CN113388538A - 重组酵母、构建方法和其在制备酪醇及衍生物中的应用 - Google Patents

重组酵母、构建方法和其在制备酪醇及衍生物中的应用 Download PDF

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CN113388538A
CN113388538A CN202110653988.1A CN202110653988A CN113388538A CN 113388538 A CN113388538 A CN 113388538A CN 202110653988 A CN202110653988 A CN 202110653988A CN 113388538 A CN113388538 A CN 113388538A
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方诩
郭伟
侯少莉
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Abstract

本发明涉及重组酵母、构建方法和其在制备酪醇及衍生物中的应用，所述重组酵母为将外源性的果糖‑6‑磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中构建获得，所述经过改造的酵母细胞为具有经4‑磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。本发明首次公开了在酵母中表达果糖‑6‑磷酸盐磷酸酮酶的过程中，果糖‑6‑磷酸在合成1,6‑二磷酸果糖的同时，被催化为赤藓糖‑4‑磷酸和乙酰磷酸，木酮糖‑5‑磷酸被催化为甘油醛‑3‑磷酸和乙酰磷酸，这改变了酵母中的碳代谢流量分布，增强酪醇生物合成重要中间物质的赤藓糖‑4‑磷酸的合成，优化了合成酪醇的代谢途径，提高酪醇及羟基酪醇等衍生物的产率。

Description

重组酵母、构建方法和其在制备酪醇及衍生物中的应用
本案为申请号202010012886.7、申请日为2020年01月07日的分案申请
技术领域
本发明涉及重组酵母、构建方法和其在制备酪醇及衍生物中的应用，特别涉及一株通过外源性的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase，fxp)的表达基因导入酵母，以及利用该菌株高效生产酪醇及其衍生物的方法，属于微生物基因工程技术领域。
背景技术
酪醇是天然的抗氧化剂，来源于橄榄油，是苯乙醇的一种衍生物。别名红景天苷元，是红景天的主要药用活性成分，是红景天苷、羟基酪醇的前体物质。可以保护细胞免受氧化伤害，是一种具有重要工业价值的酚类化合物，酪醇及其衍生物是多种有机化合物的合成前体，酪醇可被用于医药剂。酪醇的衍生物羟基酪醇是一种具有很强的抗氧化作用及多种生理医药功能，羟基酪醇的抗氧化性强于酪醇，同时可以合成很多聚合物，且没有已知毒性，在生物医药、功能食品等行业应用广泛，具有预防心血管、骨质缺乏等疾病的发生。目前，羟基酪醇的获得主要是从橄榄叶中提取，从植物中提取，成本高，占用大量的耕地。
化学法中利用苯乙醇合成法，大多先采用保护羟基，然后硝化、还原、重氮化、水解得到对羟基苯乙醇，收率为70％。苯乙醇价格高供应紧张，利用硝基甲苯合成，价格低廉但步骤较长，产率低，利用对羟基苯乙烯合成产率达到96％，纯度99％，产率和纯度都很高，具有一定的价值，但原料成本较高。化学法制备酪醇原料成本高且环境不友好，这些都直接制约了酪醇的工业化生产。因此，生物法合成酪醇及其衍生物已经成为研究热点。
酪醇(Tyrosol)具有以下特征：化学名称为4-(2-Hydroxyethyl)phenol，分子式为C8H10O2，分子量为138.164，CAS号为501-94-0，结构式为
Figure BDA0003113048580000011
中国专利文献CN108753636A(申请号201810601213.8)公开了一种生产酪醇及羟基酪醇的酵母及构建方法，将PcAAS和ADH序列导入酵母BY4741，得到生产酪醇的PcAAS-ADH重组酵母；在所述PcAAS-ADH重组酵母中导入pdc1基因敲除盒、tyrA表达盒得到生产酪醇的PcAAS-ADH-Δpdc1-tyrA重组酵母；将HpaBC的DNA序列导入PcAAS-ADH-Δpdc1-tyrA重组酵母，得到生产羟基酪醇的PcAAS-ADH-HpaBC-Δpdc1-tyrA重组酵母。在酵母BY4741中构建酪醇或羟基酪醇生物合成途径，提高酪醇或羟基酪醇的产量。该技术虽然可以提高酵母中酪醇的产量，但酪醇产量仍然无法达到工业化生产的要求。受限于酵母中酪醇的合成受多种代谢途径的影响，且相关代谢途径并未完全研究清楚，因此，如何实现酪醇酵母发酵工业化仍然是目前亟需解决的技术难题。
发明内容
本发明针对现有技术的不足，提供重组酵母、构建方法和其在制备酪醇及衍生物中的应用。
本发明的目的之一是在发明专利申请(申请号201810601213.8)的基础上，在酵母中表达来源于青春双岐杆菌(Bifidobacterium adolescentis)菌种编号为ATCC 15703的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC 4.1.2.22)(氨基酸序列如GenBank:BAF39468.1所示，SEQ ID NO.1)或短双歧杆菌(Bifidobacteriumbreve)BBRI4的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC4.1.2.22)(氨基酸序列如GenBank:KND53308.1所示，SEQ ID NO.2)的基因片段等，通过构建由果糖-6-磷酸催化生成酪醇生物合成重要前体物质赤藓糖-4-磷酸的新途径，提高酪醇的产率。
本发明的目的之二是提供一种生产羟基酪醇的方法。
本发明的目的之三是提供一种生产酪醇的酵母的构建方法。
本发明的目的之四是提供所述一种生产酪醇的酵母或构建方法在生产酪醇中的应用。
本发明的目的之五是提供所述一种生产酪醇的酵母或构建方法在生产羟基酪醇中的应用。
为了解决以上技术问题，本发明的技术方案如下：
重组酵母在生产酪醇中的应用，所述重组酵母为将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中构建获得，所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。
根据本发明优选的，所述经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
根据本发明进一步优选的，所述芳香醛合成酶来源于香芹，酶的系统编号EC4.1.1.25；所述分支酸变位酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12；所述预苯酸脱水酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12，EC 5.4.99.5。
根据本发明优选的，所述果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacterium animalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、短双歧杆菌(Bifidobacterium breve)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、齿双歧杆菌(Bifidobacterium dentium)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)、类植物乳杆菌(Lactobacillusparaplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)等。
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.1或SEQ IDNO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示。
根据本发明优选的，所述酵母细胞为：酿酒酵母(Saccharomyces cerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candida glabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candidaauris)。
进一步优选的，所述酵母细胞为酿酒酵母(Saccharomyces cerevisiae)，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777。
根据本发明更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinumcrispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得。
一种高产酪醇的重组酵母的构建方法，包括如下步骤：
(1)构建表达盒，表达盒由启动子、终止子、同源臂以及果糖-6-磷酸盐磷酸酮酶的表达基因经融合后获得；
(2)将步骤(1)构建的表达盒转化至经过改造的酵母细胞，制得高产酪醇的重组酵母；
所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。
根据本发明更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinumcrispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得；
根据本发明更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinumcrispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
根据本发明优选的，所述步骤(1)中果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacteriumanimalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、短双歧杆菌(Bifidobacterium breve)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、齿双歧杆菌(Bifidobacterium dentium)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillusplantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candidatropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidusnecator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorulaglutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomycespastorianus)等。
更优的，所述果糖-6-磷酸盐磷酸酮酶的的氨基酸序列如SEQ ID NO.1或SEQ IDNO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示。
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示。
根据本发明优选的，所述步骤(1)中同源臂为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板，利用引物扩增预苯酸脱水酶(prephenatedehydratase)基因pha2上游及下游500bp的基因片段，所述上游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；下游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.7和SEQ ID NO.8所示；
根据本发明优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的启动子tpi1，所述启动子tpi1的扩增引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；
根据本发明优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的终止子gpm1，所述终止子gpm1的扩增引物的核苷酸序列分别如SEQ ID NO.9和SEQ ID NO.10所示；
根据本发明优选的，所述步骤(2)中，经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
根据本发明进一步优选的，所述步骤(2)中，芳香醛合成酶来源于香芹(Petroselinum crispum)，酶的系统编号EC4.1.1.25；所述分支酸变位酶/预苯酸脱水酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12，EC 5.4.99.5。
根据本发明优选的，所述步骤(2)中所述酵母细胞为：酿酒酵母(Saccharomycescerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candidaglabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candida auris)等。
进一步优选的，所述酵母细胞为酿酒酵母，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777。
进一步优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，获得。
根据本发明更优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得。
进一步优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，获得。
根据本发明更优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
上述方法构建的高产酪醇的重组酵母。
上述高产酪醇的重组酵母在发酵制备酪醇中的应用。
根据本发明优选的，所述发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
上述高产酪醇的重组酵母在发酵制备羟基酪醇中的应用。
根据本发明优选的，将上述高产酪醇的重组酵母通过发酵制备酪醇后，经过羟化酶反应获得羟基酪醇。
根据本发明优选的，将上述高产酪醇的重组酵母经发酵所得酪醇，利用过表达4-羟基苯乙酸羟化酶的大肠杆菌催化，获得羟基酪醇。
根据本发明进一步优选的，所述的发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
本发明的有益效果：
1、本发明首次公开了在酵母中表达过程中，果糖-6-磷酸(Fructose-6-phosphate)在合成1,6-二磷酸果糖(beta-D-Fructose 1,6-bisphosphate)的同时，被催化为赤藓糖-4-磷酸(Erythrose-4-phosphate)和乙酰磷酸(Acetyl-phosphate)，木酮糖-5-磷酸(Xylulose-5-phosphate)被催化为甘油醛-3-磷酸(Glyceraldehydes-3-phosphate)和乙酰磷酸，这改变了酵母中的碳代谢流量分布，增强酪醇生物合成重要中间物质的赤藓糖-4-磷酸的合成，优化了合成酪醇的代谢途径，提高酪醇及羟基酪醇等衍生物的产率；
2、本发明将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中，获得了一种重组酵母，该重组酵母可以提高酪醇的产率，并利用过表达4-羟基苯乙酸羟化酶的大肠杆菌催化酪醇后，获得羟基酪醇。
3、本发明提供了一种新型且环境友好型酪醇及羟基酪醇生物制备技术，为酪醇及羟基酪醇的大规模工业生产奠定了基础，具有重要的经济价值和社会效益。
附图说明
图1为实施例1中所述的重组质粒pUG6的结构图；
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
下面结合实施例对本发明进一步说明：
表1中英文名称的对照
Figure BDA0003113048580000071
Figure BDA0003113048580000081
以下实施例中，大肠杆菌BL21和表达载体pET-28a为市售常用。下列实施例中未注明具体条件的实验方法，按照常规条件进行，例如《分子克隆：实验室手册》中所述的条件，或按照相应生物学试剂的制造厂商所建议的条件。PCR扩增反应的反应程序可以为常规的PCR扩增反应程序。
酿酒酵母CICC1964，购自中国工业微生物菌种保藏管理中心，菌种编号CICC1964，为已知非保藏菌株；
按照发明专利申请(申请号201810601213.8)的方法，将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenatedehydrogenase，EC1.3.1.12，EC 5.4.99.5)替换酿酒酵母CICC1964的pdc1基因，获得SC-1菌株。
实施例1
Bafxpk和Bbfxpk表达盒的构建
将SEQ ID NO.1和SEQ ID NO.2的氨基酸序列根据宿主酿酒酵母密码子偏好性进行密码子优化，获得SEQ ID NO.1和SEQ ID NO.2对应的优化后核苷酸序列SEQ ID NO.3和SEQ ID NO.4后，进行基因合成。选用引物对Bafxpk-F/Bafxpk-R和Bbfxpk-F/Bbfxpk-R，用Vazyme公司的Phanta Max High-Fidelity DNA聚合酶扩增获得目标基因果糖-6-磷酸盐磷酸酮酶的表达基因(Bafxpk片段核苷酸序列如SEQ ID NO.3所示，Bbfxpk片段核苷酸序列如SEQ ID NO.4所示)。以酿酒酵母CICC1964的基因组为模板，以引物对U-F/U-R和D-F/D-RPCR扩增上下游同源臂的DNA片段，以引物对Ptpi1-F/Ptpi1-R和Tgpm1-F/Tgpm1-R扩增启动子tpi1和终止子gpm1片段。以带有遗传霉素(Geneticin)抗性基因KanMX4(核苷酸序列如SEQ ID NO.24所示)的重组质粒pUG6见附图1的DNA为模板，以引物对G418-F/G418-R经PCR扩增抗性基因KanMX4的DNA片段。琼脂糖凝胶电泳验证条带大小正确后切取条带，用OMEGA凝胶提取试剂盒回收基因片段。PCR扩增引物如下：
引物对Bafxpk-F/Bafxpk-R的序列为SEQ ID NO.13和SEQ ID NO.14；引物对Bbfxpk-F/Bbfxpk-R的序列为SEQ ID NO.15和SEQ ID NO.16；引物对U-F/U-R的序列为SEQID NO.5和SEQ ID NO.6；引物对D-F/D-R的序列为SEQ ID NO.7和SEQ ID NO.8；引物对Ptpi1-F/Ptpi1-R的序列为SEQ ID NO.9和SEQ ID NO.10；引物对Tgpm1-F/Tgpm1-R的序列为SEQ ID NO.11和SEQ ID NO.12；引物对G418-F/G418-R的序列为SEQ ID NO.17和SEQ IDNO.18；
使用高保真性Phanta Max High-Fidelity DNA聚合酶扩增目的基因片段，并保证每条片段都与相邻片段存在50bp的同源序列，PCR产物经凝胶电泳后使用DNA片段胶回收试剂盒回收，并测定DNA浓度。随后将获得的纯化后的带有同源序列的DNA片段采用融合PCR的方法进行融合：
(1)使用Phanta Max High-Fidelity DNA聚合酶对片段进行连接。反应体系如表2所示：
表2反应体系
Figure BDA0003113048580000091
将上述试剂加入PCR管中，反应条件如表3所示：
表3反应条件
Figure BDA0003113048580000092
Figure BDA0003113048580000101
(2)以步骤1的PCR反应产物作为PCR扩增模板，选用引物对Yzaw-F/Yzaw-R，用Vazyme公司的Phanta Max High-Fidelity DNA聚合酶扩增获得目标片段。琼脂糖凝胶电泳验证条带大小正确后切取条带，用OMEGA凝胶提取试剂盒回收DNA片段即Bafxpk和Bbfxpk表达盒的DNA片段。PCR扩增引物如下：
Yzaw-F的序列为SEQ ID NO.19；Yzaw-R的序列为SEQ ID NO.20；
(3)将步骤2所获得的Bafxpk和Bbfxpk表达盒的DNA片段，测序验证。
实施例2
Bafxpk和Bbfxpk异源表达菌株的构建，以酿酒酵母为例：
采用PEG/LiAc法转化酿酒酵母酪醇合成菌株CICC1964，通过在培养基中添加G418抗性进行筛选挑取单克隆，提取基因组，利用引物对Yzaw-F/Yzaw-R进行PCR验证，获得SC-bafxpk和SC-bbfxpk菌株。
实施例3
合成酪醇微生物的发酵，以酿酒酵母为例：
在产酪醇的菌株CICC1964和SC-bafxpk和SC-bbfxpk的平板上挑取单克隆，接种到5mL YPD液体培养基中，在30～32℃，200rpm条件下培养24h，转接到50mL YPD液体培养基中，初始接种OD600为0.2，30℃，200rpm条件下培养12h后，转接到100mL YPD液体培养基中，初始接种OD600为0.2，培养基中分别含有2％葡萄糖或者2％蔗糖或者2％葡萄糖和1％酪氨酸等碳源，培养24小时后，再次加入2％葡萄糖或者2％蔗糖或者2％葡萄糖和1％酪氨酸等碳源，共进行72小时发酵。采用文献(Satoh et al.,Journal of Agricultural and FoodChemistry,60,979-984,2012)报道的HPLC法检测发酵液中酪醇的浓度。不同碳源培养条件下酪醇产量如表4所示。
表4不同碳源下发酵72小时后酪醇的产量
Figure BDA0003113048580000102
Figure BDA0003113048580000111
实施例4
基因HpaBC及大肠杆菌表达载体的获得方法
将来源于大肠杆菌的4-羟基苯乙酸羟化酶(HpaBC，4-hydroxyphenylacetate3-hydroxylase，酶的系统编号EC 1.5.1.37)的DNA序列基因簇在大肠杆菌中过表达，利用大肠杆菌全细胞将酪醇催化为羟基酪醇。
所述4-羟基苯乙酸羟化酶的氨基酸序列基因簇包括4-羟基苯乙酸羟化酶(HpaB)的氨基酸序列为SEQ ID NO.21，对应的核苷酸序列为SEQ ID NO.23；4-羟基苯乙酸羟化酶(HpaC)的氨基酸序列为SEQ ID NO.22，对应的核苷酸序列为SEQ ID NO.24。
利用细菌基因组试剂盒提取大肠杆菌DE3基因组作为模板，分别使用引物对hpaB-F/hpaB-R和hpaC-F/hpaC-R来扩增SEQ ID NO.23和SEQ ID NO.24，测序验证。hpaB-F的序列为SEQ ID NO.25；hpaB-R的序列为SEQ ID NO.26；hpaC-F的序列为SEQ ID NO.27；hpaC-R的序列为SEQ ID NO.28。
以pET-28a作为表达载体，培养含有pET-28a空载体的大肠杆菌，利用细菌质粒提取试剂盒提取pET-28a质粒，采用常规分子生物学方法，构建表达载体pEThpaBC(SEQ IDNO.29)。然后将pEThpaBC转化大肠杆菌表达型载体BL21，以卡那霉素为筛选标记，获得单克隆BL21-pEThpaBC。
摇瓶培养BL21-pEThpaBC，使用1mM IPTG诱导表达HPAB/C的表达。
将获得的BL21-pEThpaBC菌培养液添加到实施例3的培养液中，反应3小时后，检测获得的羟基酪醇产量结果如表5所示：
表5在表4所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure BDA0003113048580000112
实施例5
采用马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777，按照发明专利申请(申请号201810601213.8)的方法，将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到马克斯克鲁维酵母的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC 5.4.99.5)替换马克斯克鲁维酵母的pdc1基因，获得具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的马克斯克鲁维酵母菌株。
按照本发明实施例1-2所述方法，将果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC 4.1.2.22)制备获得的KM-bafxp和KM-bbfxp菌株，不同之处在于，马克斯克鲁维酵母细胞的培养温度为42～49℃。
实施例6
采用来源于齿双歧杆菌(Bifidobacterium dentium)的果糖-6-磷酸盐磷酸酮酶(bdfxp，GenBank：BAQ26957.1)，氨基酸序列如SEQ ID NO.30所示，按照实施例3和实施例4所述的方法进行，所用引物对Bdfxp-F/Bdfxp-R的序列为SEQ ID NO.32和SEQ ID NO.33；结果与实施例3和实施例4的相似，也提高了酪醇和羟基酪醇的产量。具体结果如下：
表6不同碳源下发酵72小时后酪醇的产量
Figure BDA0003113048580000121
表7在表6所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure BDA0003113048580000122
实施例7
按照实施例1-2所述的方法，将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到酿酒酵母CICC1964的delta12位点，并且将如SEQ ID NO.4所示的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC4.1.2.22)导入上述酵母中，获得SC-bbfxpk-AAS菌株。结果与实施例3中SC-bbfxpk菌株的酪醇产量相似。
将获得的BL21-pEThpaBC菌培养液添加到上述制得的含有酪醇的培养液中，反应3小时后，检测获得的羟基酪醇产量也和实施例4中所述SC-bbfxpk菌株的产量相似。说明酵母中没有分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenatedehydrogenase，EC1.3.1.12，EC 5.4.99.5)基因导入，也可以达到相近的酪醇和羟基酪醇产量；具体结果如表8和表9所示：
表8不同碳源下发酵72小时后酪醇的产量
Figure BDA0003113048580000131
表9在表8所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure BDA0003113048580000132
实施例8
按照实施例6所述的方法，分别将来源于动物双歧杆菌(Bifidobacteriumanimalis)的果糖-6-磷酸盐磷酸酮酶(baifxp，GenBank:WP_052826255.1)氨基酸序列如SEQ ID NO.34所示，两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)的果糖-6-磷酸盐磷酸酮酶(bbifxp，GenBank:WP_047289945.1)氨基酸序列如SEQ ID NO.35所示，乳酸双歧杆菌(Bifidobacterium lactis)的果糖-6-磷酸盐磷酸酮酶(blafxp，GenBank:CAC29121.1)氨基酸序列如SEQ ID NO.36所示，长双歧杆菌(Bifidobacterium longum)果糖-6-磷酸盐磷酸酮酶(blofxp，GenBank:PWH09343.1)氨基酸序列如SEQ ID NO.37所示，摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)的果糖-6-磷酸盐磷酸酮酶(bmfxp，GenBank:CAC29121.1)氨基酸序列如SEQ ID NO.38所示，假长双歧杆菌(Bifidobacteriumpseudolongum)的果糖-6-磷酸盐磷酸酮酶(bpfxp，GenBank:WP_034883174.1)氨基酸序列如SEQ ID NO.39所示，构巢曲霉(Aspergillus nidulans)的果糖-6-磷酸盐磷酸酮酶(Anfxp，GenBank:CBF76492.1)氨基酸序列如SEQ ID NO.40所示，丙酮丁醇梭菌(Clostridium acetobutylicum)的果糖-6-磷酸盐磷酸酮酶(Cafxp，GenBank:KHD36088.1)氨基酸序列如SEQ ID NO.41所示，肠系膜明串珠菌(Leuconostoc mesenteroides)的果糖-6-磷酸盐磷酸酮酶(Lmfxp，GenBank:AAV66077.1)氨基酸序列如SEQ ID NO.42所示，类植物乳杆菌(Lactobacillus paraplantarum)的果糖-6-磷酸盐磷酸酮酶(Lprfxp，GenBank:ALO04878.1)氨基酸序列如SEQ ID NO.43所示，胚芽乳杆菌(Lactobacillus plantarum)的果糖-6-磷酸盐磷酸酮酶(Lplfxp，GenBank:KRU19755.1)氨基酸序列如SEQ ID NO.44所示对应的基因片段转化改造的酿酒酵母；分别获得改造菌株SC-baifxp，SC-bbifxp，SC-blafxp，SC-blofxp，SC-bmfxp，SC-bpfxp，SC-Anfxp，SC-Cafxp，SC-Lmfxp，SC-Lprfxp，SC-Lplfxp；所述改造的酿酒酵母为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点。结果与实施例3和实施例4的相似，也提高了酪醇和羟基酪醇的产量。具体结果如表8和表9所示：
表8不同碳源下发酵72小时后酪醇的产量
Figure BDA0003113048580000141
表9在表8所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure BDA0003113048580000151
从以上的结果来看，当酵母细胞中表达以上外源性的果糖-6-磷酸盐磷酸酮酶时，虽然产量有所不同，和实施例3、实施例4和实施例5的来源于青春双岐杆菌、短双歧杆菌以及齿双歧杆菌的果糖-6-磷酸盐磷酸酮酶相比，产量较低，但是和出发菌株相比，酪醇和羟基酪醇的产量均有显著提高。
基于实施例7的结果，发明人同时对以上果糖-6-磷酸盐磷酸酮酶对应的基因片段转化按照发明专利申请(申请号201810601213.8)的方法获得的SC-1菌株，即：将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenatedehydrogenase，EC1.3.1.12，EC5.4.99.5)替换酿酒酵母CICC1964的pdc1基因获得；结果显示baifxp，bbifxp，blafxp，blofxp，bmfxp，bpfxp，Anfxp，Cafxp，Lmfxp，Lprfxp，Lplfxp与实施例7中bbfxpk的结果类似，从而说明酵母中有没有分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC 5.4.99.5)基因的导入，对酪醇和羟基酪醇产量影响不大具有普遍性。
结果分析
通过上述结果本领域技术人员可以发现，当酵母细胞中表达外源性的果糖-6-磷酸盐磷酸酮酶时，可以显著提高酪醇的产量，并且该现象并非仅限于一种来源的果糖-6-磷酸盐磷酸酮酶和特异的酵母细胞，本领域技术人员通过本发明的技术教导可以预期，具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞中通过表达果糖-6-磷酸盐磷酸酮酶后，由于代谢外源性的果糖-6-磷酸盐磷酸酮酶的表达影响了酵母细胞的该糖代谢途径，从而促进了酪醇产物的生成，因此均能够产生本申请所述的技术效果。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
序列表
<110> 山东恒鲁生物科技有限公司
<120> 重组酵母、构建方法和其在制备酪醇及衍生物中的应用
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gctcaaactg atgatatgac tagaccattt taccccatga tcattttcag aactccaaaa 900
ggttggacct gtccaaagtt tatcgatggt aaaaagactg aaggctcttg gagatctcat 960
caagttccat tggcatctgc tagagatact gaagctcatt tcgaagtttt gaagaactgg 1020
ttggaaagct acaaacctga agagttgttt gacgaaaatg gtgcagttaa gcctgaagtt 1080
actgctttta tgccaacagg tgaattgaga attggtgaaa atccaaatgc taacggtggt 1140
agaatcaggg aagaattgaa attgccaaag ttggaagatt acgaggtcaa agaagttgct 1200
gaatacggtc atggttgggg tcaattggaa gctactagaa gattgggtgt ttacaccaga 1260
gacatcatca agaacaatcc agactccttc agaatttttg gtccagacga aactgcttct 1320
aacagattgc aagctgctta cgatgttacc aacaaacaat gggatgctgg ttatttgtct 1380
gcccaagttg atgaacatat ggctgttact ggtcaagtta ccgaacaatt gtcagaacat 1440
caaatggaag gtttcttgga gggttacttg ttgactggta gacatggtat ttggtcctct 1500
tacgaatctt tcgttcacgt tatcgactct atgttgaatc aacacgctaa gtggttagaa 1560
gctaccgtta gagaaattcc ttggagaaag ccaatctcct ctatgaactt gttggtttct 1620
tcacacgttt ggagacaaga tcataacggt ttttcacatc aagatccagg tgttacttct 1680
gtcttgttga acaagtgttt caacaacgat catgtcatcg gtatctactt cccagttgac 1740
tctaatatgt tgttggctgt tgctgaaaag tgctacaaat ctaccaacaa gatcaacgct 1800
attatcgctg gtaagcaacc agctgctact tggttgactt tagatgaagc tagagctgaa 1860
ttggaaaaag gtgctgctga atggaaatgg gcttctaatg ttaagtccaa cgatgaagct 1920
caaatcgttt tggctgctac aggtgatgtt ccaactcaag aaattatggc tgcagctgat 1980
aagttggatg ctatgggtat taagttcaag gttgttaacg ttgtcgacct ggttaagttg 2040
caatcagcta aagaaaacaa cgaagccttg tccgacgaag aatttgctga gttgtttact 2100
gaagataagc cagttttgtt cgcctaccat tcttacgcta gagatgttag aggtttgata 2160
tacgatagac caaaccacga taacttcaac gttcatggtt atgaagaaca aggttctact 2220
actaccccat acgatatggt tagagttaac aacatcgaca ggtacgaatt acaagctgaa 2280
gccttgagaa tgattgatgc agataagtac gccgacaaga ttaacgaact agaagctttt 2340
agacaagagg cctttcaatt cgctgttgat aatggttacg atcacccaga ttataccgat 2400
tgggtttatt ctggtgtcaa cactaacaag caaggtgcta tttctgctac tgctgcaact 2460
gctggtgata atgaataa 2478
<210> 4
<211> 2475
<212> DNA
<213> 人工序列(人工序列)
<400> 4
atgacctctc cagttattgg tactccatgg aaaaaattga acgccccagt ttctgaagaa 60
tccttggaag gtgttgataa gtattggaga gttgccaact acttgtccat tggtcaaatc 120
tacttgaggt ctaacccatt gatgaaggct ccattcacta gagaagatgt taagcacaga 180
ttggttggtc attggggtac tactccaggt ttgaattttt tgatcggtca catcaacaga 240
ttcattgctg atcatggtca aaacaccgtt attatcatgg gtccaggtca tggtggtcca 300
gctggtactt ctcaatctta tttggatggt acttacaccg agactttccc aaagattaca 360
aaagatgaag ctggcttgca gaagttcttc agacaatttt cttatccagg tggtatccca 420
tctcattttg ctccagaaac accaggttct attcatgaag gtggtgaatt gggttatgct 480
ttgtctcatg cttatggtgc cattatggat aacccatctt tgtttgttcc agccatagtt 540
ggtgatggtg aagctgaaac tggtccattg gctactggtt ggcaatctaa caaattggtt 600
aacccaagaa ccgatggtat cgttttgcca atcttgcatt tgaacggtta caagattgct 660
aacccaacca tcttgtccag aatctctgat gaagaattgc atgagttctt ccatggtatg 720
ggttatgaac catacgaatt tgttgctggt ttcgatgatg aagatcacat gtccattcat 780
agaagattcg ctgaattgtg ggaaaccatt tgggacgaaa tttgcgatat taaggctgct 840
gctcaaaccg ataatgttca tagaccattt taccccatgc tgattttcag aactccaaaa 900
ggttggactt gcccaaagta cattgatggt aaaaagactg aaggttcttg gagagcacat 960
caagttccat tggcatctgc tagagatact gaagctcatt tcgaagtttt gaagaactgg 1020
ttggaaagct acaaacctga agagttgttt gatgctaatg gtgctgttaa ggatgatgtt 1080
ttggctttta tgccaaaggg tgaattgaga attggtgcta atccaaatgc aaacggtggt 1140
gttattagag atgatctgaa gttgccaaac ttggaagatt acgaagtcaa agaagttgcc 1200
gaatacggtc atggttgggg tcaattggaa gctactagaa ctttgggtgc ttacaccaga 1260
gatatcatta gaaacaaccc aagagacttc agaattttcg gtccagacga aactgcttct 1320
aatagattgc aagcttctta cgaggttacc aacaaacaat gggatgctgg ttacatttcc 1380
gatgaagttg atgaacatat gcacgtttct ggtcaagttg tcgaacaatt gtcagaacat 1440
caaatggaag gttttttgga ggcttacttg ttgactggta gacatggtat ttggtcctct 1500
tacgaatctt tcgttcacgt tatcgactct atgttgaatc aacacgctaa atggttggaa 1560
gccaccgtta gagaaattcc ttggagaaaa cctattgcct ccatgaactt gttggtttct 1620
tcacatgttt ggagacaaga tcacaacggt ttctctcatc aagatccagg tgttacttct 1680
gtcttgttga acaagtgttt ccacaacgat catgtcatcg gtatctactt tgctactgat 1740
gctaacatgt tgttggctat tgctgaaaag tgttacaagt ccaccaacaa gattaacgct 1800
attattgctg gtaaacaacc agctgctact tggttgactt tggatgaagc tagagctgaa 1860
ttggctaaag gtgctgctgc ttgggattgg gcttctactg ctaaaaacaa tgatgaagcc 1920
gaagttgttt tagctgctgc tggtgatgtt ccaactcaag aaattatggc tgcttccgac 1980
aagttgaaag aattgggtgt taagttcaag gttgttaacg ttgccgattt gttgtcattg 2040
caatccgcta aagaaaacga cgaagctttg tcagacgaag aatttgctga tatcttcact 2100
gctgataagc cagttttgtt cgcttatcat tcttacgccc atgatgtcag aggtttgata 2160
tacgatagac caaaccacga taacttcaac gttcatggtt atgaagaaga aggttctact 2220
actaccccat acgatatggt tagagttaac agaatcgaca ggtacgaatt gactgctgaa 2280
gctttgagaa tgattgatgc agataagtac gccgataaga tcgacgaatt ggaaaagttc 2340
agagatgaag cctttcaatt cgcagttgat aagggttatg atcacccaga ttataccgat 2400
tgggtttact ctggtgttaa taccgataag aaaggtgcag ttactgctac tgctgcaaca 2460
gctggtgata atgaa 2475
<210> 5
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 5
atatttccgc attcatcctt caattgtgtt taaagattac ggatatttaa 50
<210> 6
<211> 47
<212> DNA
<213> 人工序列(人工序列)
<400> 6
accaataact ggagaggtca tttttagttt atgtatgtgt tttttgt 47
<210> 7
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 7
gctatacgaa gttattaggt gatatatgtt atatagtctt tttatttatg 50
<210> 8
<211> 20
<212> DNA
<213> 人工序列(人工序列)
<400> 8
ctatgttcac tctggcgtgc 20
<210> 9
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 9
atatttccgc attcatcctt caattgtgtt taaagattac ggatatttaa 50
<210> 10
<211> 47
<212> DNA
<213> 人工序列(人工序列)
<400> 10
accaataact ggagaggtca tttttagttt atgtatgtgt tttttgt 47
<210> 11
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 11
tgcaactgct ggtgataatg aataagtctg aagaatgaat gatttgatga 50
<210> 12
<211> 46
<212> DNA
<213> 人工序列(人工序列)
<400> 12
cattatacga agttatatta agggttattc gaactgccca ttcagc 46
<210> 13
<211> 47
<212> DNA
<213> 人工序列(人工序列)
<400> 13
acaaaaaaca catacataaa ctaaaaatga cctctccagt tattggt 47
<210> 14
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 14
tcatcaaatc attcattctt cagacttatt cattatcacc agcagttgca 50
<210> 15
<211> 47
<212> DNA
<213> 人工序列(人工序列)
<400> 15
acaaaaaaca catacataaa ctaaaaatga cctctccagt tattggt 47
<210> 16
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 16
tcatcaaatc attcattctt cagacttatt cattatcacc agctgttgca 50
<210> 17
<211> 46
<212> DNA
<213> 人工序列(人工序列)
<400> 17
gctgaatggg cagttcgaat aacccttaat ataacttcgt ataatg 46
<210> 18
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 18
cataaataaa aagactatat aacatatatc acctaataac ttcgtatagc 50
<210> 19
<211> 24
<212> DNA
<213> 人工序列(人工序列)
<400> 19
ctccgtttat gataagatga tggc 24
<210> 20
<211> 21
<212> DNA
<213> 人工序列(人工序列)
<400> 20
gttaattgcc tccgcatcta a 21
<210> 21
<211> 520
<212> PRT
<213> 大肠杆菌(E Coli.)
<400> 21
Met Lys Pro Glu Asp Phe Arg Ala Ser Thr Gln Arg Pro Phe Thr Gly
1 5 10 15
Glu Glu Tyr Leu Lys Ser Leu Gln Asp Gly Arg Glu Ile Tyr Ile Tyr
20 25 30
Gly Glu Arg Val Lys Asp Val Thr Thr His Pro Ala Phe Arg Asn Ala
35 40 45
Ala Ala Ser Val Ala Gln Leu Tyr Asp Ala Leu His Lys Pro Glu Met
50 55 60
Gln Asp Ser Leu Cys Trp Asn Thr Asp Thr Gly Ser Gly Gly Tyr Thr
65 70 75 80
His Lys Phe Phe Arg Val Ala Lys Ser Ala Asp Asp Leu Arg His Glu
85 90 95
Arg Asp Ala Ile Ala Glu Trp Ser Arg Leu Ser Tyr Gly Trp Met Gly
100 105 110
Arg Thr Pro Asp Tyr Lys Ala Ala Phe Gly Cys Ala Leu Gly Gly Thr
115 120 125
Pro Gly Phe Tyr Gly Gln Phe Glu Gln Asn Ala Arg Asn Trp Tyr Thr
130 135 140
Arg Ile Gln Glu Thr Gly Leu Tyr Phe Asn His Ala Ile Val Asn Pro
145 150 155 160
Pro Ile Asp Arg His Leu Pro Thr Asp Lys Val Lys Asp Val Tyr Ile
165 170 175
Lys Leu Glu Lys Glu Thr Asp Ala Gly Ile Ile Val Ser Gly Ala Lys
180 185 190
Val Val Ala Thr Asn Ser Ala Leu Thr His Tyr Asn Met Ile Gly Phe
195 200 205
Gly Ser Ala Gln Val Met Gly Glu Asn Pro Asp Phe Ala Leu Met Phe
210 215 220
Val Ala Pro Met Asp Ala Asp Gly Val Lys Leu Ile Ser Arg Ala Ser
225 230 235 240
Tyr Glu Met Val Ala Gly Ala Thr Gly Ser Pro Tyr Asp Tyr Pro Leu
245 250 255
Ser Ser Arg Phe Asp Glu Asn Asp Ala Ile Leu Val Met Asp Asn Val
260 265 270
Leu Ile Pro Trp Glu Asn Val Leu Leu Tyr Arg Asp Phe Asp Arg Cys
275 280 285
Arg Arg Trp Thr Met Glu Gly Gly Phe Ala Arg Met Tyr Pro Leu Gln
290 295 300
Ala Cys Val Arg Leu Ala Val Lys Leu Asp Phe Ile Thr Ala Leu Leu
305 310 315 320
Lys Lys Ser Leu Glu Cys Thr Gly Thr Leu Glu Phe Arg Gly Val Gln
325 330 335
Ala Asp Leu Gly Glu Val Val Ala Trp Arg Asn Thr Phe Trp Ala Leu
340 345 350
Ser Asp Ser Met Cys Ser Glu Ala Thr Pro Trp Val Asn Gly Ala Tyr
355 360 365
Leu Pro Asp His Ala Ala Leu Gln Thr Tyr Arg Val Leu Ala Pro Met
370 375 380
Ala Tyr Ala Lys Ile Lys Asn Ile Ile Glu Arg Asn Val Thr Ser Gly
385 390 395 400
Leu Ile Tyr Leu Pro Ser Ser Ala Arg Asp Leu Asn Asn Pro Gln Ile
405 410 415
Asp Gln Tyr Leu Ala Lys Tyr Val Arg Gly Ser Asn Gly Met Asp His
420 425 430
Val Gln Arg Ile Lys Ile Leu Lys Leu Met Trp Asp Ala Ile Gly Ser
435 440 445
Glu Phe Gly Gly Arg His Glu Leu Tyr Glu Ile Asn Tyr Ser Gly Ser
450 455 460
Gln Asp Glu Ile Arg Leu Gln Cys Leu Arg Gln Ala Gln Ser Ser Gly
465 470 475 480
Asn Met Asp Lys Met Met Ala Met Val Asp Arg Cys Leu Ser Glu Tyr
485 490 495
Asp Gln Asn Gly Trp Thr Val Pro His Leu His Asn Asn Asp Asp Ile
500 505 510
Asn Met Leu Asp Lys Leu Leu Lys
515 520
<210> 22
<211> 170
<212> PRT
<213> 大肠杆菌(E Coli.)
<400> 22
Met Gln Leu Asp Glu Gln Arg Leu Arg Phe Arg Asp Ala Met Ala Ser
1 5 10 15
Leu Ser Ala Ala Val Asn Ile Ile Thr Thr Glu Gly Asp Ala Gly Gln
20 25 30
Cys Gly Ile Thr Ala Thr Ala Val Cys Ser Val Thr Asp Thr Pro Pro
35 40 45
Ser Leu Met Val Cys Ile Asn Ala Asn Ser Ala Met Asn Pro Val Phe
50 55 60
Gln Gly Asn Gly Lys Leu Cys Val Asn Val Leu Asn His Glu Gln Glu
65 70 75 80
Leu Met Ala Arg His Phe Ala Gly Met Thr Gly Met Ala Met Glu Glu
85 90 95
Arg Phe Ser Leu Ser Cys Trp Gln Lys Gly Pro Leu Ala Gln Pro Val
100 105 110
Leu Lys Gly Ser Leu Ala Ser Leu Glu Gly Glu Ile Arg Asp Val Gln
115 120 125
Ala Ile Gly Thr His Leu Val Tyr Leu Val Glu Ile Lys Asn Ile Ile
130 135 140
Leu Ser Ala Glu Gly His Gly Leu Ile Tyr Phe Lys Arg Arg Phe His
145 150 155 160
Pro Val Met Leu Glu Met Glu Ala Ala Ile
165 170
<210> 23
<211> 1563
<212> DNA
<213> 人工序列(人工序列)
<400> 23
atgaaaccag aagatttccg cgccagtacc caacgtccgt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcact 120
actcatccgg catttcgtaa tgcggctgcg tctgttgccc aactgtacga cgcgctacac 180
aaaccggaga tgcaggactc tctgtgctgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccacgaacg cgatgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagctgct 360
ttcggttgcg cactgggcgg aactccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtaaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggctcg gcacaagtaa tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtca aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctca ccgtatgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga tcccatggga aaacgtgctg 840
ctctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt cgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaactcg acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtgg cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttctgaagcg 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaacaatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgccattgg cagcgagttt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aagctccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccagaacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taa 1563
<210> 24
<211> 513
<212> DNA
<213> 人工序列(人工序列)
<400> 24
atgcaattag atgaacaacg cctgcgcttt cgtgacgcaa tggccagcct gtcggcagcg 60
gtaaatatta tcaccaccga gggcgacgcc ggacaatgcg ggattacggc aacggccgtc 120
tgctcggtca cggatacacc accatcgctg atggtgtgca ttaacgccaa cagtgcgatg 180
aacccggttt ttcagggcaa cggtaagttg tgcgtcaacg tcctcaacca tgagcaggaa 240
ctgatggcac gccacttcgc gggcatgaca ggcatggcga tggaagagcg ttttagcctc 300
tcatgctggc aaaaaggtcc gctggcgcag ccggtgctaa aaggttcgct ggccagtctt 360
gaaggtgaga tccgcgatgt gcaggcaatt ggcacacatc tggtgtatct ggtggagatt 420
aaaaacatca tcctcagtgc agaaggtcac ggacttatct actttaaacg ccgtttccat 480
ccggtgatgc tggaaatgga agctgcgatt taa 513
<210> 25
<211> 22
<212> DNA
<213> 人工序列(人工序列)
<400> 25
atgaaaccag aagatttccg cg 22
<210> 26
<211> 22
<212> DNA
<213> 人工序列(人工序列)
<400> 26
ttatttcagc agcttatcca gc 22
<210> 27
<211> 23
<212> DNA
<213> 人工序列(人工序列)
<400> 27
atgcaattag atgaacaacg cct 23
<210> 28
<211> 23
<212> DNA
<213> 人工序列(人工序列)
<400> 28
ttaaatcgca gcttccattt cca 23
<210> 29
<211> 7347
<212> DNA
<213> 人工序列(人工序列)
<400> 29
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
atgaaaccag aagatttccg cgccagtacc caacgtccgt tcaccgggga agagtatctg 5160
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcact 5220
actcatccgg catttcgtaa tgcggctgcg tctgttgccc aactgtacga cgcgctacac 5280
aaaccggaga tgcaggactc tctgtgctgg aacaccgaca ccggcagcgg cggctatacc 5340
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccacgaacg cgatgccatc 5400
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagctgct 5460
ttcggttgcg cactgggcgg aactccgggc ttttacggtc agttcgagca gaacgcccgt 5520
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 5580
ccgatcgatc gtcatttgcc gaccgataaa gtaaaagacg tttacatcaa gctggaaaaa 5640
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 5700
actcactaca acatgattgg cttcggctcg gcacaagtaa tgggcgaaaa cccggacttc 5760
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtca aattaatctc ccgcgcctct 5820
tatgagatgg tcgcgggtgc taccggctca ccgtatgact acccgctctc cagccgcttc 5880
gatgagaacg atgcgattct ggtgatggat aacgtgctga tcccatggga aaacgtgctg 5940
ctctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt cgcccgtatg 6000
tatccgctgc aagcctgtgt gcgcctggca gtgaaactcg acttcattac ggcactgctg 6060
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 6120
gaagtggtgg cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttctgaagcg 6180
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 6240
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 6300
ctgatctatc tcccttccag tgcccgtgac ctgaacaatc cgcagatcga ccagtatctg 6360
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 6420
ctgatgtggg atgccattgg cagcgagttt ggtggtcgtc acgaactgta tgaaatcaac 6480
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aagctccggc 6540
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccagaacggc 6600
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 6660
taacgcagca ggaggttaag atgcaattag atgaacaacg cctgcgcttt cgtgacgcaa 6720
tggccagcct gtcggcagcg gtaaatatta tcaccaccga gggcgacgcc ggacaatgcg 6780
ggattacggc aacggccgtc tgctcggtca cggatacacc accatcgctg atggtgtgca 6840
ttaacgccaa cagtgcgatg aacccggttt ttcagggcaa cggtaagttg tgcgtcaacg 6900
tcctcaacca tgagcaggaa ctgatggcac gccacttcgc gggcatgaca ggcatggcga 6960
tggaagagcg ttttagcctc tcatgctggc aaaaaggtcc gctggcgcag ccggtgctaa 7020
aaggttcgct ggccagtctt gaaggtgaga tccgcgatgt gcaggcaatt ggcacacatc 7080
tggtgtatct ggtggagatt aaaaacatca tcctcagtgc agaaggtcac ggacttatct 7140
actttaaacg ccgtttccat ccggtgatgc tggaaatgga agctgcgatt caccaccacc 7200
accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 7260
ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt 7320
tgctgaaagg aggaactata tccggat 7347
<210> 30
<211> 824
<212> PRT
<213> 齿双歧杆菌(Bifidobacterium dentium)
<400> 30
Thr Ser Pro Val Ile Gly Thr Pro Thr Leu Leu Leu Ala Ala Pro Val
1 5 10 15
Ser Gly Gly Ala Ile Gly Gly Val Ala Leu Thr Thr Ala Ala Ala Ala
20 25 30
Thr Leu Ser Ile Gly Gly Ile Thr Leu Ala Ser Ala Pro Leu Met Leu
35 40 45
Gly Pro Pro Thr Ala Gly Ala Val Leu His Ala Leu Val Gly His Thr
50 55 60
Gly Thr Thr Pro Gly Leu Ala Pro Leu Ile Gly His Ile Ala Ala Leu
65 70 75 80
Ile Ala Ala His Gly Gly Ala Thr Val Ile Ile Met Gly Pro Gly His
85 90 95
Gly Gly Pro Ala Gly Thr Ala Gly Ser Thr Leu Ala Gly Thr Thr Thr
100 105 110
Gly Thr Pro Pro Ala Ile Thr Leu Ala Gly Ala Gly Leu Gly Leu Pro
115 120 125
Pro Ala Gly Pro Ser Thr Pro Gly Gly Ile Pro Ser His Thr Ala Pro
130 135 140
Gly Thr Pro Gly Ser Ile His Gly Gly Gly Gly Leu Gly Thr Ala Leu
145 150 155 160
Ser His Ala Thr Gly Ala Val Met Ala Ala Pro Ser Leu Pro Val Pro
165 170 175
Ala Ile Val Gly Ala Gly Gly Ala Gly Thr Gly Pro Leu Ala Thr Gly
180 185 190
Thr Gly Ser Ala Leu Leu Val Ala Pro Ala Thr Ala Gly Ile Val Leu
195 200 205
Pro Ile Leu His Leu Ala Gly Thr Leu Ile Ala Ala Pro Thr Ile Leu
210 215 220
Ser Ala Ile Ser Ala Gly Gly Leu His Gly Pro Pro His Gly Met Gly
225 230 235 240
Thr Gly Pro Thr Gly Pro Val Ala Gly Pro Ala Ala Gly Ala His Leu
245 250 255
Ser Ile His Ala Ala Pro Ala Gly Leu Pro Gly Thr Val Pro Ala Gly
260 265 270
Ile Cys Ala Ile Leu Ala Ala Ala Gly Thr Ala Ala Met Thr Ala Pro
275 280 285
Pro Thr Pro Met Ile Ile Pro Ala Thr Pro Leu Gly Thr Thr Cys Pro
290 295 300
Leu Pro Ile Ala Gly Leu Leu Thr Gly Gly Ser Thr Ala Ser His Gly
305 310 315 320
Val Pro Leu Ala Ser Ala Ala Ala Thr Gly Ala His Pro Gly Val Leu
325 330 335
Leu Ala Thr Leu Gly Ser Thr Leu Pro Gly Gly Leu Pro Ala Ala Ala
340 345 350
Gly Ala Val Leu Pro Gly Val Thr Ala Pro Met Pro Thr Gly Gly Leu
355 360 365
Ala Ile Gly Gly Ala Pro Ala Ala Ala Gly Gly Ala Ile Ala Gly Gly
370 375 380
Leu Ala Leu Pro Ala Leu Gly Ala Thr Gly Val Leu Gly Val Ala Gly
385 390 395 400
Thr Gly His Gly Thr Gly Gly Leu Gly Ala Thr Ala Ala Leu Gly Val
405 410 415
Thr Thr Ala Ala Ile Ile Leu Ala Ala Pro Ala Ser Pro Ala Ile Pro
420 425 430
Gly Pro Ala Gly Thr Ala Ser Ala Ala Leu Gly Ala Ala Thr Ala Val
435 440 445
Thr Ala Leu Gly Thr Ala Ala Gly Thr Leu Ser Ala Gly Val Ala Gly
450 455 460
His Met Ala Val Thr Gly Gly Val Thr Gly Gly Leu Ser Gly His Gly
465 470 475 480
Met Gly Gly Pro Leu Gly Ala Thr Leu Leu Thr Gly Ala His Gly Ile
485 490 495
Thr Ser Ser Thr Gly Ser Pro Val His Val Ile Ala Ser Met Leu Ala
500 505 510
Gly His Ala Leu Thr Leu Gly Ala Thr Val Ala Gly Ile Pro Thr Ala
515 520 525
Leu Pro Ile Ser Ser Met Ala Leu Leu Val Ser Ser His Val Thr Ala
530 535 540
Gly Ala His Ala Gly Pro Ser His Gly Ala Pro Gly Val Thr Ser Val
545 550 555 560
Leu Leu Ala Leu Cys Pro Ala Ala Ala His Val Ile Gly Ile Thr Pro
565 570 575
Pro Val Ala Ser Ala Met Leu Leu Ala Val Ala Gly Leu Cys Thr Leu
580 585 590
Ser Thr Ala Met Ile Ala Ala Ile Ile Ala Gly Leu Gly Pro Ala Ala
595 600 605
Thr Thr Leu Thr Leu Ala Gly Ala Ala Ala Gly Leu Gly Leu Gly Ala
610 615 620
Ala Gly Thr Gly Thr Ala Ser Thr Ala Leu Ser Ala Ala Gly Ala Gly
625 630 635 640
Ile Val Leu Ala Ser Ala Gly Ala Val Pro Ala Gly Gly Ile Met Ala
645 650 655
Ala Ala Ala Leu Leu Ala Ala Met Gly Ile Leu Pro Leu Val Val Ala
660 665 670
Val Val Ala Leu Val Leu Leu Gly Ser Thr Leu Gly Ala Ala Gly Ala
675 680 685
Ile Ser Ala Ala Ala Pro Ala Ala Leu Pro Thr Gly Ala Leu Pro Val
690 695 700
Leu Pro Ala Thr His Ser Thr Ala Ala Ala Val Ala Gly Leu Ile Thr
705 710 715 720
Ala Ala Pro Ala His Ala Ala Pro Ala Val His Gly Thr Gly Gly Gly
725 730 735
Gly Ser Thr Thr Thr Pro Thr Ala Met Val Ala Val Ala Ala Ile Ala
740 745 750
Ala Thr Gly Leu Val Ala Gly Ala Leu Ala Met Ile Ala Ala Ala Leu
755 760 765
Thr Ala Ala Leu Ile Ala Gly Leu Gly Ala Pro Ala Leu Gly Ala Pro
770 775 780
Gly Pro Ala Val Ala Ala Gly Thr Ala His Pro Ala Thr Thr Ala Thr
785 790 795 800
Val Thr Ser Gly Val Ala Thr Ala Leu Gly Gly Ala Val Ser Ala Thr
805 810 815
Ala Ala Thr Ala Gly Ala Ala Gly
820
<210> 31
<211> 2478
<212> DNA
<213> 人工序列(人工序列)
<400> 31
atgacctctc cagttattgg tactccatgg aaaaaattga acgccccagt ttctgaagag 60
gctattgaag gtgttgataa gtattggaga gctgccaact atttgtccat tggtcaaatc 120
tacttgaggt ctaacccatt gatgaaggaa ccattcacta gggaagatgt taagcacaga 180
ttggttggtc attggggtac tactccaggt ttgaattttt tgatcggtca catcaacaga 240
ttgatcgctg atcatcaaca aaacaccgtt attatcatgg gtccaggtca tggtggtcca 300
gctggtactg ctcaatctta tttggatggt acttacaccg aatacttccc aaacattact 360
aaggatgaag ctggcttgca aaagttcttc agacaatttt cttacccagg tggtatccca 420
tctcattatg ctccagaaac accaggttct attcatgaag gtggtgaatt gggttatgct 480
ttgtctcatg cttatggtgc tgttatgaac aacccatctt tgtttgttcc agctatagtt 540
ggtgatggtg aagctgaaac tggtccattg gctactggtt ggcaatctaa caaattggtt 600
aacccaagaa ccgatggtat cgttttgcca atcttgcatt tgaacggtta caagattgct 660
aacccaacca tcttgtccag aatctctgat gaagaattgc atgagttctt ccatggtatg 720
ggttatgaac catacgaatt tgttgctggt ttcgacaacg aagatcattt gtctatccat 780
agaagattcg ccgagttgtt cgaaactgtt ttcgacgaaa tttgcgatat taaggctgct 840
gctcaaactg atgatatgac tagaccattt taccccatga tcattttcag aactccaaaa 900
ggttggacct gtccaaagtt tatcgatggt aaaaagactg aaggttcttg gagatcacat 960
caagttccat tggcatctgc tagagatact gaagctcatt tcgaagtttt gaagaactgg 1020
ttggaaagct acaaacctga agagttgttt gatgctaatg gtgcagtaaa gcctgaagtt 1080
actgctttta tgccaacagg tgaattgaga attggtgaaa atccaaatgc taacggtggt 1140
agaatcaggg aagaattgaa tttgccagct ttggaagatt acgaagtcaa agaagttgct 1200
gaatacggtc atggttgggg tcaattggaa gctactagaa gattgggtgt ttacaccaga 1260
gatatcatca agaacaatcc agactccttc agaatttttg gtccagacga aactgcttct 1320
aacagattgc aagctgctta cgatgttacc aacaaacaat gggatgctgg ttatttgtct 1380
gcccaagttg atgaacatat ggctgttact ggtcaagtta ccgaacaatt gtcagaacat 1440
caaatggaag gttttttgga ggcttacttg ttgactggta gacatggtat ttggtcctct 1500
tacgaatctt tcgttcacgt tatcgactct atgttgaatc aacacgctaa atggttggaa 1560
gccactgtta gagaaattcc ttggagaaag ccaatctcct ctatgaactt gttggtttct 1620
tcacacgttt ggagacaaga tcataacggt ttctctcatc aagatccagg tgttacttcc 1680
gtcttgttga acaagtgttt caacaacgat catgtcatcg gtatctactt cccagttgac 1740
tctaatatgt tgttggctgt tgctgaaaag tgctacaagt ctaccgatat gattaacgct 1800
attatcgctg gtaagcaacc agctgctact tggttgactt tagatgaagc tagagctgaa 1860
ttggaaaaag gtgctgctga atgggaatgg gcttctactg ctaaatcaaa tgatgaagcc 1920
caaatcgttt tggcttctgc tggtgatgtt ccagcacaag aaattatggc tgcagctgat 1980
aagttggatg ctatgggtat taagttcaag gttgttaacg ttgtcgacct ggttaagttg 2040
caatctacca aagaaaacga tgaagccatt tccgatgcag attttgctga tttgttcacc 2100
gaagataagc cagttttgtt cgcttatcat tcctacgcta gagatgtcag aggtttgata 2160
tacgatagac caaaccacga taacttcaac gttcatggtt atgaagaaca aggttctact 2220
actaccccat acgatatggt tagagttaac aacatcgaca ggtatgaatt ggttgctgaa 2280
gccttgagaa tgattgatgc agataagtac gctgacaaga tcgatgaatt agaggctttt 2340
agaaaagagg ccttccaatt cgctgttgat aatggttacg atcatccaga ttacaccgat 2400
tgggtttatt ctggtgttaa taccaacaag caaggtgctg tttctgctac tgctgctaca 2460
gctggtgata atgaataa 2478
<210> 32
<211> 47
<212> DNA
<213> 人工序列(人工序列)
<400> 32
acaaaaaaca catacataaa ctaaaaatga cctctccagt tattggt 47
<210> 33
<211> 50
<212> DNA
<213> 人工序列(人工序列)
<400> 33
tcatcaaatc attcattctt cagacttatt cattatcacc agctgtagca 50
<210> 34
<211> 825
<212> PRT
<213> Bifidobacterium animalis
<400> 34
Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp Arg Pro
1 5 10 15
Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg Val Ala
20 25 30
Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile Asn Arg
65 70 75 80
Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Ile Asp Gly Thr Tyr
100 105 110
Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val
165 170 175
Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile
210 215 220
Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg Gly Met
225 230 235 240
Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His
245 250 255
Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Ile Phe Asp
260 265 270
Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asp Met Thr Arg
275 280 285
Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His
305 310 315 320
Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val
325 330 335
Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala
340 345 350
Asp Gly Ser Ile Lys Glu Asp Val Thr Ala Phe Met Pro Lys Gly Glu
355 360 365
Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu
370 375 380
Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Ile Thr Gly Val Lys
385 390 395 400
Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ser Leu Gly
405 410 415
Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Val
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr Tyr Glu
435 440 445
Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu Ser Ala Leu Val Asp
450 455 460
Glu Asn Met Ala Val Thr Gly Gln Val Val Glu Gln Leu Ser Glu His
465 470 475 480
Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn Ile Tyr
565 570 575
Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Phe
580 585 590
Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala Gly Lys Gln Pro Ala
595 600 605
Ala Thr Trp Ile Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Ala Gly
610 615 620
Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys Ser Asn Asp Glu Val
625 630 635 640
Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met
645 650 655
Ala Ala Ser Asp Ala Leu Asn Lys Met Gly Ile Lys Phe Lys Val Val
660 665 670
Asn Val Val Asp Leu Ile Lys Leu Gln Ser Ser Lys Glu Asn Asp Glu
675 680 685
Ala Met Ser Asp Glu Asp Phe Ala Asp Leu Phe Thr Ala Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe Thr Val Val Gly Tyr Lys Glu
725 730 735
Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn Asp Met
740 745 750
Asp Arg Tyr Ala Leu Gln Ala Lys Ala Leu Glu Leu Ile Asp Ala Asp
755 760 765
Lys Tyr Ala Asp Lys Ile Asn Glu Leu Asn Glu Phe Arg Lys Thr Ala
770 775 780
Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe Thr Asp
785 790 795 800
Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Ser Met Leu Ser Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 35
<211> 825
<212> PRT
<213> Bifidobacterium bifidum
<400> 35
Met Thr Ser Pro Val Ile Gly Thr Pro Trp Lys Lys Leu Asn Ala Pro
1 5 10 15
Val Ser Glu Glu Ala Leu Glu Gly Val Asp Lys Tyr Trp Arg Val Ala
20 25 30
Asn Tyr Leu Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Glu Asp Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Ile Gly His Ile Asn Arg
65 70 75 80
Phe Ile Ala Asp His Gly Gln Asn Thr Val Phe Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ser Gln Ser Tyr Leu Asp Gly Thr Tyr
100 105 110
Thr Glu Thr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val
165 170 175
Pro Cys Ile Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile
210 215 220
Leu Ser Arg Ile Ser Asp Glu Glu Leu His Glu Phe Phe His Gly Met
225 230 235 240
Gly Tyr Glu Pro Tyr Glu Phe Val Ala Gly Phe Asp Asp Glu Asp His
245 250 255
Met Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Ser Val Trp Asp
260 265 270
Glu Ile Cys Asp Ile Lys Ala Ala Ala Asn Thr Asp Asn Met His Arg
275 280 285
Pro Phe Tyr Pro Met Ile Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Tyr Ile Asp Gly Lys Lys Thr Glu Ala Pro Gly Ala Pro Thr
305 310 315 320
Arg Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val
325 330 335
Leu Lys Asn Trp Leu Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asp Ala
340 345 350
Asn Gly Ala Val Lys Asp Asp Val Leu Ala Phe Met Pro Lys Gly Glu
355 360 365
Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Val Ile Arg Lys
370 375 380
Asp Leu Val Leu Pro Ala Leu Glu Asp Tyr Glu Val Lys Glu Val Lys
385 390 395 400
Glu Phe Gly His Gly Trp Gly Gln Leu Glu Ala Thr Arg Arg Leu Gly
405 410 415
Val Tyr Thr Arg Asp Ile Ile Lys Asn Asn Met His Asp Phe Arg Ile
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gln Ala Ser Tyr Glu
435 440 445
Val Thr Asn Lys Gln Trp Asp Ala Gly Tyr Ile Ser Asp Glu Val Asp
450 455 460
Glu His Met His Val Ser Gly Gln Val Val Glu Gln Leu Ser Glu His
465 470 475 480
Gln Met Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ala Ser Met Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Val Leu Leu Asn Lys Cys Phe His Asn Asp His Val Ile Gly Ile Tyr
565 570 575
Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Tyr
580 585 590
Lys Ser Thr Asn Lys Ile Asn Ala Ile Ile Ala Gly Lys Gln Pro Ala
595 600 605
Ala Thr Trp Leu Thr Leu Asp Glu Ala Arg Ala Glu Leu Ala Lys Gly
610 615 620
Ala Ala Ala Trp Asp Trp Ala Ser Thr Ala Lys Thr Asn Asp Glu Ala
625 630 635 640
Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met
645 650 655
Ala Ala Ser Asp Lys Leu Lys Ala Leu Gly Ile Lys Phe Lys Val Val
660 665 670
Asn Val Ala Asp Leu Leu Ser Leu Gln Ser Ala Lys Glu Asn Asp Glu
675 680 685
Ala Leu Thr Asp Glu Glu Phe Ala Asp Ile Phe Thr Ala Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala His Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val His Gly Tyr Glu Glu
725 730 735
Glu Gly Ser Thr Thr Thr Pro Tyr Asp Met Val Arg Val Asn Glu Leu
740 745 750
Asp Arg Tyr Glu Leu Thr Ala Glu Ala Leu Arg Met Ile Asp Ala Asp
755 760 765
Lys Tyr Ala Asp Glu Ile Gln Lys Leu Glu Asp Phe Arg Gln Glu Ala
770 775 780
Phe Gln Phe Ala Val Asp Lys Gly Tyr Asp His Pro Asp Tyr Thr Asp
785 790 795 800
Trp Val Tyr Ser Gly Val Lys Thr Asp Lys Lys Gly Ala Val Thr Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 36
<211> 825
<212> PRT
<213> Bifidobacterium lactis
<400> 36
Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp Arg Pro
1 5 10 15
Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg Val Ala
20 25 30
Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile Asn Arg
65 70 75 80
Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Ile Asp Gly Thr Tyr
100 105 110
Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val
165 170 175
Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile
210 215 220
Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg Gly Met
225 230 235 240
Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His
245 250 255
Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Ile Phe Asp
260 265 270
Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asp Met Thr Arg
275 280 285
Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His
305 310 315 320
Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val
325 330 335
Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala
340 345 350
Asp Gly Ser Ile Lys Glu Asp Val Thr Ala Phe Met Pro Lys Gly Glu
355 360 365
Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu
370 375 380
Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Ile Thr Gly Val Lys
385 390 395 400
Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ser Leu Gly
405 410 415
Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Val
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr Tyr Glu
435 440 445
Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu Ser Ala Leu Val Asp
450 455 460
Glu Asn Met Ala Val Thr Gly Gln Val Val Glu Gln Leu Ser Glu His
465 470 475 480
Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn Ile Tyr
565 570 575
Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Phe
580 585 590
Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala Gly Lys Gln Pro Ala
595 600 605
Ala Thr Trp Ile Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Ala Gly
610 615 620
Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys Ser Asn Asp Glu Val
625 630 635 640
Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met
645 650 655
Ala Ala Ser Asp Ala Leu Asn Lys Met Gly Ile Lys Phe Lys Val Val
660 665 670
Asn Val Val Asp Leu Ile Lys Leu Gln Ser Ser Lys Glu Asn Asp Glu
675 680 685
Ala Met Ser Asp Glu Asp Phe Ala Asp Leu Phe Thr Ala Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe Thr Val Val Gly Tyr Lys Glu
725 730 735
Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn Asp Met
740 745 750
Asp Arg Tyr Ala Leu Gln Ala Lys Ala Leu Glu Leu Ile Asp Ala Asp
755 760 765
Lys Tyr Ala Asp Lys Ile Asn Glu Leu Asn Glu Phe Arg Lys Thr Ala
770 775 780
Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe Thr Asp
785 790 795 800
Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Ser Met Leu Ser Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 37
<211> 825
<212> PRT
<213> Bifidobacterium longum
<400> 37
Met Thr Ser Pro Val Ile Gly Thr Pro Trp Lys Lys Leu Asn Ala Pro
1 5 10 15
Val Ser Glu Glu Ala Leu Glu Gly Val Asp Lys Tyr Trp Arg Val Ala
20 25 30
Asn Tyr Leu Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Glu Asp Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Ile Gly His Ile Asn Arg
65 70 75 80
Phe Ile Ala Asp His Gly Gln Asn Thr Val Ile Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ser Gln Ser Tyr Leu Asp Gly Thr Tyr
100 105 110
Thr Glu Thr Phe Pro Lys Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val
165 170 175
Pro Ala Ile Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile
210 215 220
Leu Ser Arg Ile Ser Asp Glu Glu Leu His Glu Phe Phe His Gly Met
225 230 235 240
Gly Tyr Glu Pro Tyr Glu Phe Val Ala Gly Phe Asp Asp Glu Asp His
245 250 255
Met Ser Ile His Arg Arg Phe Ala Glu Leu Trp Glu Thr Ile Trp Asp
260 265 270
Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asn Val His Arg
275 280 285
Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Tyr Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His
305 310 315 320
Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val
325 330 335
Leu Lys Asn Trp Leu Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asp Ala
340 345 350
Asn Gly Ala Val Lys Asp Asp Val Leu Ala Phe Met Pro Lys Gly Glu
355 360 365
Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Val Ile Arg Asp
370 375 380
Asp Leu Lys Leu Pro Asn Leu Glu Asp Tyr Glu Val Lys Glu Val Ala
385 390 395 400
Glu Tyr Gly His Gly Trp Gly Gln Leu Glu Ala Thr Arg Thr Leu Gly
405 410 415
Val Tyr Thr Arg Asp Ile Ile Lys Asn Asn Pro Arg Asp Phe Arg Ile
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gln Ala Ser Tyr Glu
435 440 445
Val Thr Asn Lys Gln Trp Asp Ala Gly Tyr Ile Ser Asp Glu Val Asp
450 455 460
Glu His Met His Val Ser Gly Gln Val Val Glu Gln Leu Ser Glu His
465 470 475 480
Gln Met Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ala Ser Met Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Val Leu Leu Asn Lys Cys Phe His Asn Asp His Val Ile Gly Ile Tyr
565 570 575
Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Tyr
580 585 590
Lys Ser Thr Asn Lys Ile Asn Ala Ile Ile Ala Gly Lys Gln Pro Ala
595 600 605
Ala Thr Trp Leu Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Lys Gly
610 615 620
Ala Ala Ala Trp Asp Trp Ala Ser Thr Ala Lys Asn Asn Asp Glu Ala
625 630 635 640
Glu Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met
645 650 655
Ala Ala Ser Asp Lys Leu Lys Glu Leu Gly Val Lys Phe Lys Val Val
660 665 670
Asn Val Val Asp Leu Leu Ser Leu Gln Ser Ala Lys Glu Asn Asp Glu
675 680 685
Ala Leu Thr Asp Glu Glu Phe Ala Asp Ile Phe Thr Ala Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala His Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val His Gly Tyr Glu Glu
725 730 735
Glu Gly Ser Thr Thr Thr Pro Tyr Asp Met Val Arg Val Asn Arg Ile
740 745 750
Asp Arg Tyr Glu Leu Thr Ala Glu Ala Leu Arg Met Ile Asp Ala Asp
755 760 765
Lys Tyr Ala Asp Lys Ile Asp Glu Leu Glu Lys Phe Arg Asp Glu Ala
770 775 780
Phe Gln Phe Ala Val Asp Lys Gly Tyr Asp His Pro Asp Tyr Thr Asp
785 790 795 800
Trp Val Tyr Ser Gly Val Asn Thr Asp Lys Lys Gly Ala Val Thr Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 38
<211> 825
<212> PRT
<213> Bifidobacterium mongoliense
<400> 38
Met Thr Ser Pro Val Ile Gly Thr Pro Trp Thr Lys Leu Asp Glu Pro
1 5 10 15
Val Ser Glu Glu Thr Leu Glu Gly Ile Asp Lys Tyr Trp Arg Ala Ala
20 25 30
Asn Tyr Leu Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Asp Asn Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Gly His Ile Asn His
65 70 75 80
Leu Ile Ala Ala His Lys Gln Asn Thr Val Ile Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Leu Asp Gly Thr Tyr
100 105 110
Thr Glu Tyr Tyr Pro Lys Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Val Met Asn Asn Pro Ser Leu Phe Val
165 170 175
Pro Ala Ile Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Ser Ile
210 215 220
Leu Ser Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe His Gly Met
225 230 235 240
Gly Tyr Glu Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His
245 250 255
Leu Ser Ile His Arg Arg Phe Ala Asp Leu Phe Glu Ser Ile Phe Asp
260 265 270
Glu Ile Cys Asp Ile Lys Ala Gln Ala Gln Thr Asp Asp Met Thr Arg
275 280 285
Pro Phe Tyr Pro Met Ile Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Tyr Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His
305 310 315 320
Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val
325 330 335
Leu Lys His Trp Leu Glu Ser Tyr Lys Pro Glu Glu Leu Phe Ala Ala
340 345 350
Asp Gly Ser Ile Lys Pro Glu Val Thr Ser Phe Met Pro Glu Gly Glu
355 360 365
Leu Arg Ile Gly Glu Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu
370 375 380
Asp Leu Lys Leu Pro Lys Leu Glu Asp Tyr Glu Val Asn Glu Val Lys
385 390 395 400
Glu Tyr Gly His Gly Trp Gly Gln Leu Glu Ala Thr Arg Arg Met Gly
405 410 415
Val Tyr Thr Arg Asp Val Ile Lys Leu Asn Pro Asp Ser Phe Arg Ile
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Ala Ala Ala Tyr Glu
435 440 445
Val Thr Asn Lys Gln Trp Asp Ala Gly Tyr Leu Ser Ala Gln Val Asp
450 455 460
Glu His Met Ala Val Thr Gly Gln Val Thr Glu Gln Leu Ser Glu His
465 470 475 480
Gln Met Glu Gly Phe Leu Glu Gly Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Val Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His Val Val Asn Ile Tyr
565 570 575
Phe Pro Val Asp Ala Asn Met Leu Leu Ala Val Thr Glu Arg Cys Tyr
580 585 590
Lys Ser Thr Asn Glu Ile Asn Ala Ile Ile Ala Gly Lys Gln Pro Ala
595 600 605
Ala Thr Trp Leu Thr Leu Asp Glu Ala Arg Ala Glu Leu Asp Lys Gly
610 615 620
Ala Ala Glu Trp Thr Trp Ala Ser Asn Thr Asp Asn Asn Asp Asp Val
625 630 635 640
Gln Val Val Leu Ala Cys Ala Gly Asp Val Pro Thr Gln Glu Ile Met
645 650 655
Ala Ala Ala Asp Lys Leu Ser Ala Phe Gly Ile Lys Phe Lys Val Val
660 665 670
Asn Val Val Asp Leu Leu Lys Leu Gln Ser Ala Lys Glu Asn Asp Glu
675 680 685
Ala Leu Ser Asp Ala Glu Phe Ser Asp Leu Phe Thr Ala Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala His Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val His Gly Tyr Lys Glu
725 730 735
Gln Gly Ser Thr Thr Thr Pro Tyr Asp Met Val Arg Val Asn Asp Met
740 745 750
Asp Arg Tyr Ala Leu Thr Ala Glu Ala Leu Arg Met Val Asp Lys Asp
755 760 765
Lys Tyr Ala Ala Asp Ile Asp Lys Leu Glu Ala Phe Arg Lys Glu Ala
770 775 780
Phe Gln Phe Ala Val Asp Lys Gly Tyr Asp His Pro Asp Tyr Thr Asp
785 790 795 800
Trp Leu Trp His Gly Val Lys Ser Asp Lys Ala Gly Ala Val Thr Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 39
<211> 825
<212> PRT
<213> Bifidobacterium pseudolongum
<400> 39
Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp Arg Pro
1 5 10 15
Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg Val Thr
20 25 30
Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile Asn Arg
65 70 75 80
Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ser Gln Ser Tyr Val Asp Gly Thr Tyr
100 105 110
Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Val Met Asn Asn Pro Ser Leu Phe Val
165 170 175
Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile
210 215 220
Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg Gly Met
225 230 235 240
Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His
245 250 255
Met Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Ile Phe Asp
260 265 270
Glu Ile Cys Asp Ile Lys Leu Arg Pro Gln Thr Asp Asp Met Thr Arg
275 280 285
Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His
305 310 315 320
Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Glu His Phe Glu Val
325 330 335
Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala
340 345 350
Asp Gly Ser Ile Lys Asp Asp Val Thr Ala Phe Met Pro Lys Gly Glu
355 360 365
Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Val Ile Arg Glu
370 375 380
Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Val Thr Gly Val Lys
385 390 395 400
Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ala Leu Gly
405 410 415
Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Ile
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr Tyr Glu
435 440 445
Val Thr Asp Lys Gln Trp Asp Asn Gly Tyr Leu Ser Gly Leu Val Asp
450 455 460
Glu His Met Ala Val Thr Gly Gln Val Thr Glu Gln Leu Ser Glu His
465 470 475 480
Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Leu Leu Ile Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn Ile Tyr
565 570 575
Phe Ala Thr Asp Thr Asn Met Leu Leu Ala Ile Ser Glu Lys Cys Phe
580 585 590
Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala Gly Lys Gln Pro Ala
595 600 605
Pro Thr Trp Val Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Ala Gly
610 615 620
Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Glu Asn Asn Asp Glu Val
625 630 635 640
Gln Val Val Leu Ala Ser Ala Gly Asp Val Pro Thr Gln Glu Leu Met
645 650 655
Ala Ala Ser Asp Ala Leu Asn Lys Met Gly Ile Lys Phe Lys Val Val
660 665 670
Asn Val Val Asp Leu Leu Lys Leu Gln Ser Arg Glu Asn Asn Asp Glu
675 680 685
Ala Leu Thr Asp Glu Glu Phe Thr Glu Leu Phe Thr Ala Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe His Val Val Gly Tyr Lys Glu
725 730 735
Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn Asp Met
740 745 750
Asp Arg Tyr Ala Leu Gln Ala Ala Ala Leu Lys Leu Ile Asp Ala Asp
755 760 765
Lys Tyr Ala Asp Lys Ile Asp Glu Leu Asn Ala Phe Arg Lys Lys Ala
770 775 780
Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe Thr Asp
785 790 795 800
Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Gln Met Leu Ser Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 40
<211> 780
<212> PRT
<213> Aspergillus nidulans
<400> 40
Met Pro Gly Glu Val Ile Glu Arg Pro Asn Pro Ala Pro Lys Pro Ser
1 5 10 15
His Val Pro Asp Leu Val Glu Lys Leu Ile Ile Pro Ala Gln Lys Thr
20 25 30
Lys Leu Glu Lys Ser Asp Cys Asp Ala Leu His Lys Tyr Arg Arg Ala
35 40 45
Ala Ala Tyr Ile Ala Ala Gly His Trp Gly Thr Cys Pro Gly Leu Ile
50 55 60
Leu Val Tyr Ser His Leu Asn Tyr Leu Ile Lys Lys Gln Asn Leu Asp
65 70 75 80
Met Leu Tyr Val Val Gly Pro Gly His Gly Ala Pro Gly Leu Leu Ala
85 90 95
Ser Leu Trp Leu Glu Gly Ser Leu Gly Lys Phe Tyr Pro Gln Tyr Thr
100 105 110
Lys Asp Lys Glu Gly Leu His Asn Leu Ile Ser Thr Phe Ser Thr Ser
115 120 125
Ala Gly Leu Pro Ser His Ile Asn Ala Glu Thr Pro Gly Ala Ile His
130 135 140
Glu Gly Gly Glu Leu Gly Tyr Ala Leu Ser Val Ser Phe Gly Ala Val
145 150 155 160
Met Asp Asn Pro Asp Leu Ile Val Thr Cys Val Val Gly Asp Gly Glu
165 170 175
Ala Glu Thr Gly Pro Thr Ala Thr Ser Trp His Ala Ile Lys Tyr Ile
180 185 190
Asp Pro Ala Glu Ser Gly Ala Val Leu Pro Ile Leu His Val Asn Gly
195 200 205
Phe Lys Ile Ser Glu Arg Thr Ile Phe Gly Cys Met Asp Asn Arg Glu
210 215 220
Ile Val Cys Leu Phe Thr Gly Tyr Gly Tyr Gln Val Arg Ile Val Glu
225 230 235 240
Asp Leu Glu Asp Ile Asp Asn Asp Leu His Ser Ala Met Ser Trp Ala
245 250 255
Val Glu Glu Ile Arg Asn Ile Gln Lys Ala Ala Arg Ser Gly Lys Pro
260 265 270
Ile Met Lys Pro Gln Trp Pro Met Ile Val Leu Arg Thr Pro Lys Gly
275 280 285
Trp Ser Gly Pro Lys Glu Leu His Gly Gln Phe Ile Glu Gly Ser Phe
290 295 300
His Ser His Gln Val Pro Leu Pro Asn Ala Lys Lys Asp Asp Glu Glu
305 310 315 320
Leu Gln Ala Leu Gln Lys Trp Leu Ser Ser Tyr Lys Pro Asp Glu Leu
325 330 335
Phe Thr Glu Ser Gly Asp Val Ile Asp Glu Ile Leu Ser Ile Ile Pro
340 345 350
Ser Asp Asp Lys Lys Leu Gly Met Arg Pro Glu Ala Tyr Lys Thr His
355 360 365
Leu Pro Pro Asp Leu Pro Asp Trp Arg Gln Phe Cys Val Lys Lys Gly
370 375 380
Asp Gln Phe Ser Ala Met Lys Ala Ile Gly Ser Phe Ile Asp Gln Val
385 390 395 400
Phe Val Lys Asn Pro His Thr Val Arg Leu Phe Ser Pro Asp Glu Leu
405 410 415
Glu Ser Asn Lys Leu Ser Ala Ala Leu Ser His Thr Gly Arg Asn Phe
420 425 430
Gln Trp Asp Glu Phe Ser Asn Ala Lys Gly Gly Arg Val Ile Glu Val
435 440 445
Leu Ser Glu His Leu Cys Gln Gly Phe Met Gln Gly Tyr Thr Leu Thr
450 455 460
Gly Arg Thr Gly Ile Phe Pro Ser Tyr Glu Ser Phe Leu Gly Ile Ile
465 470 475 480
His Thr Met Met Val Gln Tyr Ala Lys Phe Ala Lys Met Ala Lys Glu
485 490 495
Thr Ala Trp His His Asp Val Ser Ser Ile Asn Tyr Ile Glu Thr Ser
500 505 510
Thr Trp Ala Arg Gln Glu His Asn Gly Phe Ser His Gln Asn Pro Ser
515 520 525
Phe Ile Gly Ala Val Leu Lys Leu Lys Pro Tyr Ala Ala Arg Val Tyr
530 535 540
Leu Pro Pro Asp Ala Asn Thr Phe Leu Thr Thr Leu His His Cys Leu
545 550 555 560
Lys Ser Lys Asn Tyr Ile Asn Leu Met Val Gly Ser Lys Gln Pro Thr
565 570 575
Pro Val Tyr Leu Ser Pro Glu Glu Ala Glu Ser His Cys Arg Ala Gly
580 585 590
Ala Ser Ile Phe Lys Phe Cys Ser Thr Asp Gly Gly Leu Arg Pro Asp
595 600 605
Val Val Leu Val Gly Ile Gly Val Glu Val Met Phe Glu Val Ile Lys
610 615 620
Ala Ala Ala Ile Leu Arg Glu Arg Cys Pro Glu Leu Arg Val Arg Val
625 630 635 640
Val Asn Val Thr Asp Leu Phe Ile Leu Glu Asn Glu Gly Ala His Pro
645 650 655
His Ala Leu Lys His Glu Ala Phe Asp Asn Leu Phe Thr Glu Asp Arg
660 665 670
Ser Ile His Phe Asn Tyr His Gly Tyr Val Asn Glu Leu Gln Gly Leu
675 680 685
Leu Phe Gly Arg Pro Arg Leu Asp Arg Ala Thr Ile Lys Gly Tyr Lys
690 695 700
Glu Glu Gly Ser Thr Thr Thr Pro Phe Asp Met Met Leu Val Asn Glu
705 710 715 720
Val Ser Arg Tyr His Val Ala Lys Ala Ala Val Thr Gly Gly Ala Arg
725 730 735
Phe Asn Glu Lys Val Lys Leu Arg His Gln Glu Leu Cys Ser Glu Phe
740 745 750
Asp His Asn Ile Ala Glu Thr Arg Lys Tyr Ile Met Asn Asn His Gln
755 760 765
Asp Pro Glu Asp Thr Tyr Asn Met Pro Ser Phe Asn
770 775 780
<210> 41
<211> 796
<212> PRT
<213> Clostridium acetobutylicum
<400> 41
Met Gln Ser Ile Ile Gly Lys His Lys Asp Glu Gly Lys Ile Thr Pro
1 5 10 15
Glu Tyr Leu Lys Lys Ile Asp Ala Tyr Trp Arg Ala Ala Asn Phe Ile
20 25 30
Ser Val Gly Gln Leu Tyr Leu Leu Asp Asn Pro Leu Leu Arg Glu Pro
35 40 45
Leu Lys Pro Glu His Leu Lys Arg Lys Val Val Gly His Trp Gly Thr
50 55 60
Ile Pro Gly Gln Asn Phe Ile Tyr Ala His Leu Asn Arg Val Ile Lys
65 70 75 80
Lys Tyr Asp Leu Asp Met Ile Tyr Val Ser Gly Pro Gly His Gly Gly
85 90 95
Gln Val Met Val Ser Asn Ser Tyr Leu Asp Gly Thr Tyr Ser Glu Val
100 105 110
Tyr Pro Asn Val Ser Arg Asp Leu Asn Gly Leu Lys Lys Leu Cys Lys
115 120 125
Gln Phe Ser Phe Pro Gly Gly Ile Ser Ser His Met Ala Pro Glu Thr
130 135 140
Pro Gly Ser Ile Asn Glu Gly Gly Glu Leu Gly Tyr Ser Leu Ala His
145 150 155 160
Ser Phe Gly Ala Val Phe Asp Asn Pro Asp Leu Ile Thr Ala Cys Val
165 170 175
Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr Ser Trp Gln
180 185 190
Ala Asn Lys Phe Leu Asn Pro Val Thr Asp Gly Ala Val Leu Pro Ile
195 200 205
Leu His Leu Asn Gly Tyr Lys Ile Ser Asn Pro Thr Val Leu Ser Arg
210 215 220
Ile Pro Lys Asp Glu Leu Glu Lys Phe Phe Glu Gly Asn Gly Trp Lys
225 230 235 240
Pro Tyr Phe Val Glu Gly Glu Asp Pro Glu Ala Met His Lys Leu Met
245 250 255
Ala Glu Thr Leu Asp Ile Val Thr Glu Glu Ile Leu Asn Ile Gln Lys
260 265 270
Asn Ala Arg Glu Asn Asn Asp Cys Ser Arg Pro Lys Trp Pro Met Ile
275 280 285
Val Leu Arg Thr Pro Lys Gly Trp Thr Gly Pro Lys Phe Val Asp Gly
290 295 300
Val Pro Asn Glu Gly Ser Phe Arg Ala His Gln Val Pro Leu Ala Val
305 310 315 320
Asp Arg Tyr His Thr Glu Asn Leu Asp Gln Leu Glu Glu Trp Leu Lys
325 330 335
Ser Tyr Lys Pro Glu Glu Leu Phe Asp Glu Asn Tyr Arg Leu Ile Pro
340 345 350
Glu Leu Glu Glu Leu Thr Pro Lys Gly Asn Lys Arg Met Ala Ala Asn
355 360 365
Leu His Ala Asn Gly Gly Leu Leu Leu Arg Glu Leu Arg Thr Pro Asp
370 375 380
Phe Arg Asp Tyr Ala Val Asp Val Pro Thr Pro Gly Ser Thr Val Lys
385 390 395 400
Gln Asp Met Ile Glu Leu Gly Lys Tyr Val Arg Asp Val Val Lys Leu
405 410 415
Asn Glu Asp Thr Arg Asn Phe Arg Ile Phe Gly Pro Asp Glu Thr Met
420 425 430
Ser Asn Arg Leu Trp Ala Val Phe Glu Gly Thr Lys Arg Gln Trp Leu
435 440 445
Ser Glu Ile Lys Glu Pro Asn Asp Glu Phe Leu Ser Asn Asp Gly Arg
450 455 460
Ile Val Asp Ser Met Leu Ser Glu His Leu Cys Glu Gly Trp Leu Glu
465 470 475 480
Gly Tyr Leu Leu Thr Gly Arg His Gly Phe Phe Ala Ser Tyr Glu Ala
485 490 495
Phe Leu Arg Ile Val Asp Ser Met Ile Thr Gln His Gly Lys Trp Leu
500 505 510
Lys Val Thr Ser Gln Leu Pro Trp Arg Lys Asp Ile Ala Ser Leu Asn
515 520 525
Leu Ile Ala Thr Ser Asn Val Trp Gln Gln Asp His Asn Gly Tyr Thr
530 535 540
His Gln Asp Pro Gly Leu Leu Gly His Ile Val Asp Lys Lys Pro Glu
545 550 555 560
Ile Val Arg Ala Tyr Leu Pro Ala Asp Ala Asn Thr Leu Leu Ala Val
565 570 575
Phe Asp Lys Cys Leu His Thr Lys His Lys Ile Asn Leu Leu Val Thr
580 585 590
Ser Lys His Pro Arg Gln Gln Trp Leu Thr Met Asp Gln Ala Val Lys
595 600 605
His Val Glu Gln Gly Ile Ser Ile Trp Asp Trp Ala Ser Asn Asp Lys
610 615 620
Gly Gln Glu Pro Asp Val Val Ile Ala Ser Cys Gly Asp Thr Pro Thr
625 630 635 640
Leu Glu Ala Leu Ala Ala Val Thr Ile Leu His Glu His Leu Pro Glu
645 650 655
Leu Lys Val Arg Phe Val Asn Val Val Asp Met Met Lys Leu Leu Pro
660 665 670
Glu Asn Glu His Pro His Gly Leu Ser Asp Lys Asp Tyr Asn Ala Leu
675 680 685
Phe Thr Thr Asp Lys Pro Val Ile Phe Ala Phe His Gly Phe Ala His
690 695 700
Leu Ile Asn Gln Leu Thr Tyr His Arg Glu Asn Arg Asn Leu His Val
705 710 715 720
His Gly Tyr Met Glu Glu Gly Thr Ile Thr Thr Pro Phe Asp Met Arg
725 730 735
Val Gln Asn Lys Leu Asp Arg Phe Asn Leu Val Lys Asp Val Val Glu
740 745 750
Asn Leu Pro Gln Leu Gly Asn Arg Gly Ala His Leu Val Gln Leu Met
755 760 765
Asn Asp Lys Leu Val Glu His Asn Gln Tyr Ile Arg Glu Val Gly Glu
770 775 780
Asp Leu Pro Glu Ile Thr Asn Trp Gln Trp His Val
785 790 795
<210> 42
<211> 813
<212> PRT
<213> Leuconostoc mesenteroides
<400> 42
Met Ala Asp Phe Asp Ser Lys Glu Tyr Leu Glu Leu Val Asp Lys Trp
1 5 10 15
Trp Arg Ala Thr Asn Tyr Leu Ser Ala Gly Met Ile Phe Leu Lys Ser
20 25 30
Asn Pro Leu Phe Ser Val Thr Asn Thr Pro Ile Lys Ala Glu Asp Val
35 40 45
Lys Val Lys Pro Ile Gly His Trp Gly Thr Ile Ser Gly Gln Thr Phe
50 55 60
Leu Tyr Ala His Ala Asn Arg Leu Ile Asn Lys Tyr Gly Leu Asn Met
65 70 75 80
Phe Tyr Val Gly Gly Pro Gly His Gly Gly Gln Val Met Val Thr Asn
85 90 95
Ala Tyr Leu Asp Gly Ala Tyr Thr Glu Asp Tyr Pro Glu Ile Thr Gln
100 105 110
Asp Ile Glu Gly Met Ser His Leu Phe Lys Arg Phe Ser Phe Pro Gly
115 120 125
Gly Ile Gly Ser His Met Thr Ala Gln Thr Pro Gly Ser Leu His Glu
130 135 140
Gly Gly Glu Leu Gly Tyr Ser Leu Ser His Ala Phe Gly Ala Val Leu
145 150 155 160
Asp Asn Pro Asp Gln Val Ala Phe Ala Val Val Gly Asp Gly Glu Ala
165 170 175
Glu Thr Gly Pro Ser Met Ala Ser Trp His Ser Ile Lys Phe Leu Asn
180 185 190
Ala Lys Asn Asp Gly Ala Val Leu Pro Val Leu Asp Leu Asn Gly Phe
195 200 205
Lys Ile Ser Asn Pro Thr Ile Phe Ser Arg Met Ser Asp Glu Glu Ile
210 215 220
Thr Lys Phe Phe Glu Gly Leu Gly Tyr Ser Pro Arg Phe Ile Glu Asn
225 230 235 240
Asp Asp Ile His Asp Tyr Ala Thr Tyr His Gln Leu Ala Ala Asn Ile
245 250 255
Leu Asp Gln Ala Ile Glu Asp Ile Gln Ala Ile Gln Asn Asp Ala Arg
260 265 270
Glu Asn Gly Lys Tyr Gln Asp Gly Glu Ile Pro Ala Trp Pro Val Ile
275 280 285
Ile Ala Arg Leu Pro Lys Gly Trp Gly Gly Pro Thr His Asp Ala Ser
290 295 300
Asn Asn Pro Ile Glu Asn Ser Phe Arg Ala His Gln Val Pro Leu Pro
305 310 315 320
Leu Glu Gln His Asp Leu Ala Thr Leu Pro Glu Phe Glu Asp Trp Met
325 330 335
Asn Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala Asp Gly Ser Leu Lys
340 345 350
Asp Glu Leu Lys Ala Ile Ala Pro Lys Gly Asp Lys Arg Met Ser Ala
355 360 365
Asn Pro Ile Thr Asn Gly Gly Ala Asp Arg Ser Asp Leu Lys Leu Pro
370 375 380
Asn Trp Arg Glu Phe Ala Asn Asp Ile Asn Asp Asp Thr Arg Gly Lys
385 390 395 400
Glu Phe Ala Asp Ser Lys Arg Asn Met Asp Met Ala Thr Leu Ser Asn
405 410 415
Tyr Leu Gly Ala Val Ser Gln Leu Asn Pro Thr Arg Phe Arg Phe Phe
420 425 430
Gly Pro Asp Glu Thr Met Ser Asn Arg Leu Trp Gly Leu Phe Asn Val
435 440 445
Thr Pro Arg Gln Trp Met Glu Glu Ile Lys Glu Pro Gln Asp Gln Leu
450 455 460
Leu Ser Pro Thr Gly Arg Ile Ile Asp Ser Gln Leu Ser Glu His Gln
465 470 475 480
Ala Glu Gly Trp Leu Glu Gly Tyr Thr Leu Thr Gly Arg Val Gly Ile
485 490 495
Phe Ala Ser Tyr Glu Ser Phe Leu Arg Val Val Asp Thr Met Val Thr
500 505 510
Gln His Phe Lys Trp Leu Arg His Ala Ser Glu Gln Ala Trp Arg Asn
515 520 525
Asp Tyr Pro Ser Leu Asn Leu Ile Ala Thr Ser Thr Ala Phe Gln Gln
530 535 540
Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly Met Leu Thr His Leu
545 550 555 560
Ala Glu Lys Lys Ser Asn Phe Ile Arg Glu Tyr Leu Pro Ala Asp Gly
565 570 575
Asn Ser Leu Leu Ala Val Gln Glu Arg Ala Phe Ser Glu Arg His Lys
580 585 590
Val Asn Leu Leu Ile Ala Ser Lys Gln Pro Arg Gln Gln Trp Phe Thr
595 600 605
Val Glu Glu Ala Glu Val Leu Ala Asn Glu Gly Leu Lys Ile Ile Asp
610 615 620
Trp Ala Ser Thr Ala Pro Ser Ser Asp Val Asp Ile Thr Phe Ala Ser
625 630 635 640
Ala Gly Thr Glu Pro Thr Ile Glu Thr Leu Ala Ala Leu Trp Leu Ile
645 650 655
Asn Gln Ala Phe Pro Asp Val Lys Phe Arg Tyr Val Asn Val Val Glu
660 665 670
Leu Leu Arg Leu Gln Lys Lys Ser Glu Pro Asn Met Asn Asp Glu Arg
675 680 685
Glu Leu Ser Ala Glu Glu Phe Asn Lys Tyr Phe Gln Ala Asp Thr Pro
690 695 700
Val Ile Phe Gly Phe His Ala Tyr Glu Asn Leu Ile Glu Ser Phe Phe
705 710 715 720
Phe Glu Arg Lys Phe Thr Gly Asp Val Tyr Val His Gly Tyr Arg Glu
725 730 735
Asp Gly Asp Ile Thr Thr Thr Tyr Asp Met Arg Val Tyr Ser His Leu
740 745 750
Asp Arg Phe His Gln Ala Lys Glu Ala Ala Glu Ile Leu Ser Ala Asn
755 760 765
Gly Lys Ile Asp Gln Ala Ala Ala Asp Thr Phe Ile Ala Lys Met Asp
770 775 780
Asp Thr Leu Ala Lys His Phe Gln Val Thr Arg Asn Glu Gly Arg Asp
785 790 795 800
Ile Glu Glu Phe Thr Asp Trp Thr Trp Ser Pro Leu Lys
805 810
<210> 43
<211> 788
<212> PRT
<213> Lactobacillus paraplantarum
<400> 43
Met Thr Thr Asp Tyr Ser Ser Pro Ala Tyr Leu Gln Lys Val Asp Lys
1 5 10 15
Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Tyr Leu Lys
20 25 30
Asp Asn Pro Leu Leu Gln Gln Pro Leu Lys Ala Ser Asp Val Lys Val
35 40 45
His Pro Ile Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr
50 55 60
Ala His Leu Asn Arg Val Ile Asn Lys Tyr Gly Leu Lys Met Phe Tyr
65 70 75 80
Val Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr
85 90 95
Leu Asp Gly Thr Tyr Thr Asp Ile Tyr Pro Glu Ile Thr Gln Asp Val
100 105 110
Glu Gly Met Gln Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val
115 120 125
Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly
130 135 140
Glu Leu Gly Tyr Ser Ile Ser His Gly Val Gly Ala Ile Leu Asp Asn
145 150 155 160
Pro Asp Glu Ile Ala Ala Val Val Val Gly Asp Gly Glu Ser Glu Thr
165 170 175
Gly Pro Leu Ala Thr Ser Trp Gln Ser Thr Lys Phe Ile Asn Pro Ile
180 185 190
Asn Asp Gly Ala Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile
195 200 205
Ser Asn Pro Thr Ile Phe Gly Arg Thr Ser Asp Ala Lys Ile Lys Glu
210 215 220
Tyr Phe Glu Ser Met Asn Trp Glu Pro Ile Phe Val Glu Gly Asp Asp
225 230 235 240
Pro Glu Lys Val His Pro Ala Leu Ala Lys Ala Met Asp Glu Ala Val
245 250 255
Glu Lys Ile Lys Ala Ile Gln Lys His Ala Arg Glu Asn Asp Asp Ala
260 265 270
Thr Leu Pro Val Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp
275 280 285
Thr Gly Pro Lys Ser Trp Asp Gly Asp Lys Ile Glu Gly Ser Phe Arg
290 295 300
Ala His Gln Ile Pro Ile Pro Val Asp Gln Asn Asp Met Glu His Ala
305 310 315 320
Asp Ala Leu Val Asp Trp Leu Glu Ser Tyr Gln Pro Lys Glu Leu Phe
325 330 335
Asn Glu Asp Gly Ser Leu Lys Asp Asp Ile Lys Glu Ile Ile Pro Thr
340 345 350
Gly Asp Ser Arg Met Ala Ala Asn Pro Ile Thr Asn Gly Gly Val Asp
355 360 365
Pro Lys Ala Leu Asn Leu Pro Asn Phe Arg Asp Tyr Ala Val Asp Thr
370 375 380
Ser Lys Glu Gly Ala Asn Val Lys Gln Asp Met Leu Val Trp Ser Asp
385 390 395 400
Tyr Leu Arg Asp Val Ile Lys Lys Asn Pro Asp Asn Phe Arg Leu Phe
405 410 415
Gly Pro Asp Glu Thr Met Ser Asn Arg Leu Tyr Gly Val Phe Glu Thr
420 425 430
Thr Asn Arg Gln Trp Met Glu Asp Ile His Pro Asp Ser Asp Gln Tyr
435 440 445
Glu Ala Pro Ala Gly Arg Val Leu Asp Ala Gln Leu Ser Glu His Gln
450 455 460
Ala Glu Gly Trp Leu Glu Gly Tyr Val Leu Thr Gly Arg His Gly Leu
465 470 475 480
Phe Ala Ser Tyr Glu Ala Phe Leu Arg Val Val Asp Ser Met Leu Thr
485 490 495
Gln His Phe Lys Trp Leu Arg Lys Ala Asn Glu Leu Asp Trp Arg Lys
500 505 510
Lys Tyr Pro Ser Leu Asn Ile Ile Ala Ala Ser Thr Val Phe Gln Gln
515 520 525
Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly Ala Leu Thr His Leu
530 535 540
Ala Glu Lys Lys Pro Glu Tyr Ile Arg Glu Tyr Leu Pro Ala Asp Ala
545 550 555 560
Asn Thr Leu Leu Ala Val Gly Asp Val Ile Phe Arg Ser Gln Glu Lys
565 570 575
Ile Asn Tyr Val Val Thr Ser Lys His Pro Arg Gln Gln Trp Phe Ser
580 585 590
Ile Glu Glu Ala Lys Gln Leu Val Asp Asn Gly Leu Gly Ile Ile Asp
595 600 605
Trp Ala Ser Thr Asp Gln Gly Ser Glu Pro Asp Ile Val Phe Ala Ala
610 615 620
Ala Gly Thr Glu Pro Thr Leu Glu Thr Leu Ala Ala Ile Gln Leu Leu
625 630 635 640
His Asp Ser Phe Pro Glu Met Lys Ile Arg Phe Val Asn Val Val Asp
645 650 655
Ile Leu Lys Leu Arg Ser Pro Glu Lys Asp Pro Arg Gly Leu Ser Asp
660 665 670
Ala Glu Phe Asp His Tyr Phe Thr Lys Asp Lys Pro Val Val Phe Ala
675 680 685
Phe His Gly Tyr Glu Asp Leu Val Arg Asp Ile Phe Phe Asp Arg His
690 695 700
Asn His Asn Leu Tyr Val His Gly Tyr Arg Glu Asn Gly Asp Ile Thr
705 710 715 720
Thr Pro Phe Asp Val Arg Val Met Asn Gln Met Asp Arg Phe Asp Leu
725 730 735
Ala Lys Ser Ala Ile Ala Ala Gln Pro Ala Met Glu Asn Thr Gly Ala
740 745 750
Ala Phe Val Gln Ser Met Asp Asn Met Leu Ala Lys His Asn Ala Tyr
755 760 765
Ile Arg Asp Ala Gly Thr Asp Leu Pro Glu Val Asn Asp Trp Gln Trp
770 775 780
Lys Gly Leu Lys
785
<210> 44
<211> 796
<212> PRT
<213> Lactobacillus plantarum
<400> 44
Met Ser Glu Ala Ile Lys Ser Lys Thr Val Asp Tyr Ser Ser Asp Glu
1 5 10 15
Tyr Leu Lys Arg Val Asp Glu Tyr Trp Arg Ala Ala Asn Tyr Ile Ser
20 25 30
Val Gly Gln Leu Tyr Leu Leu Asn Asn Pro Leu Leu Arg Glu Pro Leu
35 40 45
Lys Ala Thr Asp Val Lys Val His Pro Ile Gly His Trp Gly Thr Ile
50 55 60
Ala Gly Gln Asn Phe Ile Tyr Ala His Leu Asn Arg Ala Ile Asn Lys
65 70 75 80
Tyr Gly Leu Asn Met Phe Tyr Ile Glu Gly Pro Gly His Gly Gly Gln
85 90 95
Val Met Val Ser Asn Ser Tyr Leu Asp Gly Thr Tyr Thr Glu Thr Tyr
100 105 110
Pro Lys Ile Thr Gln Asp Lys Ala Gly Met Lys Arg Leu Phe Lys Gln
115 120 125
Phe Ser Phe Pro Gly Gly Val Ala Ser His Ala Asp Pro Lys Thr Pro
130 135 140
Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ser Ile Leu His Gly
145 150 155 160
Ala Gly Ala Val Leu Asp Asn Pro Gly Leu Ile Ala Ala Thr Val Val
165 170 175
Gly Asp Gly Glu Ser Glu Thr Gly Pro Leu Ala Thr Ser Trp Gln Val
180 185 190
Asn Lys Phe Leu Asn Pro Ile Thr Asp Gly Thr Val Leu Pro Ile Leu
195 200 205
Asn Leu Asn Gly Phe Lys Ile Ser Asn Pro Thr Val Leu Ser Arg Glu
210 215 220
Ser His Glu Glu Leu Glu Asp Tyr Phe Lys Gly Leu Gly Trp Asp Pro
225 230 235 240
His Phe Val Glu Gly Thr Asp Pro Ala Lys Met His Lys Ile Met Ala
245 250 255
Glu Glu Leu Asp Lys Val Ile Glu Glu Ile His Ala Ile Arg Lys Asn
260 265 270
Ala Lys Asp Asn Asn Asp Glu Ser Arg Pro Lys Trp Pro Met Ile Val
275 280 285
Phe Arg Ala Pro Lys Gly Trp Thr Gly Pro Lys Ser Trp Asp Gly Glu
290 295 300
Pro Ile Glu Gly Ser Phe Arg Ala His Gln Ile Pro Ile Pro Val Asp
305 310 315 320
Arg Asn His Met Glu His Ala Asp Lys Leu Val Asp Trp Leu Lys Ser
325 330 335
Tyr Lys Pro Glu Glu Leu Phe Asp Glu Asn Gly Thr Leu Lys Pro Glu
340 345 350
Ile Ala Ala Ile Ile Pro Glu Gly Gln Ala Arg Met Ala Ala Asn Pro
355 360 365
Val Thr Asn Gly Gly Lys Leu Thr Lys Asp Leu Ile Thr Pro Asn Ile
370 375 380
Asp Asp Tyr Ala Leu Asp Asn Lys Asp His Gly Lys Glu Asp Gly Ser
385 390 395 400
Asp Met Thr Glu Leu Gly Lys Tyr Ile Arg Asp Leu Ile Glu Leu Asn
405 410 415
Lys Asp Asn Lys Asn Phe Arg Gly Trp Gly Pro Asp Glu Thr Leu Ser
420 425 430
Asn Lys Leu Gly Ala Ala Phe Glu Asp Thr Lys Arg Gln Trp Met Glu
435 440 445
Pro Ile His Glu Pro Asn Asp Ala Leu Leu Ala Pro Gln Gly Arg Ile
450 455 460
Ile Asp Ser Met Leu Ser Glu His Met Asp Glu Gly Met Leu Glu Ala
465 470 475 480
Tyr Asn Leu Thr Gly Arg Tyr Gly Phe Phe Ala Ser Tyr Glu Ser Phe
485 490 495
Leu Arg Val Val Asp Ser Met Leu Thr Gln His Phe Lys Trp Leu Arg
500 505 510
Asn Ser His Glu Glu Thr Pro Trp Arg Ala Asp Val Pro Ser Leu Asn
515 520 525
Val Ile Ala Ser Ser Thr Ala Phe Gln Gln Asp His Asn Gly Tyr Ser
530 535 540
His Gln Asp Pro Gly Ile Ile Ser His Leu Ala Glu Lys Lys Thr Glu
545 550 555 560
Tyr Val Arg Ala Tyr Leu Pro Gly Asp Ala Asn Thr Leu Ile Ala Thr
565 570 575
Phe Asp Lys Ala Ile Gln Ser Lys Gln Leu Ile Asn Leu Ile Ile Ala
580 585 590
Ser Lys His Pro Arg Pro Gln Trp Phe Thr Met Asp Glu Ala Lys Arg
595 600 605
Leu Val Arg Asp Gly Leu Gly Val Ile Asp Trp Ala Ser Thr Asp His
610 615 620
Gly Glu Glu Pro Asp Val Val Phe Ala Thr Ala Gly Ser Glu Pro Thr
625 630 635 640
Thr Glu Ser Leu Ala Ala Val Ser Ile Leu His Ala Arg Phe Pro Glu
645 650 655
Met Lys Ile Arg Phe Ile Asn Val Val Asp Leu Leu Lys Leu Lys Lys
660 665 670
Asp Asp Pro Arg Gly Leu Ser Asp Ala Glu Phe Asp Ala Phe Phe Thr
675 680 685
Lys Asp Lys Pro Val Ile Phe Ala Tyr His Ala Tyr Asp Asp Leu Val
690 695 700
Lys Thr Ile Phe Phe Asp Arg His Asn His Asn Leu His Val His Gly
705 710 715 720
Tyr Arg Glu Glu Gly Asp Ile Thr Thr Pro Phe Asp Met Arg Val Arg
725 730 735
Asn Glu Leu Asp Arg Phe His Leu Val Lys Ala Ala Leu Leu Ala Thr
740 745 750
Pro Ala Tyr Ala Glu Lys Gly Ala His Val Ile Gln Glu Met Asn Ser
755 760 765
Ile Leu Asp Lys His His Asp Tyr Ile Arg Ala Glu Gly Thr Asp Ile
770 775 780
Pro Glu Val Glu Asn Trp Lys Trp Thr Ala Leu Lys
785 790 795

Claims (10)

1.重组酵母在生产酪醇中的应用，所述重组酵母为将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中构建获得，所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞；
所述果糖-6-磷酸盐磷酸酮酶的表达基因来源于动物双歧杆菌(Bifidobacteriumanimalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacteriummongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)；所述经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
或者，所述果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、短双歧杆菌(Bifidobacterium breve)、齿双歧杆菌(Bifidobacterium dentium)；所述经过改造的酵母细胞为将芳香醛合成酶整合后获得。
2.如权利要求1所述的应用，其特征在于，所述芳香醛合成酶来源于香芹，酶的系统编号EC4.1.1.25；所述分支酸变位酶来源于大肠杆菌，酶的系统编号EC1.3.1.12；所述预苯酸脱水酶来源于大肠杆菌，酶的系统编号5.4.99.5。
3.如权利要求1所述的应用，其特征在于，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示。
4.如权利要求1所述的应用，其特征在于，所述酵母细胞为：酿酒酵母(Saccharomycescerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candidaglabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candida auris)等；
进一步优选的，所述酵母细胞为酿酒酵母(Saccharomyces cerevisiae)，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777；
进一步优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinumcrispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，获得。
更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得；
进一步优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，获得。
更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
5.一种高产酪醇的重组酵母的构建方法，其特征在于，包括如下步骤：
(1)构建表达盒，表达盒由启动子、终止子、同源臂以及果糖-6-磷酸盐磷酸酮酶的表达基因经融合后获得；
(2)将步骤(1)构建的表达盒转化至经过改造的酵母细胞，制得高产酪醇的重组酵母；
所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞；
所述步骤(1)中果糖-6-磷酸盐磷酸酮酶的表达基因来源于动物双歧杆菌(Bifidobacterium animalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacteriummongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)；所述步骤(2)中，经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
或者，所述步骤(1)中果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、短双歧杆菌(Bifidobacterium breve)、齿双歧杆菌(Bifidobacterium dentium)；所述步骤(2)中，经过改造的酵母细胞为将芳香醛合成酶整合后获得。
6.如权利要求5所述的构建方法，其特征在于，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ IDNO.4所示；
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示；
优选的，所述步骤(1)中同源臂为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板，利用引物扩增预苯酸脱水酶(prephenate dehydratase)基因pha2上游及下游500bp的基因片段，所述上游同源臂扩增引物的核苷酸序列分别如SEQ IDNO.5和SEQ ID NO.6所示；下游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.7和SEQ IDNO.8所示；
优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的启动子tpi1，所述启动子tpi1的扩增引物的核苷酸序列分别如SEQ ID NO.9和SEQ ID NO.10所示；
优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的终止子gpm1，所述终止子gpm1的扩增引物的核苷酸序列分别如SEQ ID NO.11和SEQ ID NO.12所示；
进一步优选的，所述步骤(2)中，芳香醛合成酶来源于香芹(Petroselinum crispum)，酶的系统编号EC4.1.1.25；所述分支酸变位酶/预苯酸脱水酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12，所述预苯酸脱水酶来源于大肠杆菌，酶的系统编号EC 5.4.99.5；
优选的，所述步骤(2)中所述酵母细胞为：酿酒酵母(Saccharomyces cerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candida glabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candidaauris)等；
进一步优选的，所述酵母细胞为酿酒酵母，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777；
进一步优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinumcrispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，获得。
更优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinumcrispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得；
进一步优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，获得。
更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
7.权利要求5所述方法构建的高产酪醇的重组酵母。
8.权利要求7所述高产酪醇的重组酵母在发酵制备酪醇中的应用；
优选的，所述发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
9.权利要求7所述高产酪醇的重组酵母在发酵制备羟基酪醇中的应用。
10.如权利要求9所述的应用，其特征在于，将权利要求7所述高产酪醇的重组酵母通过发酵制备酪醇后，经过4-羟基苯乙酸羟化酶反应获得羟基酪醇；
优选的，利用过表达4-羟基苯乙酸羟化酶大肠杆菌催化权利要求8所述制备的酪醇后，获得羟基酪醇；
进一步优选的，所述的发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
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