CN1178834A - 生物素生物合成基因ⅱ - Google Patents
生物素生物合成基因ⅱ Download PDFInfo
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- CN1178834A CN1178834A CN97119679A CN97119679A CN1178834A CN 1178834 A CN1178834 A CN 1178834A CN 97119679 A CN97119679 A CN 97119679A CN 97119679 A CN97119679 A CN 97119679A CN 1178834 A CN1178834 A CN 1178834A
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Abstract
本发明涉及利用基因工程生物体通过发酵产生生物素的方法、DNA序列以及用于这种方法的载体。
Description
本发明涉及利用基因工程有机体通过发酵产生生物素的方法。
生物素是动物、植物和微生物营养的必需维生素之一,而且对于医药或者是食品添加剂也是十分重要的。
充分研究了大肠杆菌的生物素生物合成,并且阐明生物素是从pimelyl辅酶A经7-酮基-8-氨基壬酸(KAPA)、7,8-二氨基壬酸(DAPA)和脱硫生物素(DTB)合成的[大肠杆菌和沙门氏菌,细胞和分子生物学,544,(1987)]。在大肠杆菌[生物化学杂志,263,19577,(1988)]和圆形芽孢杆菌上(美国专利5096823)和生物素的生物合成相关的遗传信息分析已有很好的进展。至少知道了这个生物合成途径包括的四种酶。这四种酶是由bioA、bioB、bioD和bioF基因编码。bioF基因编码催化把pimelyl辅酶A转化为KAPA的KAPA合成酶。bioA基因编码把KAPA转化成为DAPA的DAPA转氨酶。bioD基因编码把DAPA转化成为DTB的DTB合成酶。bioB基因编码把DTB转化成为生物素的生物素合酶。同时也报告了在大肠杆菌中参与pimelyl辅酶A合成的bioC和bioH基因。
进行了许多发酵产生生物素的研究。利用了大肠杆菌(日本专利公开149091/1986和日本专利公开155081/1987)、圆形芽孢杆菌(日本专利公开180174/1991)、粘质沙雷氏菌(日本专利公开27980/1990)和Brevibacterium flavum(日本专利公开240489/1991)。但是由于它们的生产力低,这些方法还不适合用于工业生产过程。此外,大量的DTB(生物素前体)在这些细菌的发酵中积累。因此,已经假定生物素生物合成的最后步骤(从DTB到生物素)是一个限速步骤。
另一方面,发现属于库尔特氏杆菌属的细菌菌株产生DBT和少量的生物素。而且通过对生物素抗代谢物酸酶素(ACM)的抗性、5-(2-噻吩基)-颉草酸(TVA)和α-甲基脱硫生物素(MeDTB)的抗性的选择,从库尔特氏杆菌属的野生型菌株中分离出产生大量生物素的突变体。然而,鉴于仍然低的生物素值,最好是运用基因工程提高这种突变体的生物素产量。
因此本发明涉及携带和Kurthia sp.生物素生物合成相关的基因的染色体DNA片段。此分离的染色体DNA片段携带bioA,bioB,bioC,bioD,bioF,bioFII,bioH和bioHII等8个基因和转录调节序列。bioFII基因编码bioF基因产物的酶。
本发明还涉及Kurthia sp.菌株,其中至少扩增了一个参与生物素生物合成的基因,同时也涉及通过基因工程Kurthia sp.菌株产生生物素的方法。
虽然以上提及的DNA片段可以具有各种起源,但优选的是利用属于库尔特氏杆菌属的菌株。这些菌株的特定例子包括,例如,Kurthia sp.538-6(DSM 9454)和通过选择对生物素抗代谢物的抗性的突变菌株,如Kurthiasp.538-KA26(DSM 10609)。
一般地说,本发明针对编码以SED ID NO.2、4、6、8、10、12、14或16为代表的多肽和这些多肽的包含一个或多个氨基酸残基的加成、插入、缺失和/或取代的功能性衍生物的DNA序列,以及包含一个或多个这些DNA序列的载体,其中所说的DNA序列和启动子序列功能性地连接。本发明的另一个目的是研究由以上所限定的一个或多个DNA序列或载体转化的细胞和产生生物素的方法(包括在培养基中培养以上限定的细胞和用本领域公知的方法从培养基中分离最后产生的生物素),并且具体地说,这种方法是在pH值5至9,优选的是6至8下,于10℃至45℃,优选的是25℃至30℃的温度范围内将培养进行1至10天,优选的是2至7天。
最后本发明的目的也在于研究制备药学、食品或饲料组合物的方法,这种方法的特征在于把通过这些方法获得的生物素与一种或多种通常使用的添加剂混合,本领域的技术人员熟知这种方法。
分离携带编码参与这些细菌菌株进行生物素生物合成的酶之基因的DNA片段的详尽的方法在以下的部分进行描述。
因此通过已知的苯酚法可以把DNA从Kurthia sp.538-KA26提取出来。然后,用SauSAI部分消化这种DNA,并将其连接到用BamHI消化的pBR322;以构建一个Kurthia sp.538-KA26的基因文库。
用以上获得的基因文库转化缺乏生物合成能力的生物素营养缺陷型突变体,以产生生物素的,选择表现为生物素原养型的转化体。在生物素营养缺陷型突变体中选择的转化体具有基因组的DNA片段(这种DNA片断补充了缺失的基因)。大肠杆菌R875(bioB-)、R877(bioD)、BM7086(bioH)和R878(bioC)(细菌学杂志,112,830-839,(1972),和细菌学杂志,143,789-800,(1980))都能用作生物素素营养缺陷型突变体。按照常规的方法,例如感受态细胞法,进行这些大肠杆菌菌株的转化[分子克隆,冷泉港实验室出版社,252,(1982)]。
在本发明中,通过以上所描述的方法获得了补充大肠杆菌bioB缺失突变体的杂交质粒。获得的杂交质粒命名pKB100。pKB100携带一个Kurthiasp.538-KA26的5.58 Kb基因组DNA片段的质粒pBR322相对应,它的限制切割图谱如图1和图2所示。
通过以上所述的方法也获得了命名为pKB200的补充大肠杆菌的bioD缺失突变体的杂交质粒。PKB200和Kurthia sp.538-KA26的携带一个7.87Kb基因组DNA片段的质粒pBR322相对应,它的限制切割图谱如图1和图3所示。pKB200中的基因组DNA片段和pKB100的插入片段完全重叠,并且携带4bioF、bioB、bioD、ORF1和ORF2基因和如图9-A所示的Kurthiasp.538-KA26的一部分bioA基因。通过常规的方法(如菌落杂交)利用在pKB200中一部分基因组DNA片段作为探针,能够分离Kurthia sp.538-KA26的整个bioA基因。用限制性内切酶如HindIII消化Kurthia sp.538-KA26的总DNA,并且用相同的限制性内切酶切割的质粒载体连接。然后,大肠杆菌用携带Kurthia sp.538-KA26的基因组DNA片段的杂交质粒转化以构建基因组文库。pUC19[Takara Shuzo公司(Higashiira,Higashinotohin,Shimogyo-Mu,Kyoto-shi,日本)]和大肠杆菌JM109(Takara Shuzo公司)都能分别用作载体与大肠杆菌菌株。
通过菌落杂交获得了命名为pKB300的携带Kurthia sp.538-KA26的8.44 Kb基因组DNA片段的杂交质粒,它的限制切割图谱如图1所示。在pKB300中的携带参与Kurthia sp.538-KA26的生物素生物合成的两个基因簇的基因组DNA片段如图9-A所示。一个族由ORF1、bioD和bioA基因组成。另一个族由ORF2、bioF和bioB基因组成。bioD和bioA基因的核苷酸序列分别显示在SEQ ID NO.1和SEQ ID NO.3中。推测的bioD和bioA基因的氨基酸序列产物分别显示在SEQ ID NO.2和SEQ ID NO.4中。编码236个氨基酸残基的多肽(分子量为26,642)。bioA基因编码一个460个氨基酸残基的多肽(分子量为51,731)。ORF1基因编码一个194个氨基酸残基的多肽(分子量为21,516),但是此基因的产物的生物功能还不清楚。
BioF和bioB基因的氨基酸序列分别显示在SEQ ID NO.5和SEQ ID NO.7中。推测的BioF和bioB基因产物的氨基酸序列分别显示在SEQ ID NO.6和SEQ ID NO.8中。bioF基因编码一个387个氨基酸残基的多肽(分子量为42,619)。bioB基因编码一个338个氨基酸残基的多肽(分子量为37,438)。ORF2基因编码一个63个氨基酸残基的多肽(分子量为7,447),但是此基因产物的生物功能还不清楚。在bioA和bioB基因的下游发现作为转录终止区信号的反向重复序列。发现在ORF1和ORF2基因之间有两个在两个方向开始转录的转录启动子序列(如图10所示)。而且,在每种启动子序列和翻译起始密码子之间有两个命名为Box1和Box2、参与转录负调节的反向重复序列。
另外,通过以上描述的相同方式得到了补充大肠杆菌生物素营养缺陷型突变体的两个杂交质粒。命名为pKH100的杂交质粒补充bioH缺失突变体,命名为pKC100的杂交质粒补体bioC突变体。pKH100(图4和图5)有一个Kurthia sp.538-KA26的1.91 Kb的基因组DNA片段,其携带在图9-B中所示的由bioH和ORF3基因组成的的基因簇。bioH基因的核苷酸序列和这种基因产物的推测的氨基酸序列分别显示在SEQ ID NO.9和SEQID NO.10中。bioH基因编码一个267个氨基酸残基的多肽(分子量为29,423)。ORF3基因编码一个86个氨基酸残基的多肽(分子量为9,995),但是这种基因产物的生物功能还不知道。如图11所示在bioH基因的上游发现一个启动子序列,在ORF3基因的下游有作为转录终止子的反向重复序列。因为在启动子区域没有如Box1和Box2的反向重复序列,所以可以预见这些基因的表达是不被调节的。
另一方面,pKC100携带Kurthia sp.538-KA26的6.76Kb基因组DNA片段(如图6和图7所示)。在pKC100中的基因组DNA片段携带一个由bioFII、bioHII和bioC基因组成的基因簇,如图9-C所示。bioHII和bioFII基因分别为bioH和bioF基因编码酶的基因,因为bioHII和bioFII基因分别补充了大肠杆菌的bioH缺失和bioFII缺失突变体。bioHII、bioFII和bioC基因的核苷酸序列分别显示在SEQ ID No.11、SEQ ID NO.13和SEQ ID NO.15中。推测的bioHII、bioFII和bioC基因产物的氨基酸序列分别显示在SEQ ID NO.12、SEQ ID NO.14和SEQ ID NO.16中。BioFII基因编码一个398个氨基酸残基的多肽(分子量为44,776)。BioHII基因编码一个248个氨基酸残基的多肽(分子量为28,629)。BioC基因编码一个276个氨基酸残基的多肽(分子量为31,599)。如图12所示在bioFII基因的上游发现一个启动子序列,在启动子区域有一个命名为Box3的反向重复序列上。这些基因的转录终止在位于bioC基因下游的反向重复序列,因为Box3的核苷酸序列和Box1和Box2的核苷酸序列非常类似,所以估计这些基因表达的调节和bioA和bioB基因簇的相同。
更不用说,从以上基因分离出的核苷酸序列和氨基酸的序列在某些情况下可以人工改变,例如,起始密码子GTG或TTG可以转化成为ATG密码子。
因此,本发明也直接针对本发明的多肽的功能性衍生物。在本发明的氨基酸序列的基础上通过添加、插入、缺失和/或取代一个或多个这些序列的氨基酸残基来限定这些功能性衍生物(其中的这些衍生物还具有和本发明的相应的多肽相同类型的酶活性)。利用本领域公知的分析方法或本文以下的描述来测定其活性。也将以通过本领域公知的化学肽合成方法或在本文所公开的DNA序列基础上的重组方式(由Sambrook等公开的、本领域?公知的方法)(分子克隆,冷泉港实验室出版社,纽约,美国,第二版1989)合成这些功能性衍生物。本领域已知在蛋白质和多肽中一般不改变该分子的活性的氨基酸变化,并且,例如由H.Neurath和R.L.HILL在“蛋白质”中描述过(Academix出版社,纽约,1979,尤其参见14页图6)。最普遍发生的变化是:Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、AZa/Thr、Ser/Asn、AlaNal、Ser/Gly、Thy/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Nal、Ala/Glu和Asp/Gly以及相反的变化。
进而,本发明不仅直接针对于如在序列表中公开的DNA序列以及它们的互补链,而且也直接针对于那些包括以下的序列,即在标准的条件下同以上DNA序列或其片段杂交的DNA序列、和由于遗传密码的退化在标准的条件下不能和以上序列杂交但其的确编码具有相同的氨基酸序列多肽的DNA序列。
在此文中杂交的″标准条件″意味着本领域技术人员检测特异性的杂交信号普遍使用的条件,和如Sambrook等(同上)描述的条件或者优选地称为严格杂交和非严格的洗涤条件或者本领域技术人员公知的更优选地称为严格杂交和非严格洗涤条件及Sambrook等(同上)描述的条件。
源于本发明的DNA序列,因为它们和这些DNA序列(参见上文)杂交或者能通过使用以这些DNA序列为基础设计的引物利用聚合酶链反应构建而成,所以可以通过PCR反应以及位点直接诱变[参见Smith,Ann.V.Genet.19,423(1985)]或者如在EP 747 483中描述的合成方法或者通过Sambrook等(同上)中描述的分子克隆常用的方法来制备。
至于用于本发明的表达和/或DNA序列扩增的宿主菌株,任何微生物都可以利用,例如在EP 635 572中鉴别出来的,但更优选的是利用属于库尔特氏杆菌属的菌株,尤其是Kurthia sp.538-6(DSM NO.9454)4和Kurthiasp.538-51F9(DSM NO.10610)。
为了获得高生物素生产力的转化体,在这些宿主细胞中有效的启动子控制下利用了本发明的DNA序列。本发明的DNA序列能通过携带这些DNA序列的质粒转化以引入宿主细胞或者整合到宿主细胞的染色体上。
当Kurthia sp.538-51F9用作宿主细胞时,Kurthia sp.538-51F9可以用从以上Kurthia sp.菌株中分离出来的携带至少一个参与生物素生物合成的基因之杂交质粒来转化。至于用作杂交质粒的载体质粒,pUB110[细菌学杂志,154,1184-1194,(1983)]、pHP13(mol.Gen.Genet.,209,335-342,1987)或在Kurthia sp.菌株中其它的包含复制起点的功能质粒都可以使用。至于在Kurthia sp.中用作扩增和/或表达的DNA序列,本发明的任何DNA序列都能加以利用,但是与编码生物素合酶的bioB基因相应的DNA序列是优选的。这种杂交质粒的实施例是在图14中所示的pYK114。在这种质粒中,bioB基因处于bioH基因的启动子调节下并且携带pUB110的复制起点。
可以利用通过原生质体转化方法获得的以上所述的pYK114转化Kurthia sp.538-51F9[用于芽孢杆菌属的分子生物方法,150,(1990)]。然而,由于Kurthia sp.538-51F9从原生质体的再生效率很低,这种菌株的转化效率十分低。因此,优选的是利用具有由原生质体再生效率高的菌株,如本发明的实施例14中所描述的方法制备的Kurthia sp.538-51F9-RG21。
本发明也提供了通过培养所获得的转化体、分离和纯化产生的生物素来生产生物素的方法。
利用本领域公知的方法培养本发明的转化体。可以利用包含可同化的碳源、可消化的氮源、无机盐和其它的转化体的生长所必需的营养的培养基。至于碳源,如葡萄糖、果糖、乳糖、半乳糖、蔗糖、麦芽糖、淀粉、糊精或者甘油都可以使用。至于氮源,如胨、大豆粉、玉米浆、肉膏、铵硫酸盐、硝酸铵、尿素或者以上各种的混合物都可以使用。并且,至于无机盐,硫酸盐,盐酸硫胺素或者钙、镁、锌、锰、钴和铁的磷酸盐都可以使用。如果需要的话,可以添加常规的营养成分或者消泡剂(如动物油、植物油或者矿物油)。如果所获得的转化体具有抗生素抗性标记,各自的抗生素应该补充到培养基中。培养基的pH值可以在5至9之间,优选的为6-8。培养温度可以是10℃-45℃,优选的是25℃-30℃。培养时间为是1至10天,优选地2至7天。
通过本领域公知的方法很容易地回收以上所述条件下产生的生物素。因此,例如,在固体材料从溶液中除去之后,把在滤液中的生物素吸附在活性碳上,然后洗脱,用离子交换树脂进一步纯化。另外,把滤液直接用离子交换树脂纯化,在洗脱之后,把所要求的产物从酒精和水的混合物中重结晶出来。
在引用下面的实施例详细地解释本发明之前,给出附图的简要描述:
图.1:pKB100、pKB200和PKB300的限制性图谱。
图.2:pKB100的结构。
图.3:pKB200的结构。
图.4:pKH100、pKH101和pKH102的限制性图谱和互补结果。
图.5:pKH100的结构。
图.6:pKC100、pKC101和pKC102的限制性图谱。
图.7:pKC100的结构。
图.8:由pKB100、pKB200和pKB300衍生的质粒的结构。
图.9:参与Kurthia sp.538-KA26生物素生物合成的基因簇基因结构。
图.10:Kurthia sp.538-KA26中ORF1和ORF2基因之间的核苷酸序列。
图.11:bioH基因簇启动子区的核苷酸序列。
图.12:bioFII基因簇启动子区的核苷酸序列。
图.13:穿梭载体pYK1的构建体。
图.14:bioB表达质粒pYK114的构建体。
实施例
实施例1
克隆Kurthia sp.538-KA26的bioB和bioF基因。
1.基因组文库的制备。
把Kurthia sp.538-KA26(DSM NO.10609)的酸霉素抗性菌株培养在100ml的营养肉汤中(Kyokuto Seiyaku CO;Honcho31,Nihonbashi,Chuoh-Ku,东京,日本),在30℃培养过夜,通过离心回收细菌细胞。利用酚提取法[基因熔合试验,冷泉港实验室,137-138,(1984)]从细菌细胞提取总DNA,获得了1.9mg的总DNA。
将总DNA(10μg)用1.2单位的SauSAI在37℃下部分地消化1小时以产生大约10kb长的片段。通过凝胶电泳获得了5-15Kb DNA片段。
将载体pBR322(Takara Shuzo公司)用BamHI完全消化,然后利用碱性磷酸酶处理以避免自身连接。利用DNA连接试剂盒(Takara Shuzo公司)按照厂商的说明,用切割的pBR322连接DNA片段。利用感受态细胞法[分子克隆,冷泉港实验室,252-253,(1982)]把连接混合物转移到大肠杆菌JM109(Takara Shuzo公司)中,在琼脂平面LB培养基(1%Bacto-胰胨、0.5%Bacto-酵母抽提物、0.5%NaCl、pH7.5)上选择对氨苄青霉素(100μg/ml)具有抗性的菌株。大约获得了5000个具有基因组DNA片段的克隆体,把它们作为基因文库。
把Kurthia sp.538-KA26的基因组文库中氨苄青霉素抗性的菌株,在包含100μg/ml氨苄青霉素的50mlLB培养基中37℃培养过夜,通过离心收集细菌细胞。利用碱变性方法[分子克隆,冷泉港实验室,90-91,(1982)]从细菌细胞中提取质粒DNA。
2.从基因文库选择携带bioB基因的克隆。
利用感受态细胞法把质粒DNA转移到大肠杆菌的没有生物素合成酶活性的bioB缺失突变体R875(J.Bacteriol.112,830-839,1972)。转化的大肠杆菌R875细胞用0.85%NaCl洗涤两次,并且平板接种在含有100μg/ml氨苄青霉素的100M9CT培养基(0.6%NaH2PO4、0.3%KH2PO4、0.1%NH4Cl、2mMMgSO4、0.1mMCaCl2、0.2%葡萄糖、0.6%无维生素的酪蛋白氨基酸、1μg/mlthiamin)中,把此平板在37℃下培养40小时。获得了具有生物素原养型表型的转化体。把转化体培养在含有100μg/ml氨苄青霉素的LB培养基上,从细胞中提取杂交质粒。此杂交质粒携带5.58Kb的插入片断,并命名为pKB100。限制性图谱如图1和图2所示。
3.用pKB100补充大肠杆菌生物素缺失突变体。
利用感受态细胞法把pKB100转移到大肠杆菌R875(bioB-)、W602(bioA-)、R878(bioC-)、R877(bioD-)、R874(bioF-)或BM7086(bioH-)[细菌学杂志,112,830-839,(1972),和细菌学杂志,143,789-800,(1980)]的生物素缺失突变体中。转化的突变体用0.85%NaCl洗涤三次,并在包含100μg/ml氨苄青霉素和0.1ng/ml生物素的M9CT琼脂培养基中平板接种,把平板在37℃培养过夜。把平板上的菌落复制在具有100μg/ml氨苄青霉素、存在或缺乏0.1ng/ml生物素的M9CT琼脂平板中,把平板在37℃培养24小时以完成互补作用分析。如表1所示,pKB100不仅能补充bioB突变体,而且能补充bioF突变体。相反,bioA、bioC、bioD和bioH突变体不能由pKB100补充。从这个结果中可以确认pKB100携带Kurthia sp.538-KA26的bioB和bioF基因。
表1
质粒 | 大肠杆菌生物素缺失突变体 | |||||
bioA- | bioB- | bioC- | bioD- | bioF- | bioH- | |
PKB100 | - | + | - | - | + | - |
PKB200 | - | + | - | + | - | - |
PKB300 | + |
PKB100 | - | - | - | - | - | + |
PKB100 | - | - | + | - | + | + |
实施例2
携带Kurthia sp.538-KA26的bioD基因的杂交质粒之分离。
1.携带bioD基因的杂交质粒之分离。
把实施例1-1中的Kurthia sp.538-KA26的基因文库转移到大肠杆菌bioD缺失突变体R877中,以和实施例1-2中所描述的相同方式选择具有氨苄青霉素抗性和生物素原养型表型的转化体。把转化体在具有100μg/ml氨苄青霉素的LB培养基中37℃下培养过夜。然后通过离心收集细菌细胞。利用碱变性方法从细胞中提取质粒DNA。杂交质粒具有7.87Kb插入DNA片段,把它命名为pKB200。利用各种限制性核酸内切酶(HindIII、NcoI、EcoRI、BglII、SalI和PstI)分析pKB200的切割模型,并把它和pKB100的相比较。限制性核酸内切酶分析表明两个杂交质粒有完全相同的切割位点,并且1.5Kb DNA片段延伸到pKB100左侧,0.8Kb片段延伸到pKB200的右侧(图1和图3)。
2.用pKB200补充大肠杆菌生物素缺失突变体。
把pKB200转移到大肠杆菌R875(bioB-)、W602(bioA-)、R878(bioC-)、R877(bioD-)、R874(bioF-)或BM7086(bioH-)的生物素缺失突变体中。利用实施例1-3中所描述的方法进行互补作用分析。pKB200补充bioD和bioB突变体,但是不能补充bioA、bioC、bioF和bioH突变体。尽管pKB200在整个长度的pKB100上重叠,但是它不能补充bioF突变体。
实施例3
携带Kurthia sp.538-KA26bioH基因的杂交质粒的分离。
1.携带bioH基因的杂交质粒之分离。
把实施例1-1中的Kurthia sp.538-KA26的基因文库转移到大肠杆菌bioH缺失突变体BM7086中,以和实施例1-2中所描述的相同方式选择具有bioH克隆的转化体,利用碱变性方法从转化体细胞中提取杂交质粒,并利用限制性核酸内切酶分析杂交质粒。杂交质粒具有1.91Kb的插入DNA片段,把它命名为pKH100。由于以上所利用的基因文库具有5-15Kb基因组DNA片段,所以可以认为在大肠杆菌菌株中pKH100易受到修饰(如缺失)。pKH100的限制性图谱如图4和图5所示。pKH100的断裂模式与pKB100和pKB200断裂模式是完全不同的。因此,pKH100携带了不同于pKB100和pKB200的Kurthia染色体的DNA片段。
2.用pKB200补充大肠杆菌的生物素缺失突变体。
利用实施例1-3中所描述的方法进行互补作用分析。把pKH100转移到大肠杆菌R875(bioB-)、W602(bioA-)、R878(bioC-)、R877(bioD-)、R874(bioF-)或BM7086(bioH-)的生物素缺失突变体中。pKH200只补充bioH和bioB突变体,但是不能补充bioB、bioA、bioC、bioD和bioF突变体(如表1所示)。这样,pKH100只携带了bioH基因。
实施例4
携带Kurthia sp.538-KA26 bioC基因的杂交质粒的分离。
1.携带bioC基因的杂交质粒的分离。
把实施例1-1中的Kurthia sp.538-KA26的基因文库转移到大肠杆菌bioD缺失突变体R878中,以和实施例1-2中所描述的相同的方式选择生物素原养型的、具有bioC克隆的转化体,利用碱变性方法从转化体细胞中提取杂交质粒,并利用限制性核酸内切酶分析杂交质粒。杂交质粒具有一个6.76 Kb的插入DNA片段,把它命名为pKC100。pKC100的限制性图谱如图6和图7所示。pKC100的断裂模式与pKB100、pKB200和pKH100的断裂模式是完全不同的。因此,pKC100携带了不同于pKB100、pKB100和pKH100的Kurthia染色体区域。
2.用pKC100补充大肠杆菌的生物素缺失突变体。
利用实施例1-3中所描述的方法进行互补作用分析。把pKC100转移到大肠杆菌R875(bioB-)、W602(bioA-)、R878(bioC-)、R877(bioD-)、R874(bioF-)或BM7086(bioH-)的生物素缺失突变体中。PKC100补充如表1所示的bioC、bioF和bioH突变体。由于在pKC100中插入的DNA片段不同于pKB100和pKH100中的插入片断,所以pKC100不仅携带了bioC基因,而且携带了bioF基因产物(KAPA合成酶)和bioH基因产物的同工酶基因。
实施例5
携带Kurthia sp.538-KA26 bioA基因的杂交质粒的分离。
1.pKB200中染色体DNA左区的分离。
通过杂交方法,我们从Kurthia sp.538-KA26染色体DNA中分离出pKB200的染色体DNA左区。Kurthia sp.538-KA26的总DNA用HindIII完全消化并且进行琼脂糖凝胶电泳。在凝胶上的DNA片段变性后按照厂商介绍的方法转移到尼龙膜(Hybond-N,Amersham)上。
用NcoI完全消化pKB200,通过琼脂糖凝胶电泳分离出2.1Kb的NcoI片段(图1)。通过多引物DNA标记系统(A mersham)用32P标记NcoI片段并且把它作为杂交探针。利用“快速杂交缓冲液”(Amersham)按照厂商的说明在上面制备的膜上进行杂交。探针同大约8.5Kb的HindIII片段强烈杂交。
为了分离出8.5Kb的片段,整个Kurthia sp.538-KA26的DNA用HindLII完全消化,通过琼脂糖凝胶电泳获得7.5-9.5Kb的DNA片段。pUC19(Takara Shuzo公司)载体质粒用HindIII完全消化,并且用碱性磷酸酶处理以免自身连接。7.5-9.5Kb DNA片段用切开的pUC19通过DNA连接试剂盒(Takara Shuzo公司)连接,利用感受态细胞法把反应混合物转移到大肠杆菌JM109中。获得大约1,000个携带这些基因组DNA片段的单独克隆体,并把它作为基因文库。
按照Maniatis等描述的方案利用菌落杂交的方法进行选择[分子克隆,冷泉港实验室,312-328,(1982)]。在琼脂平板上生长的菌落转移到尼龙膜上(Hybond-N,Amersham),并用碱裂解。变性的DNA固定在膜上。把以上描述制备的32P标记的NcoI片段作为杂交探针,利用“快速杂交缓冲液”(Amersham)按照厂商的说明进行杂交。获得和探针DNA杂交的三个菌落,用碱变性方法提取这些菌落中的杂交质粒。
利用限制性内切酶(BamHI、HindIII、NcoI、EcoRI、BglII、SalI和PstI)进行结构分析。所有三个杂交质粒都有8.44Kb插入的DNA片段,三个杂交质粒都具有完全相同的断裂模式。这些结果表明它们是相同的。这个杂交质粒命名为pKB300。pKB300的限制性图谱如图1所示。在pKB300中大约一半长度的基因组DNA片段和pKB200的重叠。
2.用pKB300补充大肠杆菌的bioA缺失突变体。
利用实施例1-3中所描述的方法对带有pKB300的大肠杆菌W602(bioA-)进行互补作用分析。由于pKB3100补充bioA突变体(表1),所以pKB300携带了Kurthia sp.的bioA基因。
实施例6
Kurthia sp.538-KA26的bioA、B、D和F基因的亚克隆。
1.杂交质粒pKB103和pKB104的构建。
用HindIII完全消化pKB100,分离出一个3.3Kb的HindIII片段。把3.3Kb片段通过DNA连接试剂盒连接到用HindIII切开的载体pUC18(Takara Shuzo公司)上以构建杂交质粒pKB103和pKB104。在pKB103和pKB104中,把3.3Kb片段相对于pUC18乳糖基因的启动子-操纵基因以两个方向插入。它们的限制性图谱如图8所示。
利用实施例1-3中所描述的相同方法用pKB103和pKB104进行大肠杆菌(R875或R874)互补作用分析。pKB103和pKB104补充bioB和bioF突变体(表2)。
2.pKB200衍生物的构建。
由于pKB200补充bioD突变体,并覆盖携带bioB和bioF基因之全长的pKB100,构建了pKB200的一系列缺失突变体以精确定位bioB、bioD和bioF。把pKB200的4.0Kb的SalI-HindIII片段插入到pUC18和pUC19的SalI和HindIII位点以得到pKB221和pKB222,在pKB221和pKB222中SalI-HindIII片段以两种方向存在。
用NruI完全消化pKB200,通过琼脂糖凝胶电泳分离出一个7.5Kb的NruI片段,利用DNA连接试剂盒重新环化此NruI片段。
用NruI完全消化pKB200,通过凝胶电泳分离出一个4.8Kb的NruI片段并把它在两个方向上克隆到pUC18的HindIII位点中以产生pKB224和pKB225。
用NcoI部分消化pKB200,通过琼脂糖凝胶电泳分离出一个3.1Kb的NcoI片段,利用DNA聚合酶I(Takara Shuzo公司)克列诺片段使NcoI片段的末端成为平端并且和HindIII接头(Takara Shuzo公司)连接。利用HindIII处理得到3.1Kb的HindIII片段并且把它在两个方向上克隆到pUC1S的HindIII位点上,从而得到pKB228和pKB229。
以相同的方式,通过用克列诺片段处理pKB200的2.1Kb NcoI片段的两个末端并附加HindIII接头,将该片断转移到HindIII位点上。然后把获得的HindIII片段在两个方向插入到pUC19的HindIII位点中,从而得到pKB230和pKB231。
通过把pKB230的1.6Kb HindIII-NruI片段分别插入到pUC19和pUC18的HindIII和SmaI位点中,产生pKB234和pKB235。
pKB200衍生物的限性图谱如图8所示
3.用pKB200衍生物对大肠杆菌的生物素缺失突变体的互补作用分析。
利用实施例1-3中描述的相同方式用pKB200衍生物进行互补作用分析。表2总结了把互补作用结果。bioB缺失突变体由pKB221、pKB222、pKB224和pKB225补充,但不能由pKB223、pKB228、pIB229、pKB230、pKB231、pKB234和pKB235补充。bioF缺失突变体能由pKB223、pKB224、pKB225、pKB228和pKB229补充,但不能由pKB221、pKB222、pKB230、pKB231、pK B234和pKB235补充。另一方面,bioD缺失突变体能由pKB223、pKB224和pKB225补充,但不能由KB221和pKB222补充。
连同pKB103和pKB104的互补作用分析,这些结果表明bioF基因存在于pKB103的第一个NruI位点左边,而bioB基因定位在左侧带有短重叠区的相同NruL位点上,结果还表明bioD基因存在于pKB200左边至多1.5个Kb的区域中,这样,pKB100和pKB200的各种衍生物的互补作用结果表明bioD、bioF和bioB基因依次位于pKB200的HindIII位点左边的4.4Kb区域中。
4.杂交质粒pKB361的构建。
为确定bioA基因的位置,构建了pKB300的衍生物。通过把一个pKB300的2.8Kb BamHI-SalI片段插入到pUC19的BamHI和SalI位点中产生pKB361(图8)。
把pKB361转移到大肠杆菌(W602)的bioA缺失突变体中,利用实施例1-3中描述的相同的方式进行互补作用分析。bioA突变体能由pKB361补充,这表明bioA基因位于pKB300的BamHI和SaLI位点之间的2.8Kb区域内。
表2
质粒 | 大肠杆菌生物素缺失突变体 | |||
bioA- | bioD- | bioF- | bioB- | |
pKB103,104 | + | + | ||
pKB221,222 | - | - | + | |
pKB223 | + | + | - | |
pKB224,225 | + | + | + | |
pKB228,229 | + | - | ||
pKB230,231 | - | - | ||
pKB234,235 | - | - | ||
pKB361 | + |
实施例7
Kurthia sp.538-KA26的bioH基因的亚克隆。
1.杂交质粒pKH101和pKH102的构建。
用BamHI完全消化pKH100,利用DNA连接试剂盒重新环化以产生从pKH100中缺失了0.75Kb BamHI片段的pKH101(图4)。在pKH100中,通过用HindIII处理接着用DNA连接试剂盒重新环化构建pKH102。pKH10缺乏pKH100的1.07 Kb HindIII片段(图4)。
利用实施例1-3中所描述的方法用pKH101和pKH102对大肠杆菌bioH突变体(R878)进行互补作用分析。pKH101补充bioH突变体,但pKH102不能(图4)。结果表明bioH基因位于pKH100的BamHI位点的左边区域(1.16Kb)。
实施例8
Kurthia sp.538-KA26的bioC基因的亚克隆。
用BamHI完全消化pKC100,通过凝胶电泳分离出1.81Kb BamHI片段。把BamHI片段连接到经DNA连接试剂盒用BamHI和克列诺片段处理的pBR322中。最后,得到了以两个方向插入的BamHI片段的pKC101和pKC102(图6)。
把pKC101和pKC102转化到大肠杆菌bioC突变体R878中,以与实施例1-3中相同的方式进行互补作用分析。bioC突变体与pKC101和pKC102互补,因此确认bioC基因,位于1.81Kb BamHI片段中。
实施例9
在pKB100、pKB200和pKB300上插入的DNA片段的核苷酸序列。
为了分析pKB100、pKB200和pKB300的插入的DNA片段的核苷酸序列,重复相互的若干subclones利用pUC18、pUC19、M13mp18和M13mp19(Takara Shuzo公司)构建了一些相互重叠的亚克隆,并且利用Kilo-测序缺失试剂盒(Takara Shuzo公司)获得了一系列亚克隆缺失衍生物。然后,利用双脱氧链终止法进行缺失衍生物的核苷酸序列分析(利用7-deaza-dGTP测序酶版2.0 DNA测序试剂盒,美国生物化学公司)。利用软件开发公司的计算机程序(GENETYX)分析结果。
这种序列的计算机分析揭示了克隆的DNA片段有能力编码六种开放读框(ORF)。这种基因操纵子有进行双向编码的两个基因簇(图9-A)。
在左边基因簇中的第一个ORF开始于TTG密码子(之前是和圆形芽孢杆菌16S rRNA的3′末端同源的核糖体结合位点(RBS)),且编码具有21,516分子量的194个氨基酸残基的蛋白质。不可能通过互补作用分析来确定基因产物的功能,因此,这个ORF命名为ORF1。
在左边基因簇中的第二个ORF核苷酸序列如SEQ ID NO.1所示。此基因编码一个分子量为26,642,具有236个氨基酸残基的蛋白质。这种基因产物推测的氨基酸序列在SEQ ID NO.2中所示。在ATG起始密码子的上游发现推定的RBS。互补作用分析(实施例6-3)表明此ORF是bioD基因。
在左边基因簇的第三ORF具有推定的ATG起始密码子上游RBS,这个基因的核苷酸序列如SEQ ID NO.3所示。此基因编码分子量为51,731具有460个氨基酸残基的蛋白质。这种基因产物推测的氨基酸序列在SEQ ID NO.4中所示。确认此ORF与bioA基因(实施例6-3)相对应。发现反向重复序列位于终止密码子大约3bp的下游。这种结构可能作为一个转录终止区。
在右边基因簇中的第一个ORF(命名为ORF2)开始于ATG密码子,前面是推定的RBS。此基因产物是由63个氨基酸残基组成的蛋白质,其计算的分子量为7,447。我们不能通过互补作用分析和氨基酸序列同源搜索来鉴别这种基因产物的功能。因此,此ORF命名为ORF2。
在右边基因簇中的第二个ORF的核苷酸序列如在SEQ ID NO.5中所示。此这基因中有三个潜在的ATG起始密码子,其分别于第一个、第二十五个和第三十二个氨基酸残基相对应。互补作用分析(实施例6-3)表明此ORF和bioF基因相对应。这种基因产物推测的氨基酸序列在SEQ ID NO.6中所示。推测的具有387个氨基酸残基蛋白质的分子量经计算为42,619,其开始于第一个起始密码子。
在右边基因簇中的第三个ORF的核苷酸序列如SEQ ID NO.7中所示。此基因有三个潜在的ATG起始密码子、两个ATG密码子(第一个和第十八个氨基酸残基)和一个GTG密码子(第十二个氨基酸残基)。这种基因产物推测的氨基酸序列如SEQ ID NO.8中所示。推测的具有338个氨基酸残基的从第一起始密码子翻译的蛋白质分子量经计算为37,438。互补作用分析(实施例6-3)表明此ORF和bioB基因相对应。终止密码子16bp下游的反向重复序列的存在表明其具有转录终止区的性质。
有两个可能的启动子序列面对面地形成ORF1和ORF2之间的启动子,如SEQ ID NO.10中所示。转录向左进行进入到ORF1、bioD和bioA基因簇,向右进入到ORF2、bioF和bioB基因簇。此外,两个转录终止区定位在bioA和bioB基因的终止密码子的下游。因此,在两个方向中的转录产生两个不同的mRNA。
我们发现反向重复序列的两个组分(Box1和Box2)位于ORF1和ORF2基因的开始位点之间(图10)。Box1和Box2的所有的同源部分是82.5%。,比较Box1或Box2与大肠杆菌生物素操纵子[自然,276,689-694,(1978)]的操纵区,表明具有高水平的保守性(它们有54.6%的同源性)。大肠杆菌的生物素操纵区的反向重复序列之间的相似性表明Box1和Box1一定通过生物素参与生物素合成的负调节。
实施例10
pKH100插入的DNA片段之核苷酸序列。
以实施例9中描述同样的方式对pKH100的插入DNA片段之核苷酸序列进行分析。在插入的DNA片段上发现一个包含两个ORFs的基因簇(图9-B)。此外,已经证明了一部分载体质粒pBR322和插入的DNA片段缺失了。
在SEQ ID NO.中所示的第ORF编码一个267个氨基酸残基、计算的分子量为29,423的蛋白质。推测的这种基因产物的氨基酸序列如SEQ ID NO.10所示。一个推定的RBS定位在ATG起始密码子6bp上游。如实施例7所示,互补作用分析表明此ORF和bioH基因相对应。
在bioH基因的下游发现带有潜在RBS的第二个ORF。此ORF编码一个86个氨基酸残基分子量为9,955的蛋白质。这个由ORF编码的蛋白质与大肠杆菌和圆形芽孢杆菌的生物素基因产物没有同源性。把这个ORF命名为ORF3。
如图11中所示,在bioH基因的起始密码子的上游发现一种可能的启动子序列。由于在bioH基因的5′非编码区没有发现如Box1和Box2的反向重复序列,所以这个基因簇的转录一定不受调节。此外,有一个反向重复序列和ORF3的终止密码子相重叠。由于这种结构能作为转录终止区,所以推定的bioH启动子将允许bioH和ORF3基因的转录。
实施例11
pKC100的插入DNA片段的核苷酸序列。
用在实施例9中描述的相同方式对pKC100插入的DNA片段之核苷酸序列进行分析。在插入的DNA片段上发现由三个ORF组成的基因簇(图9-C)。
第三个ORF具有起始密码子的推定的RBS上游,这个基因的核苷酸序列如SEQ ID NO.15所示。此基因编码一个有276个氨基酸残基、计算分子量为31,599的蛋白质。推测的这种基因产物的氨基酸序列如SEQ ID NO.16所示。在实施例8中所示的互补作用分析表明这个ORF与bioC基因相对应。
在SEQ ID NO.11所示的第一个ORF编码一个分子量为44,776具有398个氨基酸残基的蛋白质。推定的RBS定位在起始密码子的上游。在SEQ IDNO.12中显示的此推测的基因产物的氨基酸序列和实施例9中的Kurthiasp.538-KA26的bioF基因产物的氨基酸序列有43.0%同源性。此外,pKC100补充实施例4所示的大肠杆菌bioF突变体。因此,结论认为这个ORF为bioF基因产物的同工酶的基因,即KAPA合成酶。因此,把这个ORF命名为bioFII基因。
在SEQ ID NO.13所示的第二个ORF3具有起始密码子的上游推定的RBS。此基因编码分子量为28,629的具有248氨基酸残基的蛋白质。在SEQ ID NO.14中所示的此基因产物的推测的氨基酸序列和在实施例10中Kurthia sp.538-KA26的bioH基因产物的氨基酸序列有24.2%同源性。在实施例4中所示,pKC100也补充大肠杆菌bioH突变体。这些结果表明此ORF是bioH基因产物的同工酶基因,因此,此ORF命名为bioHII基因。
在图12中所示的bioFII基因的起始密码子上游发现一种可能的启动子序列。反向重复序列定位在启动子序列和bioFII基因的RBS之间。把此命名为Box3的这个反向重复序列与定位在ORF1和ORF2基因之间的Box1和Box2相比较(实施例9)。Box1、Box2和Box3互相之间特别类似(Box1和Box3具有80.0%同源性,Box2和Box3有77.5%同源性)。因此,bioC基因簇一定受和bioA簇及bioB簇相同的负控制调节。此外,在bioC基因的终止密码子下游具有的一个254bp的反向重复序列。认为这种结构是作为转录终止区。
实施例12
大肠杆菌和Kurthia sp.菌株穿梭载体的构建。
利用图13所示的方法构建了大肠杆菌和Kurthia sp.的穿梭载体。用EcoRI和PvuII完全消化金黄色葡萄球菌质粒pUB110(芽孢杆菌属基因贮存中心;俄亥俄州立大学,生物化学部,484西十二大街,哥伦布,俄亥俄,43210,美国)。利用琼脂糖凝胶电泳分离出一个包含Kurthia sp.的复制起点和卡那霉素抗性基因的3.5Kb EcoRI-PvuII片段。用EcoRI和DraI完全消化pUC19,同时通过琼脂糖凝胶电泳分离出具有大肠杆菌的复制起点的1.2Kb EcoRI-DraI片段。然后,把这些片段利用DNA连接试剂盒进行连接以产生穿梭载体pYK1。pYK1能在大肠杆菌和Kurthia sp.中复制,由pYK1转化的大肠杆菌或Kurthia sp.表现为卡那霉素抗性。
实施例13
Kurthia sp.的bioB基因的表达质粒构建。
通过图14所示的方法构建了pYK114(其中Kurthia bioH基因启动子的下游插入了Kurthia bioB基因)。用BanII完全消化实施例7的pKH101,利用DNA聚合酶的克列诺片段使BanII片段的末端成为平端。然后,BanII片段用EcoRI处理,通过琼脂糖凝胶电泳分离出包含bioH启动子的0.6 KbEcoRI平端片段。用KpnI完全消化实施例6中的pKB104,通过利用克列诺片段处理将KpnI末端转化成平端。在用HindIII消化之后,通过琼脂糖电泳分离出1.3Kb携带bioB基因的平端HindIII片段。利用EcoRI和HindIII消化的pYK1连接平端EcoRI和平端HindIII片段以构建pYK114。bioB基因在pYK114的bioH启动子控制下进行结构表达。
实施例14
分离具有高转化效率的Kurthia sp.538-51F9的衍生菌株。
在28℃50ml Tripticase大豆肉汤(Becton Dickinson)中培养Kurthia sp.538-51F9(DSM No.10610),一直培养到在600nm下(OD600)具有1.0光密度。通过离心收集生长的细胞并且于OD60016下在SMM中悬浮(0.5M蔗糖、0.02M顺丁烯二酸钠、0.02 MMgCl2 6H2O)。然后把溶菌酶(Sigma)加入到200μg/ml的细胞悬液中,把悬液在30℃培养90分钟以形成原生质体。原生质体用SMM洗涤两次后,把它们悬浮在0.5ml SMM中。把1.5ml PEG溶液(SMM中有30%w/v的聚乙二醇4000)加入到原生质体悬液中,此悬液在冰上培养2分钟。然后加入6ml的SMM,并且通过离心收集原生质体。把收集的原生质体培养物悬浮在SMM,并且在30℃培养90分钟。把含有0.6%琼脂糖(Sigma,VII型)的DM3培养基(0.5M琥珀酸钠pH7.3、0.5%W/V酪蛋白氨基酸、0.5%W/V酵母抽提物、0.3%W/V KH2PO4、0.7%w/v K2HPO4、0.5%W/V葡萄糖、0.02 MMgCl2 6H2O、0.01%W/V牛血清蛋白)加入到原生质体悬液中,然后把悬液搁置在DM3培养基琼脂平板上。把平板在30℃培养3天。在DM3平板上总共获得了新生的65个菌落。
用实施例12的pYK1研究再生的菌株转化效率。结果,选择了40个菌株,并把它们在30℃50mlTripticase大豆肉汤中培养,直到OD600为1.0。通过离心收集生长的细胞并把它们在OD60016下悬浮在SMM中。然后通过以上所描述的方法用溶菌酶处理细胞获得原生质体。把原生质体培养物悬浮在0.5ml的SMM中,并且把pYK1(1μg)加入到原生质体悬液中。在加入1.5ml的PEG溶液后,悬浮液在冰上培养2分钟。再加入6ml的SMM,然后通过离心收集原生质体。此后,把原生质体悬浮在SMM中,并且在30℃培养90分钟。把含有0.6%琼脂糖的DM3培养基加入到原生质体悬液中,然后把悬液搁置在DM3培养基琼脂平板上。把平板在30℃培养3天。收集包括在平板上新生菌落的DM3-琼脂糖,并且把它扩散在含有5μg/ml卡那霉素的营养肉汤琼脂平板上来选择转化体。把平板在30℃培养过夜。最后,得到了具有高转化效率特征(每μgDNA有2,000个转化体)的衍生菌株--Kurthiasp.538-51F9-RG21。
实施例15
Kurthia sp.538-51F9-RG21中的bioB基因之扩增。
1.Kurthia sp.538-51F9-RG21的转化。
如实施例13中所描述的,构建Kurthia菌株bioB基因的表达质粒--pYK114。用pYK114和实施例14中描述的转化载体质粒pYK1转化Kurthiasp.538-51F9-RG21。把携带pYK1或是pYK114的Kurthia sp.538-51F9-RG21分别命名为Kurthia sp.538-51F9-RG21(pYK1)或Kurthia sp.538-51F9-RG21(pYK114)。
2.通过发酵产生生物素。
把Kurthia sp.538-51F9-RG21(pYK1)和Kurthia sp.538-51F9-RG21(pYK114)接种在含有5μg/ml卡那霉素的50ml的产物培养基中(6%甘油、5.5%protense蛋白胨、0.1%KH2PO4、0.05%MgSO47H2O、0.05%FeSO47H2O、0.001%MnSO45H2O;pH值为7.0)。把Kurthia sp.538-51F9-RG21接种在50ml的产物培养基中作为对照。培养物在28℃培养120小时。
在培养之后,把2ml培养肉汤离心以除去细菌细胞得到上清液。通过微生物测定方法利用Lactobacillus plantarum(ATCC 8014)测定上清液中的生物素产物。所产生的生物素量列于表3。
表3
Kurthia sp.的菌株 | 生物素产物(mg/l) |
51F9-RG21 | 15.4 |
51F9-RG21(pYK1) | 14.3 |
51F9-RG21(pYK114) | 39.0 |
序列表
(1)SEQ ID NO.1
序列长度:711个碱基对
序列类型:核酸
链型:双链
拓扑结构:线性
分子类型:基因组DNA
起始来源:
生物:Kurthia sp.
菌株:538-KA25
特 征:
特征关键词:CDS
位置:1..708
定义方法:E
序列描述:ATGGGTCAAG CCTACTTTAT AACCGGAACT GGCACGGATA TCGGAAAAAC CGTCGCCACG 60AGTTTACTCT ATATGTCTCT TCAAACAATG GGAAAAAGCG TCACAATATT TAAGCCGTTT 120CAAACAGGAT TGATTCACGA AACGAATACA TACCCTGACA TCTCTTGGTT TGAGCAGGAA 180CTTGGTGTAA AGGCACCTGG GTTTTACATG CTTGAACCCG AAACATCTCC ACACTTAGCT 240ATAAAATTAA CAGGGCAACA AATCGACGAG CAAAAGGTCG TGGAACGAGT TCACGAACTC 300GAACAAATGT ATGACATCGT GTTAGTCGAG GGCGCTGGGG GATTGGCCGT ACCACTCATT 360GAACGAGCGA ACAGTTTCTA TATGACAACC GATTTAATTA GAGATTGCAA CATGCCAGTC 420ATTTTCGTTT CTACAAGCGG TTTAGGATCG ATTCATAATG TCATAACTAC GCATTCGTAT 480GCCAAATTGC ATGATATTAG CGTTAAAACT ATTTTATATA ACCATTATCG GCCCGACGAT 540GAAATTCATC GTGACAATAT CCTAACCGTT GAAAAGCTCA CAGGACTCGC TGACCTCGCC 600TGCATACCAA CATTTGTCGA CGTAAGAAAA GATCTGAGAG TCTACATACT TGATTTACTT 660AGTAATCATG AATTTACTCA ACAACTAAAA GAGGTGTTCA AGAATGAATA G 711
(1)SEQ ID NO.2
序列长度:236个残基
序列类型:氨基酸
拓扑结构:线性
分子类型:多肽 起始来源:
生物:Kurthia sp.
菌株:538-KA26特 征:
定义方法:E序列描述:Met Gly Gln Ala Tyr Phe Ile Thr Gly Thr Gly Thr Asp Ile Gly1 5 10 15Lys Thr Val Ala Thr Ser Leu Leu Tyr Met Ser Leu Gln Thr Met
20 25 30Gly Lys Ser Val Thr Ile Phe Lys Pro Phe Gln Thr Gly Leu Ile
35 40 45His Glu Thr Asn Thr Tyr Pro Asp Ile Ser Trp Phe Glu Gln Glu
50 55 60Leu Gly Val Lys Ala Pro Gly Phe Tyr Met Leu Glu Pro Glu Thr
65 70 75Ser Pro His Leu Ala Ile Lys Leu Thr Gly Gln Gln Ile Asp Glu
80 85 90Gln Lys Val Val Glu Arg Val His Glu Leu Glu Gln Met Tyr Asp
95 100 105Ile Val Leu Val Glu Gly Ala Gly Gly Leu Ala Val Pro Leu Ile
110 115 120Glu Arg Ala Asn Ser Phe Tyr Met Thr Thr Asp Leu Ile Arg Asp
125 130 135Cys Asn Met Pro Val Ile Phe Val Ser Thr Ser Gly Leu Gly Ser
140 145 150Ile His Asn Val Ile Thr Thr His Ser Tyr Ala Lys Leu His Asp
155 160 165Ile Ser Val Lys Thr Ile Leu Tyr Asn His Tyr Arg Pro Asp Asp
170 175 180Glu Ile His Arg Asp Asn Ile Leu Thr Val Glu Lys Leu Thr Gly
185 190 195Leu Ala Asp Leu Ala Cys Ile Pro Thr Phe Val Asp Val Arg Lys
200 205 210Asp Leu Arg Val Tyr Ile Leu Asp Leu Leu Ser Asn His Glu Phe
215 220 225Thr Gln Gln Leu Lys Glu Val Phe Lys Asn Glu
230 235
(3)SEQ ID NO.3
序列长度:1383个碱基对
序列类型:核酸
链型:双链
拓扑结构:线性
分子类型:基因组DNA
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特征:
特征关键词:CDS
位置:1..1380
定义方法:E
序列描述:ATGAATAGTC ATGACTTAGA AAAGTGGGAT AAGGAATATG TATGGCATCC GTTTACACAA 60ATGAAAACGT ATCGAGAAAG TAAACCGCTA ATCATTGAAC GCGGGGAAGG GAGCTACCTT 120TTTGACATAG AAGGCAATCG GTACTTGGAC GGTTATGCTT CATTATGGGT CAACGTACAT 180GGCCATAATG AACCAGAGCT AAACAACGCT CTCATTGAAC AAGTTGAAAA AGTCGCACAC 240TCAACACTAC TAGGATCTGC AAATGTACCA TCCATATTAC TGGCTAAGAA ATTAGCAGAG 300ATTACTCCTG GTCATTTATC GAAAGTCTTT TACTCGGACA CTGGATCAGC TGCTGTAGAA 360ATCTCCCTTA AAGTCGCTTA TCAATATTGG AAAAATATCG ATCCTGTAAA GTATCAACAT 420AAAAATAAAT TTGTCTCCCT GAACGAGGCG TACCACGGTG ATACAGTTGG AGCAGTGAGT 480GTCGGCGGAA TGGATTTATT CCATAGAATC TTTAAACCAC TACTATTTGA ACGGATTCCA 540ACTCCTTCTC CTTATACATA TCGCATGGCT AAACACGGGG ATCAAGAAGC AGTGAAAAAC 600TATTGTATTG ATGAGCTGGA AAAGTTGCTT CAAGACCAAG CAGAGGAAAT TGCAGGATTG 660ATTATCGAAC CGCTTGTTCA AGGAGCAGCA GGCATCATTA CCCACCCTCC TGGCTTTTTA 720AAAGCGGTCG AACAATTGTG CAAGAAGTAC AATATATTAT TGATTTGTGA CGAAGTAGCG 780GTAGGATTTG GTCGCACCGG TACATTATTT GCCTGTGAAC AAGAAGATGT CGTCCCTGAT 840ATTATGTGTA TCGGTAAAGG AATTACTGGC GGCTATATGC CTCTGGCGGC CACTATCATG 900AACGAACAAA TCTTTAATTC TTTTTTAGGA GAGCCCGATG AACATAAAAC CTTCTATCAC 960GGCCACACCT ACACAGGGAA TCAACTAGCC TGTGCCCTGG CGCTGAAGAA TATCGAACTA 1020ATAGAAAGAC GAGATCTCGT CAAAGACATC CAGAAGAAAT CCAAGCAGCT ATCTGAAAAA 1080CTGCAATCGC TATATGAACT CCCGATTGTC GGTGATATCC GCCAGCGCGG CCTCATGATT 1140GGAATAGAAA TCGTTAAAGA TCGCCAAACA AAAGAACCGT TCACAATCCA AGAAAATATC 1200GTTTCAAGCA TCATCCAAAA CGCTCGGGAA AATGGCCTGA TCATTCGGGA ACTTGGCCCT 1260GTCATCACAA TGATGCCCAT TCTTTCCATG TCAGAAAAGG AACTGAATAC TATGGTCGAA 1320ACTGTCTACC GTTCGATACA GGACGTTTCT GTGCACAACG GATTAATCCC AGCAGCAAAC 1380TGA 1383
(4)SEQ ID NO.4
序列长度:460个残基
序列类型:氨基酸
拓扑结构:线性
分子类型:多肽
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
定义方法:E
序列描述:Met Asn Ser His Asp Leu Glu Lys Trp Asp Lys Glu Tyr Val Trp1 5 10 15His Pro Phe Thr Gln Met Lys Thr Tyr Arg Glu Ser Lys Pro Leu
20 25 30Ile Ile Glu Arg Gly Glu Gly Ser Tyr Leu Phe Asp Ile Glu Gly
35 40 45Asn Arg Tyr Leu Asp Gly Tyr Ala Ser Leu Trp Val Asn Val His
50 55 60Gly His Asn Glu Pro Glu Leu Asn Asn Ala Leu Ile Glu Gln Val
65 70 75Glu Lys Val Ala His Ser Thr Leu Leu Gly Ser Ala Asn Val Pro
80 85 90Ser Ile Leu Leu Ala Lys Lys Leu Ala Glu Ile Thr Pro Gly His
95 100 105Leu Ser Lys Val Phe Tyr Ser Asp Thr Gly Ser Ala Ala Val Glu
110 115 120Ile Ser Leu Lys Val Ala Tyr Gln Tyr Trp Lys Asn Ile Asp Pro
125 130 135Val Lys Tyr Gln His Lys Asn Lys Phe Val Ser Leu Asn Glu Ala
140 145 150Tyr His Gly Asp Thr Val Gly Ala Val Ser Val Gly Gly Met Asp
155 160 165Leu Phe His Arg Ile Phe Lys Pro Leu Leu Phe Glu Arg Ile Pro
170 175 180Thr Pro Ser Pro Tyr Thr Tyr Arg Met Ala Lys His Gly Asp Gln
185 190 195Glu Ala Val Lys Asn Tyr Cys Ile Asp Glu Leu Glu Lys Leu Leu
200 205 210Gln Asp Gln Ala Glu Glu Ile Ala Gly Leu Ile Ile Glu Pro Leu
215 220 225Val Gln Gly Ala Ala Gly Ile Ile Thr His Pro Pro Gly Phe Leu
230 235 240Lys Ala Val Glu Gln Leu Cys Lys Lys Tyr Asn Ile Leu Leu Ile
245 250 255Cys Asp Glu Val Ala Val Gly Phe Gly Arg Thr Gly Thr Leu Phe
260 265 270Ala Cys Glu Gln Glu Asp Val Val Pro Asp Ile Met Cys Ile Gly
275 280 285Lys Gly Ile Thr Gly Gly Tyr Met Pro Leu Ala Ala Thr Ile Met
290 295 300Asn Glu Gln Ile Phe Asn Ser Phe Leu Gly Glu Pro Asp Glu His
305 310 315Lys Thr Phe Tyr His Gly His Thr Tyr Thr Gly Asn Gln Leu Ala
320 325 330Cys Ala Leu Ala Leu Lys Asn Ile Glu Leu Ile Glu Arg Arg Asp
335 340 345Leu Val Lys Asp Ile Gln Lys Lys Ser Lys Gln Leu Ser Glu Lys
350 355 360Leu Gln Ser Leu Tyr Glu Leu Pro Ile Val Gly Asp Ile Arg Gln
365 370 375Arg Gly Leu Met Ile Gly Ile Glu Ile Val Lys Asp Arg Gln Thr
380 385 390Lys Glu Pro Phe Thr Ile Gln Glu Asn Ile Val Ser Ser Ile Ile
395 400 405Gln Asn Ala Arg Glu Asn Gly Leu Ile Ile Arg Glu Leu Gly Pro
410 415 420Val Ile Thr Met Met Pro Ile Leu Ser Met Ser Glu Lys Glu Leu
425 430 435Asn Thr Met Val Glu Thr Val Tyr Arg Ser Ile Gln Asp Val Ser
440 445 450Val His Asn Gly Leu Ile Pro Ala Ala Asn
455 460
(5)SEQ ID NO.5
序列长度:1164个碱基对
序列类型:核酸
链型:双链
拓扑结构:线性
分子类型:基因组DNA
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特征:
特征关键词:CDS
位置:1..1161
定义方法:E
序列描述:ATGATTTGGG AGAAGGAACT AGAAAAGATT AAAGAAGGAG GGCTTTACAG ACAACTCCAA 60ACCGTTGAAA CAATGAGCGA TCAAGGGTAT GCCATGGTGA ACGGAAAAAA AATGATGATG 120TTTGCCTCCA ATAATTACTT AGGGATTGCC AATGATCAAC GATTAATTGA GGCTTCTGTC 180CAAGCGACTC AAAGATTTGG TACGGGTTCT ACTGGTTCAC GATTAACCAC TGGCAATACA 240ATTGTCCATG AAAAACTAGA GAAAAGACTT GCAGAGTTTA AGCAAACGGA TGCAGCGATA 300GTATTAAACA CAGGGTATAT GGCTAACATA GCAGCGTTAA CGACCCTTGT TGGTAGTGAC 360GATCTCATTT TATCCGATGA GATGAATCAT GCCAGTATTA TTGATGGCTG CCGTTTATCA 420CGTGCGGAAA CTATCATTTA TCGTCATGCT GATTTACTTG ACTTGGAAAT GAAACTCCAG 480ATTAATACCC GCTACAGGAA AAGAATAATT GTAACGGATG GCGTCTTTTC GATGGATGGT 540GATATTGCGC CATTGCCAGG TATTGTCGAA CTTGCCAAGC GTTATGATGC ACTTGTTATG 600GTGGATGACG CACATGCGAC GGGTGTTTTA GGTAAAGACG GAAGGGGAAC TTCTGAACAT 660TTTGGACTGA AGGGGAAGAT AGATATCGAG ATGGGGACAC TCTCCAAAGC TGTTGGTGCA 720GAAGGAGGGT ATATCGCTGG AAGCAGGTCT TTAGTTGACT ATGTCTTAAA TCGAGCCAGA 780CCGTTTGTCT TCTCTACCGC CTTATCAGCA GGAGTAGTAG CAAGTGCACT TACAGCAGTC 840GATATCATTC AATCAGAACC TGAACGCAGA GTACGCATTC GAGCCATGAG CCAGCGTCTT 900TATAATGAAT TAACCTCCCT TGGCTACACA GTTTCGGGGG GAGAAACTCC GATTCTTGCC 960ATTATTTGCG GAGAACCGGA ACAGGCCATG TTCCTTTCGA AAGAATTACA TAAGCACGGA 1020ATTTATGCAC CAGCTATCCG TTCGCCAACG GTACCTCTTG GAACTTCGCG CATTCGACTT 1080ACGTTAATGG CGACACATCA AGAAGAACAA ATGAATCATG TTATCGACGT GTTCAGAACA 1140ATCCTTACCA ATAGATACAA ATAG 1164
(6)SEQ ID NO.6
序列长度:387个残基
序列类型:氨基酸
拓扑结构:线性
分子类型:多肽
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
定义方法:E
序列描述:Met Ile Trp Glu Lys Glu Leu Glu Lys Ile Lys Glu Gly Gly Leu1 5 10 15Tyr Arg Gln Leu Gln Thr Val Glu Thr Met Ser Asp Gln Gly Tyr
20 25 30Ala Met Val Asn Gly Lys Lys Met Met Met Phe Ala Ser Asn Asn
35 40 45Tyr Leu Gly Ile Ala Asn Asp Gln Arg Leu Ile Glu Ala Ser Val
50 55 60Gln Ala Thr Gln Arg Phe Gly Thr Gly Ser Thr Gly Ser Arg Leu
65 70 75Thr Thr Gly Asn Thr Ile Val His Glu Lys Leu Glu Lys Arg Leu
80 85 90Ala Glu Phe Lys Gln Thr Asp Ala Ala Ile Val Leu Asn Thr Gly
95 100 105Tyr Met Ala Asn Ile Ala Ala Leu Thr Thr Leu Val Gly Ser Asp
110 115 120Asp Leu Ile Leu Ser Asp Glu Met Asn His Ala Ser Ile Ile Asp
125 130 135Gly Cys Arg Leu Ser Arg Ala Glu Thr Ile Ile Tyr Arg His Ala
140 145 150Asp Leu Leu Asp Leu Glu Met Lys Leu Gln Ile Asn Thr Arg Tyr
155 160 165Arg Lys Arg Ile Ile Val Thr Asp Gly Val Phe Ser Met Asp Gly
170 175 180Asp Ile Ala Pro Leu Pro Gly Ile Val Glu Leu Ala Lys Arg Tyr
185 190 195Asp Ala Leu Val Met Val Asp Asp Ala His Ala Thr Gly Val Leu
200 205 210Gly Lys Asp Gly Arg Gly Thr Ser Glu His Phe Gly Leu Lys Gly
215 220 225Lys Ile Asp Ile Glu Met Gly Thr Leu Ser Lys Ala Val Gly Ala
230 235 240Glu Gly Gly Tyr Ile Ala Gly Ser Arg Ser Leu Val Asp Tyr Val
245 250 255Leu Asn Arg Ala Arg Pro Phe Val Phe Ser Thr Ala Leu Ser Ala
260 265 270Gly Val Val Ala Ser Ala Leu Thr Ala Val Asp Ile Ile Gln Ser
275 280 285Glu Pro Glu Arg Arg Val Arg Ile Arg Ala Met Ser Gln Arg Leu
290 295 300Tyr Asn Glu Leu Thr Ser Leu Gly Tyr Thr Val Ser Gly Gly Glu
305 310 315Thr Pro Ile Leu Ala Ile Ile Cys Gly Glu Pro Glu Gln Ala Met
320 325 330Phe Leu Ser Lys Glu Leu His Lys His Gly Ile Tyr Ala Pro Ala
335 340 345Ile Arg Ser Pro Thr Val Pro Leu Gly Thr Ser Arg Ile Arg Leu
350 355 360Thr Leu Met Ala Thr His Gln Glu Glu Gln Met Asn His Val Ile
365 370 375Asp Val Phe Arg Thr Ile Leu Thr Asn Arg Tyr Lys
380 385(7)SEQ ID NO.7序列长度:1017个碱基对序列类型:核酸链型:双链拓扑结构:线性分子类型:基因组DNA起始来源:生物:Kurthia sp.
菌株:538-KA26
特征:
特征key:CDS
位置:1..1014
定义方法:E
菌株:538-KA26
序列描述:ATGAGAAAAG AGGGATTAGG TTTGGAAACA TTGGTGAAAA AGGATTGGAA GATGCTAGCG 60GAAAACGTAA TCAAAGGATA TAAAGTAACA GCGGAAGAAG CACTTGCTAT TGTACAAGCA 120CCTGACAACG AGGTTTTAGA GATTTTGAAT GCAGCTTTCC TTATTCGTCA GCACTATTAT 180GGAAAAAAGG TTAAATTGAA TATGATCATT AATACGAAGT CAGGTCTATG TCCTGAAGAT 240TGTGGCTATT GTTCGCAGTC AATCGTGTCG GAAGCTCCTA TCGATAAATA TGCTTGGCTG 300ACCAAAGAGA AGATTGTTGA AGGTGCTCAA GAATCAATTC GTCGCAAAGC TGGCACGTAT 360TGTATCGTTG CTTCTGGCCG TCGTCCGACC AATAGGGAAA TTGATCATGT CATTGAAGCT 420GTGAAAGAAA TTCGCGAGAC AACGGATCTT AAAATATGCT GCTGTCTAGG TTTCTTAAAT 480GAAACGCATG CCAGTAAGCT AGCTGAAGCT GGGGTTCATC GCTACAAGCA CAACTTAAAT 540ACATCTCAAG ATAATTATAA GAATATTACA TCCACACATA CTTATGAGGA CCGTGTAGAT 600ACAGTCGAAG CTGTAAAAGA GGCCGGAATG TCTCCATGCT CGGGTGCCAT TTTTGGTATG 660AATGAGTCTA ATGAAGAAGC AGTAGAGATT GCCCTATCCC TACGCAGTCT TGACGCGGAT 720TCTATTCCTT GTAATTTCCT CAATGCGATT GACGGTACAC CACTTGAGGG AACTTCCGAG 780TTGACTCCAA CTAAATGTTT GAAATTAATT TCGATGATGA GATTTGTTAA TCCAAGTAAG 840GAAATCCGTC TTGCTGGAGG TCGCGAGGTG AACCTCCGTT CCATGCAACC CATGGCACTT 900TATGCAGCCA ATTCTATCTT CGTCGGCGAT TATCTAACAA CAGCTGGACA AGAACCTACG 960GCGGATTGGG GCATTATCGA AGACCTTGGT TTTGAAATTG AAGAATGCGC TCTTTAA 1017
(8)SEQ ID NO.8
序列长度:338个残基
序列类型:氨基酸
拓扑结构:线性
分子类型:多肽
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
定义方法:E序列描述:Met Arg Lys Glu Gly Leu Gly Leu Glu Thr Leu Val Lys Lys Asp1 5 10 15Trp Lys Met Leu Ala Glu Asn Val Ile Lys Gly Tyr Lys Val Thr
20 25 30Ala Glu Glu Ala Leu Ala Ile Val Gln Ala Pro Asp Asn Glu Val
35 40 45Leu Glu Ile Leu Asn Ala Ala Phe Leu Ile Arg Gln His Tyr Tyr
50 55 60Gly Lys Lys Val Lys Leu Asn Met Ile Ile Asn Thr Lys Ser Gly
65 70 75Leu Cys Pro Glu Asp Cys Gly Tyr Cys Ser Gln Ser Ile Val Ser
80 85 90Glu Ala Pro Ile Asp Lys Tyr Ala Trp Leu Thr Lys Glu Lys Ile
95 100 105Val Glu Gly Ala Gln Glu Ser Ile Arg Arg Lys Ala Gly Thr Tyr
110 115 120Cys Ile Val Ala Ser Gly Arg Arg Pro Thr Asn Arg Glu Ile Asp
125 130 135His Val Ile Glu Ala Val Lys Glu Ile Arg Glu Thr Thr Asp Leu
140 145 150Lys Ile Cys Cys Cys Leu Gly Phe Leu Asn Glu Thr His Ala Ser
155 160 165Lys Leu Ala Glu Ala Gly Val His Arg Tyr Lys His Asn Leu Asn
170 175 180Thr Ser Gln Asp Asn Tyr Lys Asn Ile Thr Ser Thr His Thr Tyr
185 190 195Glu Asp Arg Val Asp Thr Val Glu Ala Val Lys Glu Ala Gly Met
200 205 210Ser Pro Cys Ser Gly Ala Ile Phe Gly Met Asn Glu Ser Asn Glu
215 220 225Glu Ala Val Glu Ile Ala Leu Ser Leu Arg Ser Leu Asp Ala Asp
230 235 240Ser Ile Pro Cys Asn Phe Leu Asn Ala Ile Asp Gly Thr Pro Leu
245 250 255Glu Gly Thr Ser Glu Leu Thr Pro Thr Lys Cys Leu Lys Leu Ile
260 265 270Ser Met Met Arg Phe Val Asn Pro Ser Lys Glu Ile Arg Leu Ala
275 280 285Gly Gly Arg Glu Val Asn Leu Arg Ser Met Gln Pro Met Ala Leu
290 295 300Tyr Ala Ala Asn Ser Ile Phe Val Gly Asp Tyr Leu Thr Thr Ala
305 310 315Gly Gln Glu Pro Thr Ala Asp Trp Gly Ile Ile Glu Asp Leu Gly
320 325 330Phe Glu Ile Glu Glu Cys Ala Leu
335(9)SEQ ID NO.9
序列长度:804个碱基对
序列类型:核酸
链型:双链
拓扑结构:线性
分子类型:基因组DNA
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
特征关键词:CDS
位置:1..801
定义方法:E
序列描述:ATGCCATTCG TAAATCATGA CAATGAAAGC CTTTACTATG AGGTTCACGG ACAAGGTGAT 60CCTTTATTGT TGATTATGGG GCTCGGCTAT AACTCTTTAT CCTGGCATAG AACGGTGCCC 120ACTTTAGCTA AGCGCTTTAA AGTAATCGTT TTTGATAATC GTGGTGTTGG TAAGAGCAGT 180AAGCCTGAAC AGCCATATTC TATTGAAATG ATGGCTGAGG ATGCAAGAGC GGTCCTTGAT 240GCTGTTTCGG TTGACTCAGC ACATGTATAT GGGATTTCAA TGGGTGGAAT GATTGCCCAA 300AGGCTGGCAA TCACATATCC AGAACGTGTT CGTTCTCTTG TTCTAGGTTG TACCACTGCG 360GGTGGTACTA CTCATATTCA ACCTTCTCCA GAAATATCTA CTTTAATGGT ATCTCGAGCC 420TCCCTTACAG GTTCTCCAAG GGATAATGCC TGGTTAGCGG CACCAATAGT TTATAGTCAA 480GCTTTTATTG AGAAGCACCC TGAATTAATT CAGGAAGATA TCCAAAAGCG AATAGAAATC 540ATTACTCCGC CAAGCGCCTA TCTGTCTCAA CTACAAGCTT GTCTAACTCA TGATACATCC 600AATGAACTTG ATAAAATAAA CATACCAACA TTGATTATAC ACGGTGATGC AGATAATTTG 660GTTCCATATG AAAACGGTAA AATGTTAGCT GAACGTATTC AGGGTTCTCA GTTTCACACC 720GTATCCTGTG CTGGCCACAT TTATTTAACA GAAGCAGCTA AGGAAGCAAA TGACAAAGTT 780ATACAGTTTC TAGCTCATCT ATAA 804(10)SEQ ID NO.10序列长度:267个残基序列类型:氨基酸拓扑结构:线性分子类型:多肽起始来源:
生物:Kurthia sp.
菌株:538-KA26特征:
定义方法:E序列描述:Met Pro Phe Val Asn His Asp Asn Glu Ser Leu Tyr Tyr Glu Val1 5 10 15His Gly Gln Gly Asp Pro Leu Leu Leu Ile Met Gly Leu Gly Tyr
20 25 30Asn Ser Leu Ser Trp His Arg Thr Val Pro Thr Leu Ala Lys Arg
35 40 45Phe Lys Val Ile Val Phe Asp Asn Arg Gly Val Gly Lys Ser Ser
50 55 60Lys Pro Glu Gln Pro Tyr Ser Ile Glu Met Met Ala Glu Asp Ala
65 70 75Arg Ala Val Leu Asp Ala Val Ser Val Asp Ser Ala His Val Tyr
80 85 90Gly Ile Ser Met Gly Gly Met Ile Ala Gln Arg Leu Ala Ile Thr
95 100 105Tyr Pro Glu Arg Val Arg Ser Leu Val Leu Gly Cys Thr Thr Ala
110 115 120Gly Gly Thr Thr His Ile Gln Pro Ser Pro Glu Ile Ser Thr Leu
125 130 135Met Val Ser Arg Ala Ser Leu Thr Gly Ser Pro Arg Asp Asn Ala
140 145 150Trp Leu Ala Ala Pro Ile Val Tyr Ser Gln Ala Phe Ile Glu Lys
155 160 165His Pro Glu Leu Ile Gln Glu Asp Ile Gln Lys Arg Ile Glu Ile
170 175 180Ile Thr Pro Pro Ser Ala Tyr Leu Ser Gln Leu Gln Ala Cys Leu
185 190 195Thr His Asp Thr Ser Asn Glu Leu Asp Lys Ile Asn Ile Pro Thr
200 205 210Leu Ile Ile His Gly Asp Ala Asp Asn Leu Val Pro Tyr Glu Asn
215 220 225Gly Lys Met Leu Ala Glu Arg Ile Gln Gly Ser Gln Phe His Thr
230 235 240Val Ser Cys Ala Gly His Ile Tyr Leu Thr Glu Ala Ala Lys Glu
245 250 255Ala Asn Asp Lys Val Ile Gln Phe Leu Ala His Leu
260 265
(11)SEQ ID NO.11
序列长度:1197个碱基对
序列类型:核酸
链型:双链
拓扑结构:线性
分子类型:基因组DNA
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
特征关键词:CDS
位置:1..1194
定义方法:E
序列描述:ATGCACAGTG AAAAACAATT ACCTTGTTGG GAAGAAAAAA TTAAGAAAGA ACTGGCTTAT 60TTAGAAGAGA TATCGCAAAA ACGTGAACTC GTTTCAACGG AATTCGCCGA GCAGCCATGG 120CTTATGATCA ACGGGTGCAA GATGCTAAAT CTAGCTTCTA ATAACTATTT AGGATATGCA 180GGGGATGAGC GGCTGAAAAA GGCTATGGTA GATGCAGTAC ATACATATGG TGCAGGAGCG 240ACGGCTTCAC GTTTAATTAT TGGCAATCAC CCTCTTTACG AGCAAGCAGA ACAAGCTCTT 300GTCAATTGGA AGAAAGCCGA AGCAGGACTC ATTATTAACA GTGGATATAA CGCGAACCTT 360GGAATTATCT CCACCTTGCT GTCCCGTAAC GATATTATTT ATAGCGATAA ATTGAATCAT 420GCAAGCATTG TCGATGGAGC TCTCTTAAGC CGTGCAAAGC ATCTACGCTA TCGTCATAAT 480GATTTAGATC ATTTAGAAGC ATTATTGAAA AAATCATCGA TGGAAGCACG TAAATTAATT 540GTGACGGATA CGGTCTTCAG CATGGACGGT GACTTTGCTT ATCTTGAAGA CCTTGTTCGG 600TTAAAAGAAC GTTATAACGC TATGTTAATG ACAGATGAAG CACACGGAAG CGGCATCTAC 660GGTAAAAACG GTGAAGGTTA TGCCGGTCAT CTCCATCTTC AAAATAAAAT AGATATCCAA 720ATGGGAACAT TCAGTAAAGC GCTCGGTTCA TTCGGGGCCT ATGTCGTCGG GAAAAAATGG 780CTCATCGACT ATTTAAAAAA TCGCATGCGC GGATTCATAT ATTCAACTGC ACTCCCCCCG 840GCCATACTCG GTGCTATGAA AACAGCGATA GAACTTGTAC AGCAAGAACC AGAACGCCGC 900TCACTGCTCC AAACACATTC AGAACACTTT AGAGAAGAAC TCACATATTA CGGGTTTAAT 960ATTTGTGGAA GTCGATCACA AATTGTTCCT ATCGTCATCG GGGAAAACGA AAAAGCGATG 1020GAATTTGCCA CACGTTTGCA GAAAGAAGGA ATTGCAGCTA TTGCTGTCAG GCCGCCGACC 1080GTTCCTGAAA ATGAGGCGAG AATCCGTTTT ACTGTAACAG CTCTCCACGA TAAAAAAGAT 1140CTTGATTGGG CAGTTGAAAA AGTTTCGATC ATTGGAAAAG AAATGGGTGT TATTTAA 1197(12)SEQ ID NO.12序列长度:398个残基序列类型:氨基酸拓扑结构:线性分子类型:多肽起始来源:
生物:Kurthia sp.
菌株:538-KA26特 征:
定义方法:E序列描述:Met His Ser Glu Lys Gln Leu Pro Cys Trp Glu Glu Lys Ile Lys1 5 10 15Lys Glu Leu Ala Tyr Leu Glu Glu Ile Ser Gln Lys Arg Glu Leu
20 25 30Val Ser Thr Glu Phe Ala Glu Gln Pro Trp Leu Met Ile Asn Gly
35 40 45Cys Lys Met Leu Asn Leu Ala Ser Asn Asn Tyr Leu Gly Tyr Ala
50 55 60Gly Asp Glu Arg Leu Lys Lys Ala Met Val Asp Ala Val His Thr
65 70 75Tyr Gly Ala Gly Ala Thr Ala Ser Arg Leu Ile Ile Gly Asn His
80 85 90Pro Leu Tyr Glu Gln Ala Glu Gln Ala Leu Val Asn Trp Lys Lys
95 100 105Ala Glu Ala Gly Leu Ile Ile Asn Ser Gly Tyr Asn Ala Asn Leu
110 115 120Gly Ile Ile Ser Thr Leu Leu Ser Arg Asn Asp Ile Ile Tyr Ser
125 130 135Asp Lys Leu Asn His Ala Ser Ile Val Asp Gly Ala Leu Leu Ser
140 145 150Arg Ala Lys His Leu Arg Tyr Arg His Asn Asp Leu Asp His Leu
155 160 165Glu Ala Leu Leu Lys Lys Ser Ser Met Glu Ala Arg Lys Leu Ile
170 175 180Val Thr Asp Thr Val Phe Ser Met Asp Gly Asp Phe Ala Tyr Leu
185 190 195Glu Asp Leu Val Arg Leu Lys Glu Arg Tyr Asn Ala Met Leu Met
200 205 210Thr Asp Glu Ala His Gly Ser Gly Ile Tyr Gly Lys Asn Gly Glu
215 220 225Gly Tyr Ala Gly His Leu His Leu Gln Asn Lys Ile Asp Ile Gln
230 235 240Met Gly Thr Phe Ser Lys Ala Leu Gly Ser Phe Gly Ala Tyr Val
245 250 255Val Gly Lys Lys Trp Leu Ile Asp Tyr Leu Lys Asn Arg Met Arg
260 265 270Gly Phe Ile Tyr Ser Thr Ala Leu Pro Pro Ala Ile Leu Gly Ala
275 280 285Met Lys Thr Ala Ile Glu Leu Val Gln Gln Glu Pro Glu Arg Arg
290 295 300Ser Leu Leu Gln Thr His Ser Glu His Phe Arg Glu Glu Leu Thr
305 310 315Tyr Tyr Gly Phe Asn Ile Cys Gly Ser Arg Ser Gln Ile Val Pro
320 325 330Ile Val Ile Gly Glu Asn Glu Lys Ala Met Glu Phe Ala Thr Arg
335 340 345Leu Gln Lys Glu Gly Ile Ala Ala Ile Ala Val Arg Pro Pro Thr
350 355 360Val Pro Glu Asn Glu Ala Arg Ile Arg Phe Thr Val Thr Ala Leu
365 370 375His Asp Lys Lys Asp Leu Asp Trp Ala Val Glu Lys Val Ser Ile
380 385 390Ile Gly Lys Glu Met Gly Val Ile
395
(13)SEQ ID NO.13
序列长度:747个碱基对
序列类型:核酸
链型:双链
拓扑结构:线性
分子类型:基因组DNA
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
特征关键词:CDS
位置:1..744
定义方法:E
序列描述:ATGAAACAGC CGAATTTAGT CATGCTTCCT GGCTGGGGAA TGGAAAAAGA TGCGTTTCAA 60CCGTTAATCA AACCGCTGTC AGAAGTATTT CACCTCTCAT TCATAGAATG GAGAGATATG 120AAAACACTAA ATGACTTTGA AGAACGAGTC ATAGACACAA TCGCTTCTAT TGATGGTCCT 180GTTTTTTTAC TTGGCTGGTC ATTAGGATCT CTATTATCAC TTGAACTTGT AAGTTCGTAT 240CGAGAAAAAA TAAAAGGTTT TATACTAATT GGCGCAACAA GTCGTTTTAC CACAGGAGAT 300AATTATTCAT TTGGCTGGGA TCCACGAATG GTCGAGCGCA TGAAGAAACA ACTGCAGCGC 360AATAAAGAGA AGACTTTGAC TTCTTTCTAT GAAGCAATGT TTTCCGAAGC TGAAAAAGAA 420GAAGGTTTTT ATCATCAATT CATCACGACA ATTCAAAGCG AGTTTCATGG GGATGACGTA 480TTTTCGCTTC TTATAGGTTT GGATTATTTA CTTCAGAAAG ATGTTAGAGT AAAGCTCGAC 540CAGATTGAAA CTCCCATTTT ATTGATCCAT GGGAGAGAAG ACAAAATTTG TCCACTCGAA 600GCCTCATCTT TCATTAAAGA AAATCTGGGT GGGAAAGCCG AGGTTCATAT TATCGAAGGC 660GCTGGTCATA TTCCATTTTT CACAAAACCA CAGGAATGTG TGCAGCTTAT AAAAACATTT 720ATTCAAAAGG AGTACATTCA TGATTGA 747
(14)SEQ ID NO.14
序列长度:248个残基
序列类型:氨基酸
链型:双链
拓扑结构:线性
分子类型:多肽起始来源:
生物:Kurthia sp.
菌株:538-KA26特 征:
定义方法:E序列描述:Met Lys Gln Pro Asn Leu Val Met Leu Pro Gly Trp Gly Met Glu1 5 10 15Lys Asp Ala Phe Gln Pro Leu Ile Lys Pro Leu Ser Glu Val Phe
20 25 30His Leu Ser Phe Ile Glu Trp Arg Asp Met Lys Thr Leu Asn Asp
35 40 45Phe Glu Glu Arg Val Ile Asp Thr Ile Ala Ser Ile Asp Gly Pro
50 55 60Val Phe Leu Leu Gly Trp Ser Leu Gly Ser Leu Leu Ser Leu Glu
65 70 75Leu Val Ser Ser Tyr Arg Glu Lys Ile Lys Gly Phe Ile Leu Ile
80 85 90Gly Ala Thr Ser Arg Phe Thr Thr Gly Asp Asn Tyr Ser Phe Gly
95 100 105Trp Asp Pro Arg Met Val Glu Arg Met Lys Lys Gln Leu Gln Arg
110 115 120Asn Lys Glu Lys Thr Leu Thr Ser Phe Tyr Glu Ala Met Phe Ser
125 130 135Glu Ala Glu Lys Glu Glu Gly Phe Tyr His Gln Phe Ile Thr Thr
140 145 150Ile Gln Ser Glu Phe His Gly Asp Asp Val Phe Ser Leu Leu Ile
155 160 165Gly Leu Asp Tyr Leu Leu Gln Lys Asp Val Arg Val Lys Leu Asp
170 175 180Gln Ile Glu Thr Pro Ile Leu Leu Ile His Gly Arg Glu Asp Lys
185 190 195Ile Cys Pro Leu Glu Ala Ser Ser Phe Ile Lys Glu Asn Leu Gly
200 205 210Gly Lys Ala Glu Val His Ile Ile Glu Gly Ala Gly His Ile Pro
215 220 225Phe Phe Thr Lys Pro Gln Glu Cys Val Gln Leu Ile Lys Thr Phe
230 235 240Ile Gln Lys Glu Tyr Ile His Asp
245(15)SEQ ID NO.15序列长度:831个碱基对序列类型:核酸链型:双链拓扑结构:线性分子类型:基因组DNA起始来源:
生物:Kurthia sp.
菌株:538-KA26特 征:
特征关键词:CDS
位置:1..828
定义方法:E序列描述:ATGATTGATA AACAATTGTT AAGTAAGCGA TTCAGTGAAC ATGCGAAAAC ATATGATGCA 60TATGCCAATG TTCAAAAAAA CATGGCGAAA CAATTAGTGG ATTTGCTCCC TCAAAAAAAC 120AGCAAACAAA GAATTAACAT CCTTGAAATT GGCTGCGGTA CTGGTTACTT AACCAGGTTA 180CTCGTTAATA CATTTCCTAA TGCTTCTATT ACCGCTGTTG ATTTAGCACC AGGGATGGTT 240GAAGTGGCGA AAGGAATAAC AATGGAAGAC CGTGTTACTT TTTTATGTGC TGATATCGAA 300GAAATGACGC TTAATGAAAA TTACGACTTA ATTATTTCTA ATGCAACGTT TCAATGGCTG 360AATAATCTTC CTGGAACCAT TGAACAATTG TTTACACGAT TAACGCCTGA AGGAAACCTG 420ATATTTTCAA CGTTTGGAAT TAAAACCTTT CAAGAGCTTC ATATGTCCTA TGAACATGCG 480AAAGAAAAGC TTCAACTTTC AATTGATAGT TCACCAGGCC AACTGTTTTA CGCTCTAGAA 540GAATTATCCC AAATTTGTGA AGAAGCAATC CCTTTTTCAT CAGCATTTCC ATTAGAGATA 600ACAAAAATAG AAAAGCTTGA ACTAGAGTAC TTTCAGACAG TACGTGAATT TTTCACTTCA 660ATTAAAAAGA TTGGTGCAGC TAACAGCAAC AAAGAAAACT ACTGCCAGCG CCCTTCTTTT 720TTTCGAGAGT TAATCAACAT ATACGAAACA AAATACCAAG ATGAATCAGG TGTGAAGGCA 780ACCTATCACT GTTTGTTTTT TAAGATAATA AAACATGCCC CCCTACCCTA A 831
(16)SEQ ID NO.16
序列长度:276个残基
序列类型:氨基酸
拓扑结构:线性
分子类型:多肽
起始来源:
生物:Kurthia sp.
菌株:538-KA26
特 征:
定义方法:E
序列描述:Met Ile Asp Lys Gln Leu Leu Ser Lys Arg Phe Ser Glu His Ala1 5 10 15Lys Thr Tyr Asp Ala Tyr Ala Asn Val Gln Lys Asn Met Ala Lys
20 25 30Gln Leu Val Asp Leu Leu Pro Gln Lys Asn Ser Lys Gln Arg Ile
35 40 45Asn Ile Leu Glu Ile Gly Cys Gly Thr Gly Tyr Leu Thr Arg Leu
50 55 60Leu Val Asn Thr Phe Pro Asn Ala Ser Ile Thr Ala Val Asp Leu
65 70 75Ala Pro Gly Met Val Glu Val Ala Lys Gly Ile Thr Met Glu Asp
80 85 90Arg Val Thr Phe Leu Cys Ala Asp Ile Glu Glu Met Thr Leu Asn
95 100 105Glu Asn Tyr Asp Leu Ile Ile Ser Asn Ala Thr Phe Gln Trp Leu
110 115 120Asn Asn Leu Pro Gly Thr Ile Glu Gln Leu Phe Thr Arg Leu Thr
125 130 135Pro Glu Gly Asn Leu Ile Phe Ser Thr Phe Gly Ile Lys Thr Phe
140 145 150Gln Glu Leu His Met Ser Tyr Glu His Ala Lys Glu Lys Leu Gln
155 160 165Leu Ser Ile Asp Ser Ser Pro Gly Gln Leu Phe Tyr Ala Leu Glu
170 175 180Glu Leu Ser Gln Ile Cys Glu Glu Ala Ile Pro Phe Ser Ser Ala
185 190 195Phe Pro Leu Glu Ile Thr Lys Ile Glu Lys Leu Glu Leu Glu Tyr
200 205 210Phe Gln Thr Val Arg Glu Phe Phe Thr Ser Ile Lys Lys Ile Gly
215 220 225Ala Ala Asn Ser Asn Lys Glu Asn Tyr Cys Gln Arg Pro Ser Phe
230 235 240Phe Arg Glu Leu Ile Asn Ile Tyr Glu Thr Lys Tyr Gln Asp Glu
245 250 255Ser Gly Val Lys Ala Thr Tyr His Cys Leu Phe Phe Lys Ile Ile
260 265 270Lys His Ala Pro Leu Pro
275
Claims (7)
1.一种DNA序列,该序列编码以SEQ ID N0.2、4、6、8、10、12、14或16为代表的多肽和包含一个或多个氨基酸残基的添加、插入、缺失和/或取代的所述多肽的功能性衍生物。
2.一种载体,该载体包含如权利要求1所限定的一个或多个DNA序列。
3.权利要求2所限定的载体,其中所说的DNA序列与启动子序列功能性地连接。
4.一种细胞,该细胞是由一种或多种权利要求1的DNA序列或权利要求2或3所限定的载体转化的细胞。
5.一种产生生物素的方法,该方法包括在培养基中培养权利要求4所限定的细胞和利用本领域公知的方法从培养基中分离形成的生物素。
6.权利要求5的方法,其中所说的培养在pH值5至9,优选的是6至8下,于10℃至45℃,优选的是25℃至30℃的温度范围内进行1至10天,优选的是2至7天。
7.一种制备食品或饲料组合物的方法,其特征在于:把用权利要求5或6的方法获得的生物素与一种或多种通常应用的添加剂混合。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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EP96115540.5 | 1996-09-27 | ||
EP96115540 | 1996-09-27 |
Publications (2)
Publication Number | Publication Date |
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CN1178834A true CN1178834A (zh) | 1998-04-15 |
CN1154722C CN1154722C (zh) | 2004-06-23 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CNB971196796A Expired - Fee Related CN1154722C (zh) | 1996-09-27 | 1997-09-26 | 生物素生物合成基因ⅱ |
Country Status (9)
Country | Link |
---|---|
US (4) | US6117669A (zh) |
EP (1) | EP0853127B1 (zh) |
JP (1) | JP4267715B2 (zh) |
CN (1) | CN1154722C (zh) |
AT (1) | ATE281527T1 (zh) |
BR (1) | BR9704908A (zh) |
DE (1) | DE69731451T2 (zh) |
DK (1) | DK0853127T3 (zh) |
ES (1) | ES2231835T3 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101848924A (zh) * | 2007-05-09 | 2010-09-29 | 马斯科马公司 | 基因敲除的嗜温和嗜热生物以及其使用方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US7423136B2 (en) * | 2005-10-19 | 2008-09-09 | Stephen F. Austin State University | Nucleic acid for biotin production |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1317245C (en) * | 1985-08-26 | 1993-05-04 | Eric F. Fisher | System for biotin synthesis |
AU616380B2 (en) * | 1986-09-30 | 1991-10-31 | Transgene S.A. | Cloning of the bioA, bioD, bioF, bioC, and bioH genes of Bacillus sphaericus, vectors and transformed cells and method for preparing biotin |
FR2640642B1 (fr) * | 1988-12-16 | 1993-08-27 | Transgene Sa | Nouvelle souche d'escherichia coli recombinante et procede pour la preparation de biotine utilisant ladite souche |
KR100200542B1 (ko) * | 1992-10-02 | 1999-06-15 | 토마스 케플러, 자비네 루츠 | 바이오틴의 생물공학적 제조방법 |
US6277609B1 (en) * | 1993-01-06 | 2001-08-21 | Basf Aktiengesellschaft | Method to produce biotin |
EP0635572A3 (en) * | 1993-06-25 | 1995-03-08 | Hoffmann La Roche | Biosynthesis of biotin in bacillus subtilis. |
ES2216027T3 (es) * | 1995-06-09 | 2004-10-16 | Dsm Ip Assets B.V. | Produccion de carotenoides fermentativos. |
JP4087919B2 (ja) | 1996-04-06 | 2008-05-21 | ディーエスエム アイピー アセッツ ビー.ブイ. | d−ビオチンの発酵による製造 |
DE69722852T2 (de) | 1996-05-06 | 2004-05-06 | F. Hoffmann-La Roche Ag | Fermentative Herstellung von Biotin |
-
1997
- 1997-09-18 ES ES97116237T patent/ES2231835T3/es not_active Expired - Lifetime
- 1997-09-18 AT AT97116237T patent/ATE281527T1/de not_active IP Right Cessation
- 1997-09-18 DK DK97116237T patent/DK0853127T3/da active
- 1997-09-18 EP EP97116237A patent/EP0853127B1/en not_active Expired - Lifetime
- 1997-09-18 DE DE69731451T patent/DE69731451T2/de not_active Expired - Fee Related
- 1997-09-22 US US08/935,263 patent/US6117669A/en not_active Expired - Fee Related
- 1997-09-25 JP JP26066197A patent/JP4267715B2/ja not_active Expired - Fee Related
- 1997-09-26 CN CNB971196796A patent/CN1154722C/zh not_active Expired - Fee Related
- 1997-09-29 BR BR9704908A patent/BR9704908A/pt not_active IP Right Cessation
-
2000
- 2000-06-14 US US09/594,185 patent/US6365388B1/en not_active Expired - Fee Related
-
2001
- 2001-12-27 US US10/033,078 patent/US6723544B2/en not_active Expired - Fee Related
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101848924A (zh) * | 2007-05-09 | 2010-09-29 | 马斯科马公司 | 基因敲除的嗜温和嗜热生物以及其使用方法 |
Also Published As
Publication number | Publication date |
---|---|
US20040137584A1 (en) | 2004-07-15 |
US6723544B2 (en) | 2004-04-20 |
JPH10179172A (ja) | 1998-07-07 |
US20020123109A1 (en) | 2002-09-05 |
US6365388B1 (en) | 2002-04-02 |
US6117669A (en) | 2000-09-12 |
EP0853127A3 (en) | 1999-01-13 |
DE69731451T2 (de) | 2005-11-24 |
DE69731451D1 (de) | 2004-12-09 |
BR9704908A (pt) | 1998-10-27 |
US6955906B2 (en) | 2005-10-18 |
DK0853127T3 (da) | 2005-03-07 |
CN1154722C (zh) | 2004-06-23 |
ATE281527T1 (de) | 2004-11-15 |
EP0853127A2 (en) | 1998-07-15 |
JP4267715B2 (ja) | 2009-05-27 |
ES2231835T3 (es) | 2005-05-16 |
EP0853127B1 (en) | 2004-11-03 |
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