagattaaactgaacattgaagctaaccacgcgacgctggcaggtcactctttccat840catgaaatagccaccgccattgcgcttggcctgttcggttctgtcgacgccaaccgtggc900gatgcgcaactgggctgggacaccgaccagttcccgaacagtgtggaagagaatgcgctg960gtgatgtatgaaattctcaaagcaggcggtttcaccaccggtggtctgaacttcgatgcc1020aaagtacgtcgtcaaagtactgataaatatgatctgttttacggtcatatcggcgcgatg1080gatacgatggcactggcgctgaaaattgcagcgcgcatgattgaagatggcgagctggat1140aaacgcatcgcgcagcgttattccggctggaatagcgaattgggccagcaaatcctgaaa1200ggccaaatgtcactggcagatttagccaaatatgctcaggaacatcatttgtctccggtg1260catcagagtggtcgccaggaacaactggaaaatctggtaaaccattatctgttcgacaaa1320taa1323<210>70<211>1455<212>dna<213>大肠杆菌(escherichiacoli)<400>70atgtatatcgggatagatcttggcacctcgggcgtaaaagttattttgctcaacgagcag60ggtgaggtggttgctgcgcaaacggaaaagctgaccgtttcgcgcccgcatccactctgg120tcggaacaagacccggaacagtggtggcaggcaactgatcgcgcaatgaaagctctgggc180gatcagcattctctgcaggacgttaaagcattgggtattgccggccagatgcacggagca240accttgctggatgctcagcaacgggtgttacgccctgccattttgtggaacgacgggcgc300tgtgcgcaagagtgcactttgctggaagcgcgagttccgcaatcgcgggtgattaccggc360aacctgatgatgcccggatttactgcgcctaaattgctatgggttcagcggcatgagccg420gagatattccgtcaaatcgacaaagtattattaccgaaagattacttgcgtctgcgtatg480acgggggagtttgccagcgatatgtctgacgcagctggcaccatgtggctggatgtcgca540aagcgtgactggagtgacgtcatgctgcaggcttgcgacttatctcgtgaccagatgccc600gcattatacgaaggcagcgaaattactggtgctttgttacctgaagttgcgaaagcgtgg660ggtatggcgacggtgccagttgtcgcaggcggtggcgacaatgcagctggtgcagttggt720gtgggaatggttgatgctaatcaggcaatgttatcgctggggacgtcgggggtctatttt780gctgtcagcgaagggttcttaagcaagccagaaagcgccgtacatagcttttgccatgcg840ctaccgcaacgttggcatttaatgtctgtgatgctgagtgcagcgtcgtgtctggattgg900gccgcgaaattaaccggcctgagcaatgtcccagctttaatcgctgcagctcaacaggct960gatgaaagtgccgagccagtttggtttctgccttatctttccggcgagcgtacgccacac1020aataatccccaggcgaagggggttttctttggtttgactcatcaacatggccccaatgaa1080ctggcgcgagcagtgctggaaggcgtgggttatgcgctggcagatggcatggatgtcgtg1140catgcctgcggtattaaaccgcaaagtgttacgttgattgggggcggggcgcgtagtgag1200tactggcgtcagatgctggcggatatcagcggtcagcagctcgattaccgtacggggggg1260gatgtggggccagcactgggcgcagcaaggctggcgcagatcgcggcgaatccagagaaa1320tcgctcattgaattgttgccgcaactaccgttagaacagtcgcatctaccagatgcgcag1380cgttatgccgcttatcagccacgacgagaaacgttccgtcgcctctatcagcaacttctg1440ccattaatggcgtaa1455<210>71<211>906<212>dna<213>大肠杆菌(escherichiacoli)<400>71atgaaaaaattcagcggcattattccaccggtatccagcacgtttcatcgtgacggaacc60cttgataaaaaggcaatgcgcgaagttgccgacttcctgattaataaaggggtcgacggg120ctgttttatctgggtaccggtggtgaatttagccaaatgaatacagcccagcgcatggca180ctcgccgaagaagctgtaaccattgtcgacgggcgagtgccggtattgattggcgtcggt240tccccttccactgacgaagcggtcaaactggcgcagcatgcgcaagcctacggcgctgat300ggtatcgtcgccatcaacccctactactggaaagtcgcaccacgaaatcttgacgactat360taccagcagatcgcccgtagcgtcaccctaccggtgatcctgtacaactttccggatctg420acgggtcaggacttaaccccggaaaccgtgacgcgtctggctctgcaaaacgagaatatc480gttggcatcaaagacaccatcgacagcgttggtcacttgcgtacgatgatcaacacagtt540aagtcggtacgcccgtcgttttcggtattctgcggttacgatgatcatttgctgaatacg600atgctgctgggcggcgacggtgcgataaccgccagcgctaactttgctccggaactctcc660gtcggcatctaccgcgcctggcgtgaaggcgatctggcgaccgctgcgacgctgaataaa720aaactactacaactgcccgctatttacgccctcgaaacaccgtttgtctcactgatcaaa780tacagcatgcagtgtgtcgggctgcctgtagagacatattgcttaccaccgattcttgaa840gcatctgaagaagcaaaagataaagtccacgtgctgcttaccgcgcagggcattttacca900gtctga906<210>72<211>909<212>dna<213>大肠杆菌(escherichiacoli)<400>72atgccgcagtccgcgttgttcacgggaatcattccccctgtctccaccatttttaccgcc60gacggccagctcgataagccgggcaccgccgcgctgatcgacgatctgatcaaagcaggc120gttgacggcctgttcttcctgggcagcggtggcgagttctcccagctcggcgccgaagag180cgtaaagccattgcccgctttgctatcgatcatgtcgatcgtcgcgtgccggtgctgatc240ggcaccggcggcaccaacgcccgggaaaccatcgaactcagccagcacgcgcagcaggcg300ggcgcggacggcatcgtggtgatcaacccctactactggaaagtgtcggaagcgaacctg360atccgctatttcgagcaggtggccgacagcgtcacgctgccggtgatgctctataacttc420ccggcgctgaccgggcaggatctgactccggcgctggtgaaaaccctcgccgactcgcgc480agcaatattatcggcatcaaagacaccatcgactccgtcgcccacctgcgcagcatgatc540cataccgtcaaaggtgcccatccgcacttcaccgtgctctgcggctacgacgatcatctg600ttcaataccctgctgctcggcggcgacggggcgatatcggcgagcggcaactttgccccg660caggtgtcggtgaatcttctgaaagcctggcgcgacggggacgtggcgaaagcggccggg720tatcatcagaccttgctgcaaattccgcagatgtatcagctggatacgccgtttgtgaac780gtgattaaagaggcgatcgtgctctgcggtcgtcctgtctccacgcacgtgctgccgccc840gcctcgccgctggacgagccgcgcaaggcgcagctgaaaaccctgctgcaacagctcaag900ctttgctga909<210>73<211>975<212>dna<213>大肠杆菌(escherichiacoli)<400>73atgaagccgtccgttatcctctacaaagccttacctgatgatttactgcaacgcctgcaa60gagcatttcaccgttcaccaggtggcaaacctcagcccacaaaccgtcgaacaaaatgca120gcaatttttgccgaagctgaaggtttactgggttcaaacgagaatgtaaatgccgcattg180ctggaaaaaatgccgaaactgcgtgccacatcaacgatctccgtcggctatgacaatttt240gatgtcgatgcgcttaccgcccgaaaaattctgctgatgcacacgccaaccgtattaaca300gaaaccgtcgccgatacgctgatggcgctggtgttgtctaccgctcgtcgggttgtggag360gtagcagaacgggtaaaagcaggcgaatggaccgcgagcataggcccggactggtacggc420actgacgttcaccataaaacactgggcattgtcgggatgggacggatcggcatggcgctg480gcacaacgtgcgcactttggcttcaacatgcccatcctctataacgcgcgccgccaccat540aaagaagcagaagaacgcttcaacgcccgctactgcgatttggatactctgttacaagag600tcagatttcgtttgcctgatcctgccgttaactgatgagacgcatcatctgtttggcgca660gaacaattcgccaaaatgaaatcctccgccattttcattaatgccggacgtggcccggtg720gttgacgaaaatgcactgatcgcagcattgcagaaaggcgaaattcacgctgccgggctg780gatgtcttcgaacaagagccactgtccgtagattcgccgttgctctcaatggccaacgtc840gtcgcagtaccgcatattggatctgccacccatgagacgcgttatggcatggccgcctgt900gccgtggataatttgattgatgcgttacaaggaaaggttgagaagaactgtgtgaatccg960cacgtcgcggactaa975<210>74<211>939<212>dna<213>大肠杆菌(escherichiacoli)<400>74atggatatcatcttttatcacccaacgttcgatacccaatggtggattgaggcactgcgc60aaagctattcctcaggcaagagtcagagcatggaaaagcggagataatgactctgctgat120tatgctttagtctggcatcctcctgttgaaatgctggcagggcgcgatcttaaagcggtg180ttcgcactcggggccggtgttgattctattttgagcaagctacaggcacaccctgaaatg240ctgaacccttctgttccactttttcgcctggaagataccggtatgggcgagcaaatgcag300gaatatgctgtcagtcaggtgctgcattggtttcgacgttttgacgattatcgcatccag360caaaatagttcgcattggcaaccgctgcctgaatatcatcgggaagattttaccatcggc420attttgggcgcaggcgtactgggcagtaaagttgctcagagtctgcaaacctggcgcttt480ccgctgcgttgctggagtcgaacccgtaaatcgtggcctggcgtgcaaagctttgccgga540cgggaagaactgtctgcatttctgagccaatgtcgggtattgattaatttgttaccgaat600acccctgaaaccgtcggcattattaatcaacaattactcgaaaaattaccggatggcgcg660tatctcctcaacctggcgcgtggtgttcatgttgtggaagatgacctgctcgcggcgctg720gatagcggcaaagttaaaggcgcaatgttggatgtttttaatcgtgaacccttaccgcct780gaaagtccgctctggcaacatccacgcgtgacgataacaccacatgtcgccgcgattacc840cgtcccgctgaagctgtggagtacatttctcgcaccattgcccagctcgaaaaaggggag900agggtctgcgggcaagtcgaccgcgcacgcggctactaa939<210>75<211>1434<212>dna<213>大肠杆菌(escherichiacoli)<400>75atgtttaagaatgcatttgctaacctgcaaaaggtcggtaaatcgctgatgctgccggta60tccgtactgcctatcgcaggtattctgctgggcgtcggttccgcgaatttcagctggctg120cccgccgttgtatcgcatgttatggcagaagcaggcggttccgtctttgcaaacatgcca180ctgatttttgcgatcggtgtcgccctcggctttaccaataacgatggcgtatccgcgctg240gccgcagttgttgcctatggcatcatggttaaaaccatggccgtggttgcgccactggta300ctgcatttacctgctgaagaaatcgcctctaaacacctggcggatactggcgtactcgga360gggattatctccggtgcgatcgcagcgtacatgtttaaccgtttctaccgtattaagctg420cctgagtatcttggcttctttgccggtaaacgctttgtgccgatcatttctggcctggct480gccatctttactggcgttgtgctgtccttcatttggccgccgattggttctgcaatccag540accttctctcagtgggctgcttaccagaacccggtagttgcgtttggcatttacggtttc600atcgaacgttgcctggtaccgtttggtctgcaccacatctggaacgtacctttccagatg660cagattggtgaatacaccaacgcagcaggtcaggttttccacggcgacattccgcgttat720atggcgggtgacccgactgcgggtaaactgtctggtggcttcctgttcaaaatgtacggt780ctgccagctgccgcaattgctatctggcactctgctaaaccagaaaaccgcgcgaaagtg840ggcggtattatgatctccgcggcgctgacctcgttcctgaccggtatcaccgagccgatc900gagttctccttcatgttcgttgcgccgatcctgtacatcatccacgcgattctggcaggc960ctggcattcccaatctgtattcttctggggatgcgtgacggtacgtcgttctcgcacggt1020ctgatcgacttcatcgttctgtctggtaacagcagcaaactgtggctgttcccgatcgtc1080ggtatcggttatgcgattgtttactacaccatcttccgcgtgctgattaaagcactggat1140ctgaaaacgccgggtcgtgaagacgcgactgaagatgcaaaagcgacaggtaccagcgaa1200atggcaccggctctggttgctgcatttggtggtaaagaaaacattactaacctcgacgca1260tgtattacccgtctgcgcgtcagcgttgctgatgtgtctaaagtggatcaggccggcctg1320aagaaactgggcgcagcgggcgtagtggttgctggttctggtgttcaggcgattttcggt1380actaaatccgataacctgaaaaccgagatggatgagtacatccgtaaccactaa1434<210>76<211>510<212>dna<213>大肠杆菌(escherichiacoli)<400>76atgggtttgttcgataaactgaaatctctggtttccgacgacaagaaggataccggaact60attgagatcattgctccgctctctggcgagatcgtcaatatcgaagacgtgccggatgtc120gtttttgcggaaaaaatcgttggtgatggtattgctatcaaaccaacgggtaacaaaatg180gtcgcgccagtagacggcaccattggtaaaatctttgaaaccaaccacgcattctctatc240gaatctgatagcggcgttgaactgttcgtccacttcggtatcgacaccgttgaactgaaa300ggcgaaggcttcaagcgtattgctgaagaaggtcagcgcgtgaaagttggcgatactgtc360attgaatttgatctgccgctgctggaagagaaagccaagtctaccctgactccggttgtt420atctccaacatggacgaaatcaaagaactgatcaaactgtccggtagcgtaaccgtgggt480gaaaccccggttatccgcatcaagaagtaa510<210>77<211>258<212>dna<213>大肠杆菌(escherichiacoli)<400>77atgttccagcaagaagttaccattaccgctccgaacggtctgcacacccgccctgctgcc60cagtttgtaaaagaagctaagggcttcacttctgaaattactgtgacttccaacggcaaa120agcgccagcgcgaaaagcctgtttaaactgcagactctgggcctgactcaaggtaccgtt180gtgactatctccgcagaaggcgaagacgagcagaaagcggttgaacatctggttaaactg240atggcggaactcgagtaa258<210>78<211>1254<212>dna<213>大肠杆菌(escherichiacoli)<400>78atgtactatttaaaaaacacaaacttttggatgttcggtttattctttttcttttacttt60tttatcatgggagcctacttcccgtttttcccgatttggctacatgacatcaaccatatc120agcaaaagtgatacgggtattatttttgccgctatttctctgttctcgctattattccaa180ccgctgtttggtctgctttctgacaaactcgggctgcgcaaatacctgctgtggattatt240accggcatgttagtgatgtttgcgccgttctttatttttatcttcgggccactgttacaa300tacaacattttagtaggatcgattgttggtggtatttatctaggcttttgttttaacgcc360ggtgcgccagcagtagaggcatttattgagaaagtcagccgtcgcagtaatttcgaattt420ggtcgcgcgcggatgtttggctgtgttggctgggcgctgtgtgcctcgattgtcggcatc480atgttcaccatcaataatcagtttgttttctggctgggctctggctgtgcactcatcctc540gccgttttactctttttcgccaaaacggatgcgccctcttctgccacggttgccaatgcg600gtaggtgccaaccattcggcatttagccttaagctggcactggaactgttcagacagcca660aaactgtggtttttgtcactgtatgttattggcgtttcctgcacctacgatgtttttgac720caacagtttgctaatttctttacttcgttctttgctaccggtgaacagggtacgcgggta780tttggctacgtaacgacaatgggcgaattacttaacgcctcgattatgttctttgcgcca840ctgatcattaatcgcatcggtgggaaaaacgccctgctgctggctggcactattatgtct900gtacgtattattggctcatcgttcgccacctcagcgctggaagtggttattctgaaaacg960ctgcatatgtttgaagtaccgttcctgctggtgggctgctttaaatatattaccagccag1020tttgaagtgcgtttttcagcgacgatttatctggtctgtttctgcttctttaagcaactg1080gcgatgatttttatgtctgtactggcgggcaatatgtatgaaagcatcggtttccagggc1140gcttatctggtgctgggtctggtggcgctgggcttcaccttaatttccgtgttcacgctt1200agcggccccggcccgctttccctgctgcgtcgtcaggtgaatgaagtcgcttaa1254<210>79<211>1545<212>dna<213>大肠杆菌(escherichiacoli)<400>79atggatgtcattaaaaagaaacattggtggcaaagcgacgcgctgaaatggtcagtgcta60ggtctgctcggcctgctggtgggttaccttgttgttttaatgtacgcacaaggggaatac120ctgttcgccattaccacgctgatattgagttcagcggggctgtatattttcgccaatcgt180aaagcctacgcctggcgctatgtttacccgggaatggctggaatgggattattcgtcctc240ttccctctggtctgcaccatcgccattgccttcaccaactacagcagcactaaccagctg300acttttgaacgtgcgcaggaagtgttgttagatcgctcctggcaagcaggcaaaacctat360aactttggtctttacccggcgggcgatgagtggcaactggcgctcagcgacggcgaaacc420ggcaaaaattacctctccgacgcttttaaatttggcggcgagcaaaaactgcaactgaaa480gaaacgaccgcccagcccgaaggcgaacgcgcgaatctgcgcgtgattacccagaatcgt540caggcgctgagtgacattaccgccattctgccggatggcaacaaagtgatgatgagctcc600ctgcgccagttttctggcacgcagccgctctacacactcgacggtgacggcacgttgacg660aataatcagagcggcgtgaaatatcgtccgaataaccaaattggcttttaccagtccatt720accgccgacggcaactggggtgatgaaaagctaagccccggttacaccgtgaccaccggc780tggaaaaactttacccgcgtctttaccgacgaaggcattcagaaaccgttcctcgccatt840ttcgtctggaccgtggtgttctcgctgatcactgtctttttaacggtggcggtcggcatg900gttctggcgtgtctggtgcagtgggaagcgttgcgcggcaaagcggtctatcgcgtcctg960ctgattctgccctacgcggtgccatcgttcatttcaatcttgattttcaaagggttgttt1020aaccagagcttcggtgaaatcaacatgatgttgagcgcgctgtttggcgtgaagcccgcc1080tggttcagcgatccgaccaccgcccgcacgatgctaattatcgtcaatacctggctgggt1140tatccgtacatgatgatcctctgcatgggcttgctgaaagcgattccggacgatttgtat1200gaagcctcagcaatggatggcgcaggtccgttccagaacttctttaagattacgctgccg1260ctgctgattaaaccgctgacgccgctgatgatcgccagcttcgcctttaactttaacaac1320ttcgtgctgattcaactgttaaccaacggcggcccggatcgtcttggcacgaccacgcca1380gccggttataccgacctgcttgttaactacacctaccgcatcgcttttgaaggcggcggg1440ggtcaggacttcggtctggcggcagcaattgccacgctgatcttcctgctggtgggtgcg1500ctggcgatagtgaacctgaaagccacgcgaatgaagtttgattaa1545<210>80<211>891<212>dna<213>大肠杆菌(escherichiacoli)<400>80atggcaatggtccaaccgaaatcgcaaaaagctcgtttatttattactcacctgctactg60ctactttttatcgcagcgattatgttcccgctgctgatggtcgtcgctatctcgctgcgt120cagggaaactttgcgaccggcagcctgatcccggagcaaatctcctgggatcactggaaa180ctggcgttaggttttagcgttgaacaggctgatggtcgcattacgccaccgccattcccg240gtactgctgtggctgtggaactcggtaaaggtcgccgggatttccgcgattggcattgtg300gcgctctccaccacctgcgcctacgctttcgcccgtatgcgctttccaggcaaagcgacg360ctgctgaaaggaatgctgattttccagatgttcccggcagtactttcactggtcgcgttg420tatgcgttgtttgatcgtctgggtgagtacattccattcattggcctgaatactcacggc480ggcgtaattttcgcgtatctgggtgggattgcgctgcatgtctggaccatcaaaggctat540ttcgaaaccatcgacagttcgctggaagaagctgctgcgctggatggtgcgacaccgtgg600caggccttccgccttgtcctgttgccgctgtcagtaccgattctggcggtggtattcatc660ctgtcgtttatcgctgccattactgaagttccggtcgcgtcgctgttactgcgtgacgta720aacagctacaccctggccgtggggatgcagcaatacctcaacccgcaaaactacctgtgg780ggtgactttgccgccgctgccgtgatgtctgcattaccgatcaccatcgtcttcttgctg840gctcaacgctggctggtcaacggcctgacggcaggtggtgtgaaaggttaa891<210>81<211>6546<212>dna<213>鼠伤寒沙门氏菌(salmonellatyphimurium)<400>81atgaatgcaaaagtttgggttctgggcgacgcggtggtggacctgctgccggagagcgaa60gggcgcctgctgcagtgccctggaggcgcgccggctaacgtggcggtaggggttgcccgc120cttggcggcaacagcggatttatcggcgccgtcggcggtgacccgtttggccgctacatg180cgtcataccctgcaacaggagcaggtcgacgtcagccatatgtatctcgacgatcagcac240cgcacgtccactgtggtcgtcgaccttgacgaccagggggaacgcacctttacctttatg300gtacgccccagcgcggacctgttcctggttgaagaagacctgccacagtttgccgccgga360cagtggttgcacgtctgctccatcgcgctcagcgccgagcccagccgtagcactaccttc420gcggcgatggagagcatcaggtctgccggcggtcgggtcagctttgaccctaatattcgt480cccgatctctggcaggatcaggctttgctgctagcctgcctcgatcgcgctttgcacatg540gccaacgtggtaaagctatcggaagaggagctggtcttcatcagcagcagtaatgattta600gcatacggaatcgccagcgtaacggagcgctatcagccagaattgctactggtgacccgg660ggcaaagcgggggtgcttgccgcgtttcagcagaagtttacccatttcaacgcccggcct720gtggccagcgtggacaccaccggcgcgggagacgcatttgtcgccggactgctcgccagc780cttgcggctaacgggatgccaacggacatgaccgcactggaaccgacactcacgcttgca840cagacctgcggcgccctggccaccacagccaaaggtgcgatgaccgccttgccttatcag900cgcgatctcaaccgtcagttttaatccttaaagccgctttgcgcggctcactttgttgca960tgcatcacatttattaaaccggtttagcatatttgttttaagaaaaacaaaggtcgggct1020taacatagcgcctaaaccggtttagcaaaaattataattttccatttttacttttgggat1080gccaacagcatgtacagaaaaagcacacttgcgatgcttatcgctttgctaaccagcgct1140gcctcagcccatgcgcaaacggatataagcaccattgaagcccgactcaacgcgctggaa1200aaacgcctgcaggaggcagaaaacagggcgcaaacggcggaaaaccgcgccggggcggcg1260gagaaaaaagttcagcaactcaccgcgcagcagcaaaaaaaccagaactcgactcaggaa1320gtggctcagcgtaccgccagacttgagaaaaaagccgatgacaaaagcggatttgagttt1380cacggttacgcccgctccggcgtgataatgaatgattccggcgccagcaccaaatccgga1440gcctacataacgccggcaggtgaaaccggcggagctatcggccgtctgggaaaccaggcc1500gatacctatgttgaaatgaatcttgaacataagcagaccctggataatggggccacgacc1560cgctttaaggtgatggtcgccgacgggcaaacctcttataacgactggactgcaagcacc1620agcgatctgaacgttcgtcaggcctttgtcgaattgggtaacctgccgacgttcgctggg1680ccatttaagggctccaccctgtgggccgggaaacgtttcgaccgcgacaatttcgatatt1740cactggattgactctgatgtcgtgttcctcgccggtaccggtggtggtatctatgacgtg1800aagtggaacgacggcctgcggagtaatttctccctgtacgggcgtaacttcggcgacatt1860gatgattccagcaacagcgtgcagaactatatcctcaccatgaatcacttcgcaggtccg1920ctgcagatgatggtcagcggtctgcgggcgaaggataacgacgagcgtaaagatagcaac1980ggcaatctggcaaaaggcgatgcggcaaacaccggcgtgcatgcgctgctcggcctgcat2040aacgacagtttctacggcctgcgcgacggtagcagtaaaaccgctctgctttatggtcat2100ggtctgggcgcagaggttaaaggtatcggatctgatggcgcacttcgtccgggagccgac2160acatggcgcattgccagttacggcaccacgccgctcagcgaaaactggtctgttgccccg2220gcaatgctggcgcaacgcagtaaagaccgctatgccgatggcgacagctatcagtgggca2280acattcaacctgcgtctgattcaggcaatcaatcagaatttcgctctcgcctacgaaggc2340agctaccagtacatggatcttaaacccgaaggttataacgatcgtcaggcggtgaacggt2400agcttctacaagctcaccttcgccccgacatttaaggtcggcagtatcggtgatttcttc2460agtcgcccggagattcgtttctatacctcctg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tgggtaacgccaacaccttctgggtg1260tatgcggctctgaacgtactgtttatcctgctgacattgtggctggtaccggaaaccaaa1320cacgtttcgctggaacatattgaacgtaatctgatgaaaggtcgtaaactgcgcgaaata1380ggcgctcacgattaa1395<210>85<211>1422<212>dna<213>运动发酵单胞菌(zymomonasmobilis)<400>85atgagttctgaaagtagtcagggtctagtcacgcgactagccctaatcgctgctataggc60ggcttgcttttcggttacgattcagcggttatcgctgcaatcggtacaccggttgatatc120cattttattgcccctcgtcacctgtctgctacggctgcggcttccctttctgggatggtc180gttgttgctgttttggtcggttgtgttaccggttctttgctgtctggctggattggtatt240cgcttcggtcgtcgcggcggattgttgatgagttccatttgtttcgtcgccgccggtttt300ggtgctgcgttaaccgaaaaattatttggaaccggtggttcggctttacaaattttttgc360tttttccggtttcttgccggtttaggtatcggtgtcgtttcaaccttgaccccaacctat420attgctgaaattcgtccgccagacaaacgtggtcagatggtttctggtcagcagatggcc480attgtgacgggtgctttaaccggttatatctttacctggttactggctcatttcggttct540atcgattgggttaatgccagtggttggtgctggtctccggcttcagaaggcctgatcggt600attgccttcttattgctgctgttaaccgcaccggatacgccgcattggttggtgatgaag660ggacgtcattccgaggctagcaaaatccttgctcgtctggaaccgcaagccgatcctaat720ctgacgattcaaaagattaaagctggctttgataaagccatggacaaaagcagcgcaggt780ttgtttgcttttggtatcaccgttgtttttgccggtgtatccgttgctgccttccagcag840ttagtcggtattaacgccgtgctgtattatgcaccgcagatgttccagaatttaggtttt900ggagctgatacggcattattgcagaccatctctatcggtgttgtgaacttcatcttcacc960atgattgcttcccgtgttgttgaccgcttcggccgtaaacctctgcttatttggggtgct1020ctcggtatggctgcaatgatggctgttttaggctgctgtttctggttcaaagtcggtggt1080gttttgcctttggcttctgtgcttctttatattgcagtctttggtatgtcatggggccct1140gtctgctgggttgttctgtcagaaatgttcccgagttccatcaagggcgcagctatgcct1200atcgctgttaccggacaatggttagctaatatcttggttaacttcctgtttaaggttgcc1260gatggttctccagcattgaatcagactttcaaccacggtttctcctatctcgttttcgca1320gcattaagtatcttaggtggcttgattgttgctcgcttcgtgccggaaaccaaaggtcgg1380agcctggatgaaatcgaggagatgtggcgctcccagaagtag1422<210>86<211>984<212>dna<213>运动发酵单胞菌(zymomonasmobilis)<400>86atggaaattgttgcgattgacatcggtggaacgcatgcgcgtttctctattgcggaagta60agcaatggtcgggttctttctcttggagaagaaacaacttttaaaacggcagaacatgct120agcttgcagttagcttgggaacgtttcggtgaaaaactgggtcgtcctctgccacgtgcc180gcagctattgcatgggctggcccggttcatggtgaagttttaaaacttaccaataaccct240tgggtattaagaccagctactctgaatgaaaagctggacatcgatacgcatgttctgatc300aatgacttcggcgcggttgcccacgcggttgcgcatatggattcttcttatctggatcat360atttgtggtcctgatgaagcgcttcctagcgatggtgttatcactattcttggtccggga420acgggcttgggtgttgcccatctgttgcggactgaaggccgttatttcgtcatcgaaact480gaaggcggtcatatcgactttgctccgcttgacagacttgaagacaaaattctggcacgt540ttacgtgaacgtttccgccgcgtttctatcgaacgcattatttctggcccgggtcttggt600aatatctacgaagcactggctgccattgaaggcgttccgttcagcttgctggatgatatt660aaattatggcagatggctttggaaggtaaagacaaccttgctgaagccgctttggatcgc720ttctgcttgagccttggcgctatcgctggtgatcttgctttggcacagggtcgaaccagt780gttgttattggcggtggtgtcggtcttcgtatcgcttcccatttgccagaatctggtttc840cgtcagcgctttgtttcaaaaggacgctttgaacgcgtcatgtccaagattccggttaag900ttgattacttatccgcagcctggactgttgggtgcgcagctgcctatgccaacaaatatt960ctgaagttgaataatattttttaa984<210>87<211>966<212>dna<213>大肠杆菌(escherichiacoli)<400>87atgacaaagtatgcattagtcggtgatgtgggcggcaccaacgcacgtcttgctctgtgt60gatattgccagtggtgaaatctcgcaggctaagacctattcagggcttgattaccccagc120ctcgaagcggtcattcgcgtttatcttgaagaacataaggtcgaggtgaaagacggctgt180attgccatcgcttgcccaattaccggtgactgggtggcgatgaccaaccatacctgggcg240ttctcaattgccgaaatgaaaaagaatctcggttttagccatctggaaattattaacgat300tttaccgctgtatcgatggcgatcccgatgctgaaaaaagagcatctgattcagtttggt360ggcgcagaaccggtcgaaggtaagcctattgcggtttacggtgccggaacggggcttggg420gttgcgcatctggtccatgtcgataagcgttgggtaagcttgccaggcgaaggcggtcac480gttgattttgcgccgaatagtgaagaagaggccattatcctcgaaatattgcgtgcggaa540attggtcatgtttcggcggagcgcgtgctttctggccctgggctggtgaatttgtatcgc600gcaattgtgaaagctgacaaccgcctgccagaaaatctcaagccaaaagatattaccgaa660cgcgcgctggctgacagctgcaccgattgccgccgcgcattgtcgctgttttgcgtcatt720atgggccgttttggcggcaatctggcgctcaatctcgggacatttggcggcgtgtttatt780gcgggcggtatcgtgccgcgcttccttgagttcttcaaagcctccggtttccgtgccgca840tttgaagataaagggcgctttaaagaatatgtccatgatattccggtgtatctcatcgtc900catgacaatccgggccttctcggttccggtgcacatttacgccagaccttaggtcacatt960ctgtaa966<210>88<211>2547<212>dna<213>大肠杆菌(escherichiacoli)<400>88ttgtacctctatattgagactctgaaacagagactggatgccataaatcaattgcgtgtg60gatcgcgcgcttgctgctatggggcctgcattccaacaggtctacagtctactgccgaca120ttgttgcactatcaccatccgctaatgccgggttaccttgatggtaacgttcccaaaggc180atttgcctttacacgcctgatgaaactcaacgccactacctgaacgagcttgaactgtat240cgtggaatgtcagtacaggatccgccgaaaggtgagcttccaattactggtgtatacacc300atgggcagcacctcgtccgtagggcaaagttgttcctctgacctggatatctgggtctgt360catcaatcctggctcgatagcgaagagcgccaattgctacaacgtaaatgtagcctgctg420gaaaactgggccgcctcgctgggtgtggaagtcagcttcttcctgattgatgaaaaccgc480ttccgtcataatgaaagcggcagcctggggggcgaagattgtggctccacccagcatata540ctgctgcttgacgaattttatcgtaccgccgtgcgtctcgccggtaagcgtattctgtgg600aatatggtgccgtgcgacgaagaagagcattacgacgactatgtgatgacgctttacgcg660cagggcgtgctgacgccaaatgaatggctggatctcggtggcttaagctcgctttctgct720gaagagtactttggtgccagcctttggcagctctacaagagtatcgattccccatacaaa780gcggtactgaaaacactgctgctggaagcctattcctgggaatacccgaacccacgtctg840ctggcgaaagatatcaaacagcgtttgcacgacggcgagattgtatcgtttggtctcgat900ccatactgcatgatgctggagcgtgttactgaatacctgacggcgattgaagattttacc960cgtctggatttagtacgtcgctgcttctatttaaaagtgtgcgaaaagctcagccgtgaa1020cgcgcctgcgtaggctggcgtcgcgcagtgttgagccagttagtgagcgagtggggttgg1080gacgaagctcgtctggcaatgctcgataaccgcgctaactggaagattgatcaggtgcgt1140gaggcgcacaacgagttgctcgacgcgatgatgcagagctaccgtaatctgatccgcttt1200gcgcgtcgcaataaccttagcgtctccgccagtccgcaggatatcggcgtgctgacgcgt1260aagctgtatgccgcgtttgaagcattaccaggtaaagtgacgctggtaaacccgcagatt1320tcacccgatctctcggaaccgaatctgacctttatttatgtgccgccgggccgggctaac1380cgttcaggttggtatctgtataaccgcgcgccaaatattgagtcgatcatcagccatcag1440ccgctggaatataaccgttacctgaataaactggtggcgtgggcatggtttaacggcctg1500ctgacctcgcgcacccgtttgtatattaaaggtaacggcattgtcgatttgcctaagttg1560caggagatggtcgccgacgtgtcgcaccatttcccgctgcgcttacctgcaccgacaccg1620aaggcgctctacagcccgtgtgagatccgccatctggcgattatcgttaacctggaatat1680gacccgacagcggcgttccgcaatcaggtggtgcatttcgatttccgtaagctggatgtc1740ttcagctttggcgagaatcaaaattgcctggtaggtagcgttgacctgctgtaccgcaac1800tcgtggaacgaagtgcgtacgctgcacttcaacggcgagcaatcgatgatcgaagccctg1860aaaactattctcggcaaaatgcatcaggacgccgcaccgccagatagcgtggaagtcttc1920tgttatagccagcatctgcgcggcttaattcgtactcgcgtgcagcaactggtttctgag1980tgtattgaattgcgtctttccagcacccgccaggaaaccgggcgtttcaaggcgctgcgc2040gtttctggtcaaacctgggggttgttcttcgaacgcctgaatgtatcggtacagaaactg2100gaaaacgccatcgagttttatggcgcgatttcgcataacaaactgcacggcctgtcagtg2160caggttgaaaccaatcacgtcaaattaccggcggtggtggacggctttgccagcgaaggg2220atcatccagttctttttcgaagaaacgcaagacgagaatggctttaatatctacattctc2280gacgaaagcaaccgggttgaggtatatcaccactgcgaaggcagcaaagaggagctggta2340cgtgacgtcagtcgcttctactcgtcatcgcatgaccgttttacctacggctcaagcttc2400atcaacttcaacctgccgcagttctatcagattgtgaaggttgatggtcgtgaacaggtg2460attccgttccgcacaaaatctatcggtaacatgccgcctgccaatcaggatcacgatacg2520ccgctattacagcaatatttttcgtga2547<210>89<211>210<212>prt<213>大肠杆菌(escherichiacoli)<400>89metvalleuglylysproglnthraspprothrleuglutrppheleu151015serhiscyshisilehislystyrproserlysserthrleuilehis202530glnglyglulysalagluthrleutyrtyrilevallysglyserval354045alavalleuilelysaspglugluglylysglumetileleusertyr505560leuasnglnglyasppheileglygluleuglyleuphegluglugly65707580glngluargseralatrpvalargalalysthralacysgluvalala859095gluilesertyrlyslyspheargglnleuileglnvalasnproasp100105110ileleumetargleuseralaglnmetalaargargleuglnvalthr115120125serglulysvalglyasnleualapheleuaspvalthrglyargile130135140alaglnthrleuleuasnleualalysglnproaspalametthrhis145150155160proaspglymetglnilelysilethrargglngluileglyglnile165170175valglycysserarggluthrvalglyargileleulysmetleuglu180185190aspglnasnleuileseralahisglylysthrilevalvaltyrgly195200205thrarg210<210>90<211>210<212>prt<213>人工的<220><223>crp*突变体<400>90metvalleuglylysproglnthraspprothrleuglutrppheleu151015serhiscyshisilehislystyrproserlysserthrleuilehis202530glnglyglulysalagluthrleutyrtyrilevallysglyserval354045alavalleuilelyshisglugluglylysglumetileleusertyr505560leuasnglnglyasppheileglygluleuglyleuphegluglugly65707580glngluargseralatrpvalargalalysthralacysgluvalala859095gluilesertyrlyslyspheargglnleuileglnvalasnproasp100105110ileleumetargleuseralaglnmetalaargargleuglnvalthr115120125serglulysvalglyasnleualapheleuaspvalthrglyargile130135140alaglnthrleuleuasnleualalysglnproaspalametthrhis145150155160proaspglymetglnilelysilethrargglngluileglyglnile165170175valglycysserarggluthrvalglyargileleulysmetleuglu180185190aspglnasnleuileseralahisglylysthrilevalvaltyrgly195200205thrarg210<210>91<211>210<212>prt<213>人工的<220><223>crp*突变体<400>91metvalleuglylysproglnthraspprothrleuglutrppheleu151015serhiscyshisilehislystyrproserlysserthrleuilehis202530glnglyglulysalagluthrleutyrtyrilevallysglyserval354045alavalleuilelysaspglugluglylysglumetileleuphetyr505560leuasnglnglyasppheileglygluleuglyleuphegluglugly65707580glngluargseralatrpvalargalalysthralacysgluvalala859095gluilesertyrlyslyspheargglnleuileglnvalasnproasp100105110ileleumetargleuseralaglnmetalaargargleuglnvalthr115120125serglulysvalglyasnleualapheleuaspvalthrglyargile130135140alaglnthrleuleuasnleualalysglnproaspalametthrhis145150155160proaspglymetglnilelysilethrargglngluileglyglnile165170175valglycysserarggluthrvalglyargileleulysmetleuglu180185190aspglnasnleuileseralahisglylysthrilevalvaltyrgly195200205thrarg210<210>92<211>210<212>prt<213>人工的<220><223>crp*突变体<400>92metvalleuglylysproglnthraspprothrleuglutrppheleu151015serhiscyshisilehislystyrproserlysserthrleuilehis202530glnglyglulysalagluthrleutyrtyrilevallysglyserval354045alavalleuilelysaspglugluglylysglumetileleusertyr505560leuasnglnglyasppheileglygluleuglyleuphegluglugly65707580glngluargseralatrpvalargalalysthralacysgluvalala859095gluilesertyrlyslyspheargglnleuileglnvalasnproasp100105110ileleumetargleuseralaglnmetalaargargleuglnvalthr115120125serglulysvalglyasnleualapheleuaspvalthraspargile130135140alaglnthrleuleuasnleualalysglnproaspalametthrhis145150155160proaspglymetglnilelysilethrargglngluileglyglnile165170175valglycysserarggluthrvalglyargileleulysmetleuglu180185190aspglnasnleuileseralahisglylysthrilevalvaltyrgly195200205thrarg210<210>93<211>210<212>prt<213>人工的<220><223>crp*突变体<400>93metvalleuglylysproglnthraspprothrleuglutrppheleu151015serhiscyshisilehislystyrproserlysserthrleuilehis202530glnglyglulysalagluthrleutyrtyrilevallysglyserval354045alavalleuilelysaspglugluglylysglumetileleusertyr505560leuasnglnglyasppheileglygluleuglyleuphegluglugly65707580glngluargseralatrpvalargalalysthralacysgluvalala859095gluilesertyrlyslyspheargglnleuileglnvalasnproasp100105110ileleumetargleuseralaglnmetalaargargleuglnvalthr115120125serglulysvalglyasnleualapheleuaspvalthrglyaspile130135140alaglnthrleuleuasnleualalysglnproaspalametthrhis145150155160proaspglymetglnilelysilethrargglngluileglyglnile165170175valglycysserarggluthrvalglyargileleulysmetleuglu180185190aspglnasnleuileseralahisglylysthrilevalvaltyrgly195200205thrarg210<210>94<211>2932<212>dna<213>大肠杆菌(escherichiacoli)<400>94atgcgaattggcataccaagagaacggttaaccaatgaaacccgtgttgcagcaacgcca60aaaacagtggaacagctgctgaaactgggttttaccgtcgcggtagagagcggcgcgggt120caactggcaagttttgacgataaagcgtttgtgcaagcgggcgctgaaattgtagaaggg180aatagcgtctggcagtcagagatcattctgaaggtcaatgcgccgttagatgatgaaatt240gcgttactgaatcctgggacaacgctggtgagttttatctggcctgcgcagaatccggaa300ttaatgcaaaaacttgcggaacgtaacgtgaccgtgatggcgatggactctgtgccgcgt360atctcacgcgcacaatcgctggacgcactaagctcgatggcgaacatcgccggttatcgc420gccattgttgaagcggcacatgaatttgggcgcttctttaccgggcaaattactgcggcc480gggaaagtgccaccggcaaaagtgatggtgattggtgcgggtgttgcaggtctggccgcc540attggcgcagcaaacagtctcggcgcgattgtgcgtgcattcgacacccgcccggaagtg600aaagaacaagttcaaagtatgggcgcggaattcctcgagctggattttaaagaggaagct660ggcagcggcgatggctatgccaaagtgatgtcggacgcgttcatcaaagcggaaatggaa720ctctttgccgcccaggcaaaagaggtcgatatcattgtcaccaccgcgcttattccaggc780aaaccagcgccgaagctaattacccgtgaaatggttgactccatgaaggcgggcagtgtg840attgtcgacctggcagcccaaaacggcggcaactgtgaatacaccgtgccgggtgaaatc900ttcactacggaaaatggtgtcaaagtgattggttataccgatcttccgggccgtctgccg960acgcaatcctcacagctttacggcacaaacctcgttaatctgctgaaactgttgtgcaaa1020gagaaagacggcaatatcactgttgattttgatgatgtggtgattcgcggcgtgaccgtg1080atccgtgcgggcgaaattacctggccggcaccgccgattcaggtatcagctcagccgcag1140gcggcacaaaaagcggcaccggaagtgaaaactgaggaaaaatgtacctgctcaccgtgg1200cgtaaatacgcgttgatggcgctggcaatcattctttttggctggatggcaagcgttgcg1260ccgaaagaattccttgggcacttcaccgttttcgcgctggcctgcgttgtcggttattac1320gtggtgtggaatgtatcgcacgcgctgcatacaccgttgatgtcggtcaccaacgcgatt1380tcagggattattgttgtcggagcactgttgcagattggccagggcggctgggttagcttc1440cttagttttatcgcggtgcttatagccagcattaatattttcggtggcttcaccgtgact1500cagcgcatgctgaaaatgttccgcaaaaattaaggggtaacatatgtctggaggattagt1560tacagctgcatacattgttgccgcgatcctgtttatcttcagtctggccggtctttcgaa1620acatgaaacgtctcgccagggtaacaacttcggtatcgccgggatggcgattgcgttaat1680cgcaaccatttttggaccggatacgggtaatgttggctggatcttgctggcgatggtcat1740tggtggggcaattggtatccgtctggcgaagaaagttgaaatgaccgaaatgccagaact1800ggtggcgatcctgcatagcttcgtgggtctggcggcagtgctggttggctttaacagcta1860tctgcatcatgacgcgggaatggcaccgattctggtcaatattcacctgacggaagtgtt1920cctcggtatcttcatcggggcggtaacgttcacgggttcggtggtggcgttcggcaaact1980gtgtggcaagatttcgtctaaaccattgatgctgccaaaccgtcacaaaatgaacctggc2040ggctctggtcgtttccttcctgctgctgattgtatttgttcgcacggacagcgtcggcct2100gcaagtgctggcattgctgataatgaccgcaattgcgctggtattcggctggcatttagt2160cgcctccatcggtggtgcagatatgccagtggtggtgtcgatgctgaactcgtactccgg2220ctgggcggctgcggctgcgggctttatgctcagcaacgacctgctgattgtgaccggtgc2280gctggtcggttcttcgggggctatcctttcttacattatgtgtaaggcgatgaaccgttc2340ctttatcagcgttattgcgggtggtttcggcaccgacggctcttctactggcgatgatca2400ggaagtgggtgagcaccgcgaaatcaccgcagaagagacagcggaactgctgaaaaactc2460ccattcagtgatcattactccggggtacggcatggcagtcgcgcaggcgcaatatcctgt2520cgctgaaattactgagaaattgcgcgctcgtggtattaatgtgcgtttcggtatccaccc2580ggtcgcggggcgtttgcctggacatatgaacgtattgctggctgaagcaaaagtaccgta2640tgacatcgtgctggaaatggacgagatcaatgatgactttgctgataccgataccgtact2700ggtgattggtgctaacgatacggttaacccggcggcgcaggatgatccgaagagtccgat2760tgctggtatgcctgtgctggaagtgtggaaagcgcagaacgtgattgtctttaaacgttc2820gatgaacactggctatgctggtgtgcaaaacccgctgttcttcaaggaaaacacccacat2880gctgtttggtgacgccaaagccagcgtggatgcaatcctgaaagctctgtaa2932<210>95<211>1401<212>dna<213>大肠杆菌(escherichiacoli)<400>95atgccacattcctacgattacgatgccatagtaataggttccggccccggcggcgaaggc60gctgcaatgggcctggttaagcaaggtgcgcgcgtcgcagttatcgagcgttatcaaaat120gttggcggcggttgcacccactggggcaccatcccgtcgaaagctctccgtcacgccgtc180agccgcattatagaattcaatcaaaacccactttacagcgaccattcccgactgctccgc240tcttcttttgccgatatccttaaccatgccgataacgtgattaatcaacaaacgcgcatg300cgtcagggattttacgaacgtaatcactgtgaaatattgcagggaaacgctcgctttgtt360gacgagcatacgttggcgctggattgcccggacggcagcgttgaaacactaaccgctgaa420aaatttgttattgcctgcggctctcgtccatatcatccaacagatgttgatttcacccat480ccacgcatttacgacagcgactcaattctcagcatgcaccacgaaccgcgccatgtactt540atctatggtgctggagtgatcggctgtgaatatgcgtcgatcttccgcggtatggatgta600aaagtggatctgatcaacacccgcgatcgcctgctggcatttctcgatcaagagatgtca660gattctctctcctatcacttctggaacagtggcgtagtgattcgtcacaacgaagagtac720gagaagatcgaaggctgtgacgatggtgtgatcatgcatctgaagtcgggtaaaaaactg780aaagctgactgcctgctctatgccaacggtcgcaccggtaataccgattcgctggcgtta840cagaacattgggctagaaactgacagccgcggacagctgaaggtcaacagcatgtatcag900accgcacagccacacgtttacgcggtgggcgacgtgattggttatccgagcctggcgtcg960gcggcctatgaccaggggcgcattgccgcgcaggcgctggtaaaaggcgaagccaccgca1020catctgattgaagatatccctaccggtatttacaccatcccggaaatcagctctgtgggc1080aaaaccgaacagcagctgaccgcaatgaaagtgccatatgaagtgggccgcgcccagttt1140aaacatctggcacgcgcacaaatcgtcggcatgaacgtgggcacgctgaaaattttgttc1200catcgggaaacaaaagagattctgggtattcactgctttggcgagcgcgctgccgaaatt1260attcatatcggtcaggcgattatggaacagaaaggtggcggcaacactattgagtacttc1320gtcaacaccacctttaactacccgacgatggcggaagcctatcgggtagctgcgttaaac1380ggtttaaaccgcctgttttaa1401<210>96<211>1434<212>dna<213>变形链球菌(streptococcusmutans)<400>96atggcaatgacaaaacaatataaaaattatgtcaatggcgagtggaagctttcagaaaat60gaaattaaaatctacgaaccagccagtggagctgaattgggttcagttccagcaatgagt120actgaagaagtagattatgtttatgcttcagccaagaaagctcaaccagcttggcgagca180ctttcatacatagaacgtgctgcctaccttcataaggtagcagatattttgatgcgtgat240aaagaaaaaataggtgctattctttccaaagaggttgctaaaggttataaatcagcagtc300agcgaagttgttcgtactgcagaaatcattaattatgcagctgaagaaggtcttcgtatg360gaaggtgaagtccttgaaggcggcagttttgaagcagccagcaagaaaaaaattgccgtt420gttcgtcgtgaaccagtaggtcttgtattagctatttcaccatttaactaccctgttaac480ttggcaggttcgaaaattgcaccggctcttattgcgggaaatgttattgcttttaaacca540ccgacgcaaggatcaatctcagggctcttacttgctgaagcatttgctgaagctggactt600cctgcaggtgtctttaataccattacaggtcgtggttctgaaattggagactatattgta660gaacatcaagccgttaactttatcaatttcactggttcaacaggaattggcgaacgtatt720ggcaaaatggctggtatgcgtccgattatgcttgaactcggtggaaaagattcagccatc780gttcttgaagatgcggaccttgaattgactgctaaaaatattattgcaggtgcttttggt840tattcaggtcaacgctgtacagcagttaaacgtgttcttgtgatggaaagtgttgctgat900gaactggtcgaaaaaatccgtgaaaaagttcttgcattaacaattggtaatccagaagac960gatgcagatattacaccgttgattgatacaaaatcagctgattatgtagaaggtcttatt1020aatgatgccaatgataaaggagccactgcccttactgaaatcaaacgtgaaggtaatctt1080atctgtccaatcctctttgataaggtaacgacagatatgcgtcttgcttgggaagaacca1140tttggtcctgttcttccgatcattcgtgtgacatctgtagaagaagccattgaaatttct1200aacaaatcggaatatggacttcaggcttctatctttacaaatgatttcccacgcgctttt1260ggtattgctgagcagcttgaagttggtacagttcatatcaataataagacacagcgcggc1320acggacaacttcccattcttaggggctaaaaaatcaggtgcaggtattcaaggggtaaaa1380tattctattgaagctatgacaactgttaaatccgtcgtatttgatatcaaataa1434<210>97<211>996<212>dna<213>大肠杆菌(escherichiacoli)<400>97atgactatcaaagtaggtatcaacggttttggccgtatcggtcgcattgttttccgtgct60gctcagaaacgttctgacatcgagatcgttgcaatcaacgacctgttagacgctgattac120atggcatacatgctgaaatatgactccactcacggccgtttcgacggtaccgttgaagtg180aaagacggtcatctgatcgttaacggtaaaaaaatccgtgttaccgctgaacgtgatccg240gctaacctgaaatgggacgaagttggtgttgacgttgtcgctgaagcaactggtctgttc300ctgactgacgaaactgctcgtaaacacatcaccgctggtgcgaagaaagtggttatgact360ggtccgtctaaagacaacactccgatgttcgttaaaggcgctaacttcgacaaatatgct420ggccaggacatcgtttccaacgcttcctgcaccaccaactgcctggctccgctggctaaa480gttatcaacgataacttcggcatcatcgaaggtctgatgaccaccgttcacgctactacc540gctactcagaaaaccgttgatggcccgtctcacaaagactggcgcggcggccgcggcgct600tcccagaacatcatcccgtcctctaccggtgctgctaaagctgtaggtaaagtactgcca660gaactgaatggcaaactgactggtatggcgttccgcgttccgaccccgaacgtatctgta720gttgacctgaccgttcgtctggaaaaagctgcaacttacgagcagatcaaagctgccgtt780aaagctgctgctgaaggcgaaatgaaaggcgttctgggctacaccgaagatgacgtagta840tctaccgatttcaacggcgaagtttgcacttccgtgttcgatgctaaagctggtatcgct900ctgaacgacaacttcgtgaaactggtatcctggtacgacaacgaaaccggttactccaac960aaagttctggacctgatcgctcacatctccaaataa996<210>98<211>1650<212>dna<213>大肠杆菌(escherichiacoli)<400>98atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgat60gaaatgaaagacgttacgatcgccgatctttttgctaaagacggcgatcgtttttctaag120ttctccgcaaccttcgacgatcagatgctggtggattactccaaaaaccgcatcactgaa180gagacgctggcgaaattacaggatctggcgaaagagtgcgatctggcgggcgcgattaag240tcgatgttctctggcgagaagatcaaccgcactgaaaaccgcgccgtgctgcacgtagcg300ctgcgtaaccgtagcaataccccgattttggttgatggcaaagacgtaatgccggaagtc360aacgcggtgctggagaagatgaaaaccttctcagaagcgattatttccggtgagtggaaa420ggttataccggcaaagcaatcactgacgtagtgaacatcgggatcggcggttctgacctc480ggcccatacatggtgaccgaagctctgcgtccgtacaaaaaccacctgaacatgcacttt540gtttctaacgtcgatgggactcacatcgcggaagtgctgaaaaaagtaaacccggaaacc600acgctgttcttggtagcatctaaaaccttcaccactcaggaaactatgaccaacgcccat660agcgcgcgtgactggttcctgaaagcggcaggtgatgaaaaacacgttgcaaaacacttt720gcggcgctttccaccaatgccaaagccgttggcgagtttggtattgatactgccaacatg780ttcgagttctgggactgggttggcggccgttactctttgtggtcagcgattggcctgtcg840attgttctctccatcggctttgataacttcgttgaactgctttccggcgcacacgcgatg900gacaagcatttctccaccacgcctgccgagaaaaacctgcctgtactgctggcgctgatt960ggcatctggtacaacaatttctttggtgcggaaactgaagcgattctgccgtatgaccag1020tatatgcaccgtttcgcggcgtacttccagcagggcaatatggagtccaacggtaagtat1080gttgaccgtaacggtaacgttgtggattaccagactggcccgattatctggggtgaacca1140ggcactaacggtcagcacgcgttctaccagctgatccaccagggaaccaaaatggtaccg1200tgcgatttcatcgctccggctatcacccataacccgctctctgatcatcaccagaaactg1260ctgtctaacttcttcgcccagaccgaagcgctggcgtttggtaaatcccgcgaagtggtt1320gagcaggaatatcgtgatcagggtaaagatccggcaacgcttgactacgtggtgccgttc1380aaagtattcgaaggtaaccgcccgaccaactccatcctgctgcgtgaaatcactccgttc1440agcctgggtgcgttgattgcgctgtatgagcacaaaatctttactcagggcgtgatcctg1500aacatcttcaccttcgaccagtggggcgtggaactgggtaaacagctggcgaaccgtatt1560ctgccagagctgaaagatgataaagaaatcagcagccacgatagctcgaccaatggtctg1620attaaccgctataaagcgtggcgcggttaa1650<210>99<211>963<212>dna<213>大肠杆菌(escherichiacoli)<400>99atgattaagaaaatcggtgtgttgacaagcggcggtgatgcgccaggcatgaacgccgca60attcgcggggttgttcgttctgcgctgacagaaggtctggaagtaatgggtatttatgac120ggctatctgggtctgtatgaagaccgtatggtacagctagaccgttacagcgtgtctgac180atgatcaaccgtggcggtacgttcctcggttctgcgcgtttcccggaattccgcgacgag240aacatccgcgccgtggctatcgaaaacctgaaaaaacgtggtatcgacgcgctggtggtt300atcggcggtgacggttcctacatgggtgcaatgcgtctgaccgaaatgggcttcccgtgc360atcggtctgccgggcactatcgacaacgacatcaaaggcactgactacactatcggtttc420ttcactgcgctgagcaccgttgtagaagcgatcgaccgtctgcgtgacacctcttcttct480caccagcgtatttccgtggtggaagtgatgggccgttattgtggagatctgacgttggct540gcggccattgccggtggctgtgaattcgttgtggttccggaagttgaattcagccgtgaa600gacctggtaaacgaaatcaaagcgggtatcgcgaaaggtaaaaaacacgcgatcgtggcg660attaccgaacatatgtgtgatgttgacgaactggcgcatttcatcgagaaagaaaccggt720cgtgaaacccgcgcaactgtgctgggccacatccagcgcggtggttctccggtgccttac780gaccgtattctggcttcccgtatgggcgcttacgctatcgatctgctgctggcaggttac840ggcggtcgttgtgtaggtatccagaacgaacagctggttcaccacgacatcatcgacgct900atcgaaaacatgaagcgtccgttcaaaggtgactggctggactgcgcgaaaaaactgtat960taa963<210>100<211>1476<212>dna<213>大肠杆菌(escherichiacoli)<400>100atggcggtaacgcaaacagcccaggcctgtgacctggtcattttcggcgcgaaaggcgac60cttgcgcgtcgtaaattgctgccttccctgtatcaactggaaaaagccggtcagctcaac120ccggacacccggattatcggcgtagggcgtgctgactgggataaagcggcatataccaaa180gttgtccgcgaggcgctcgaaactttcatgaaagaaaccattgatgaaggtttatgggac240accctgagtgcacgtctggatttttgtaatctcgatgtcaatgacactgctgcattcagc300cgtctcggcgcgatgctggatcaaaaaaatcgtatcaccattaactactttgccatgccg360cccagcacttttggcgcaatttgcaaagggcttggcgaggcaaaactgaatgctaaaccg420gcacgcgtagtcatggagaaaccgctggggacgtcgctggcgacctcgcaggaaatcaat480gatcaggttggcgaatacttcgaggagtgccaggtttaccgtatcgaccactatcttggt540aaagaaacggtgctgaacctgttggcgctgcgttttgctaactccctgtttgtgaataac600tgggacaatcgcaccattgatcatgttgagattaccgtggcagaagaagtggggatcgaa660gggcgctggggctattttgataaagccggtcagatgcgcgacatgatccagaaccacctg720ctgcaaattctttgcatgattgcgatgtctccgccgtctgacctgagcgcagacagcatc780cgcgatgaaaaagtgaaagtactgaagtctctgcgccgcatcgaccgctccaacgtacgc840gaaaaaaccgtacgcgggcaatatactgcgggcttcgcccagggcaaaaaagtgccggga900tatctggaagaagagggcgcgaacaagagcagcaatacagaaactttcgtggcgatccgc960gtcgacattgataactggcgctgggccggtgtgccattctacctgcgtactggtaaacgt1020ctgccgaccaaatgttctgaagtcgtggtctatttcaaaacacctgaactgaatctgttt1080aaagaatcgtggcaggatctgccgcagaataaactgactatccgtctgcaacctgatgaa1140ggcgtggatatccaggtactgaataaagttcctggccttgaccacaaacataacctgcaa1200atcaccaagctggatctgagctattcagaaacctttaatcagacgcatctggcggatgcc1260tatgaacgtttgctgctggaaaccatgcgtggtattcaggcactgtttgtacgtcgcgac1320gaagtggaagaagcctggaaatgggtagactccattactgaggcgtgggcgatggacaat1380gatgcgccgaaaccgtatcaggccggaacctggggacccgttgcctcggtggcgatgatt1440acccgtgatggtcgttcctggaatgagtttgagtaa1476<210>101<211>474<212>prt<213>大肠杆菌(escherichiacoli)<400>101metserthrgluilelysthrglnvalvalvalleuglyalaglypro151015alaglytyrseralaalapheargcysalaaspleuglyleugluthr202530valilevalgluargtyrasnthrleuglyglyvalcysleuasnval354045glycysileproserlysalaleuleuhisvalalalysvalileglu505560glualalysalaleualagluhisglyilevalpheglygluprolys65707580thraspileasplysileargthrtrplysglulysvalileasngln859095leuthrglyglyleualaglymetalalysglyarglysvallysval100105110valasnglyleuglylysphethrglyalaasnthrleugluvalglu115120125glygluasnglylysthrvalileasnpheaspasnalaileileala130135140alaglyserargproileglnleupropheileprohisgluasppro145150155160argiletrpaspserthraspalaleugluleulysgluvalproglu165170175argleuleuvalmetglyglyglyileileglyleuglumetglythr180185190valtyrhisalaleuglyserglnileaspvalvalglumetpheasp195200205glnvalileproalaalaasplysaspilevallysvalphethrlys210215220argileserlyslyspheasnleumetleugluthrlysvalthrala225230235240valglualalysgluaspglyiletyrvalthrmetgluglylyslys245250255alaproalagluproglnargtyraspalavalleuvalalailegly260265270argvalproasnglylysasnleuaspalaglylysalaglyvalglu275280285valaspaspargglypheileargvalasplysglnleuargthrasn290295300valprohisilephealaileglyaspilevalglyglnprometleu305310315320alahislysglyvalhisgluglyhisvalalaalagluvalileala325330335glylyslyshistyrpheaspprolysvalileproserilealatyr340345350thrgluprogluvalalatrpvalglyleuthrglulysglualalys355360365glulysglyilesertyrgluthralathrpheprotrpalaalaser370375380glyargalailealaseraspcysalaaspglymetthrlysleuile385390395400pheasplysgluserhisargvalileglyglyalailevalglythr405410415asnglyglygluleuleuglygluileglyleualaileglumetgly420425430cysaspalagluaspilealaleuthrilehisalahisprothrleu435440445hisgluservalglyleualaalagluvalphegluglyserilethr450455460aspleuproasnprolysalalyslyslys465470<210>102<211>474<212>prt<213>人工的<220><223>lpd突变体<400>102metserthrgluilelysthrglnvalvalvalleuglyalaglypro151015alaglytyrseralaalapheargcysalaaspleuglyleugluthr202530valilevalgluargtyrasnthrleuglyglyvalcysleuasnval354045glycysileproserlysalaleuleuhisvalalalysvalileglu505560glualalysalaleualagluhisglyilevalpheglygluprolys65707580thraspileasplysileargthrtrplysglulysvalileasngln859095leuthrglyglyleualaglymetalalysglyarglysvallysval100105110valasnglyleuglylysphethrglyalaasnthrleugluvalglu115120125glygluasnglylysthrvalileasnpheaspasnalaileileala130135140alaglyserargproileglnleupropheileprohisgluasppro145150155160argiletrpaspserthraspalaleugluleulysgluvalproglu165170175argleuleuvalmetglyglyglyileilealaleuglumetalathr180185190valtyrhisalaleuglyserglnileaspvalvalvalarglyshis195200205glnvalileargalaalaasplysaspilevallysvalphethrlys210215220argileserlyslyspheasnleumetleugluthrlysvalthrala225230235240valglualalysgluaspglyiletyrvalthrmetgluglylyslys245250255alaproalagluproglnargtyraspalavalleuvalalailegly260265270argvalproasnglylysasnleuaspalaglylysalaglyvalglu275280285valaspaspargglypheileargvalasplysglnleuargthrasn290295300valprohisilephealaileglyaspilevalglyglnprometleu305310315320alahislysglyvalhisgluglyhisvalalaalagluvalileala325330335glylyslyshistyrpheaspprolysvalileproserilealatyr340345350thrgluprogluvalalatrpvalglyleuthrglulysglualalys355360365glulysglyilesertyrgluthralathrpheprotrpalaalaser370375380glyargalailealaseraspcysalaaspglymetthrlysleuile385390395400pheasplysgluserhisargvalileglyglyalailevalglythr405410415asnglyglygluleuleuglygluileglyleualaileglumetgly420425430cysaspalagluaspilealaleuthrilehisalahisprothrleu435440445hisgluservalglyleualaalagluvalphegluglyserilethr450455460aspleuproasnprolysalalyslyslys465470<210>103<211>1548<212>dna<213>大肠杆菌(escherichiacoli)<400>103atgacggaccatacaatgaagaaaaaccccgtaagtataccacacaccgtctggtacgcc60gacgatatccgccgcggagaacgcgaggcggcagatgtgctggggctcacactctatgag120ctgatgcttcgcgctggcgaggccgcattccaggtgtgtcgttcggcgtatcctgacgcc180cgccactggctggtgctgtgcggtcatggtaataacggcggcgatggctacgtggtcgcg240cgactggccaaagcggtcggcattgaggtcacgttgttggcccaggagagcgacaaaccg300ttgccggaagaggccgcgctggcacgcgaagcatggttaaacgcgggtggcgagatccat360gcttcgaatattgtctggcccgaatcggtagatctgattgttgatgcgctgctcggtacc420ggtttgcggcaagcgccccgcgaatccattagccagttaatcgaccacgctaattcccat480cctgcgccgattgtggcggttgatatcccttccggcctgctggctgaaactggcgctacg540ccaggcgcggtgatcaacgccgatcacaccatcacttttattgcgctgaaaccaggcttg600ctcactggaaaagcgcgggatgttaccggacaactgcattttgactcactggggctggat660agttggctggcaggtcaggagacgaaaattcagcggttttcagcagaacaactttctcac720tggctaaaaccgcgtcgcccgacttcgcataaaggcgatcacgggcggctggtaattatc780ggtggcgatcacggcacggcgggggctattcgtatgacgggggaagcggcgctgcgtgct840ggtgctggtttagtccgagtactgacccgcagtgaaaacattgcgccgctgctgactgca900cgaccggaattgatggtgcatgaactgacgatggactctcttaccgaaagcctggaatgg960gccgatgtggtggtgattggtcccggtctgggccagcaagagtgggggaaaaaagcactg1020caaaaagttgagaattttcgcaaaccgatgttgtgggatgccgatgcattgaacctgctg1080gcaatcaatcccgataagcgtcacaatcgcgtgatcacgccgcatcctggcgaggccgca1140cggttgttaggctgttccgtcgctgaaattgaaagtgaccgcttacattgcgccaaacgt1200ctggtacaacgttatggcggcgtagcggtgctgaaaggtgccggaaccgtggtcgccgcc1260catcctgacgctttaggcattattgatgccggaaatgcaggcatggcgagcggcggcatg1320ggcgatgtgctctctggtattattggcgcattgcttgggcaaaaactgtcgccgtatgat1380gcagcctgtgcaggctgtgtcgcgcacggtgcggcagctgacgtactggcggcgcgtttt1440ggaacgcgcgggatgctggcaaccgatctcttttccacgctacagcgtattgttaacccg1500gaagtgactgataaaaaccatgatgaatcgagtaattccgctccctga1548<210>104<211>50<212>dna<213>人工的<220><223>引物缺失xylab1<400>104acgacatcatccatcacccgcggcattacctgattatggagttcaatatg50<210>105<211>50<212>dna<213>人工的<220><223>引物缺失xylab2<400>105cccccacccggtcaggcaggggataacgtttacgccattaatggcagaag50<210>106<211>50<212>dna<213>人工的<220><223>引物缺失yjhh1<400>106aatgcgcgaagttgccgacttcctgattaataaaggggtcgacgggctgt50<210>107<211>50<212>dna<213>人工的<220><223>引物缺失yfhh2<400>107gtaccgacttaactgtgttgatcatcgtacgcaagtgaccaacgctgtcg50<210>108<211>50<212>dna<213>人工的<220><223>引物缺失yage1<400>108ggcggcaccaacgcccgggaaaccatcgaactcagccagcacgcgcagca50<210>109<211>50<212>dna<213>人工的<220><223>引物缺失yage2<400>109agcacggtgaagtgcggatgggcacctttgacggtatggatcatgctgcg50<210>110<211>50<212>dna<213>人工的<220><223>引物缺失yiae1<400>110catatttcaggctaaggtgatcgccttatcagtgaatggagagaagcatg50<210>111<211>50<212>dna<213>人工的<220><223>引物缺失yiae2<400>111tatcgggctttactctacgcagtcgcggcttagtccgcgacgtgcggatt50<210>112<211>50<212>dna<213>人工的<220><223>引物缺失ycdw1<400>112aacgataagtgcgaataaatttcgcacaacgcttttcgggagtcagtatg50<210>113<211>50<212>dna<213>人工的<220><223>引物缺失ycdw2<400>113ccaaggatagcaggaatcctgatgctttattagtagccgcgtgcgcggtc50<210>114<211>100<212>dna<213>人工的<220><223>引物缺失ptsg1<400>114atgtttaagaatgcatttgctaacctgcaaaaggtcggtaaatcgctgatgctgccggta60tccgtactgcctatcgcaggtgtaggctggagctgcttcg100<210>115<211>100<212>dna<213>人工的<220><223>引物缺失ptsg2<400>115ttagtggttacggatgtactcatccatctcggttttcaggttatcggatttagtaccgaa60aatcgcctgaacaccagaaccatatgaatatcctccttag100<210>116<211>21<212>dna<213>人工的<220><223>rbs120<400>116atccggtataggaggtataga21<210>117<211>100<212>dna<213>人工的<220><223>引物缺失udha1<400>117ggtgcgcgcgtcgcagttatcgagcgttatcaaaatgttggcggcggttgcacccactgg60ggcaccatcccgtcgaaagccatatgaatatcctccttag100<210>118<211>100<212>dna<213>人工的<220><223>引物缺失udha2<400>118cccagaatctcttttgtttcccgatggaacaaaattttcagcgtgcccacgttcatgccg60acgatttgtgcgcgtgccagtgtaggctggagctgcttcg100<210>119<211>101<212>dna<213>人工的<220><223>引物缺失pfka1<400>119gttcctcggttctgcgcgtttcccggaattccgcgacgagaacatccgcgccgtggctat60cgaaaacctgaaaaaacgtggtgtaggctggagctgcttcg101<210>120<211>102<212>dna<213>人工的<220><223>引物缺失pfka2<400>120ggcctgataagcgaagcgcatcaggcatttttgcttctgtcatcggtttcagggtaaagg60aatctgcctttttccgaaatcacatatgaatatcctccttag102<210>121<211>83<212>dna<213>人工的<220><223>pr01<400>121acgttaaatctatcaccgcaagggataaatatctaacaccgtgcgtgttgacaattttac60ctctggcggtgataatggttgca83<210>122<211>714<212>dna<213>人工的<220><223>ci857<400>122atgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaa60gcaatttatgaaaaaaagaaaaatgaacttggcttatcccaggaatctgtcgcagacaag120atggggatggggcagtcaggcgttggtgctttatttaatggcatcaatgcattaaatgct180tataacgccgcattgcttacaaaaattctcaaagttagcgttgaagaatttagcccttca240atcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagt300gagtatgagtaccctgttttttctcatgttcaggcagggatgttctcacctaagcttaga360acctttaccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattct420gcattctggcttgaggttgaaggtaattccatgaccgcaccaacaggctccaagccaagc480tttcctgacggaatgttaattctcgttgaccctgagcaggctgttgagccaggtgatttc540tgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggatagcggt600caggtgtttttacaaccactaaacccacagtacccaatgatcccatgcaatgagagttgt660tccgttgtggggaaagttatcgctagtcagtggcctgaagagacgtttggctga714<210>123<211>34<212>dna<213>人工的<220><223>rbs150<400>123taccaactaacgcacgtttaagtaggaaccgtat34<210>124<211>50<212>dna<213>人工的<220><223>由gapn代替gapa<400>124acgtgactgattctaacaaaacattaacaccaactggcaaaattttgtcc50<210>125<211>50<212>dna<213>人工的<220><223>由gapn代替gapa<400>125aaaaaagagcgaccgaagtcgctctttttagatcacagtgtcatctcaac50<210>126<211>43<212>dna<213>人工的<220><223>ptrc<400>126gagctgttgacaattaatcatccggctcgtataatgtgtggaa43<210>127<211>50<212>dna<213>人工的<220><223>引物代替xylfgh启动子1<400>127ctaaaaattggttacgtttatcgcggtgattgttacttattaaaactgtc50<210>128<211>50<212>dna<213>人工的<220><223>引物代替xylfgh启动子2<400>128tgagtagaatgttctttattttcatggtgtagggccttctgtagttagag50当前第1页12