CN110637091B - 利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法 - Google Patents

利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法 Download PDF

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CN110637091B
CN110637091B CN201980002419.1A CN201980002419A CN110637091B CN 110637091 B CN110637091 B CN 110637091B CN 201980002419 A CN201980002419 A CN 201980002419A CN 110637091 B CN110637091 B CN 110637091B
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corynebacterium
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fucosyllactose
fucosyltransferase
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申哲寿
尹钟远
宋永厦
柳英善
金甫美
金镇景
李佳恩
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SNU R&DB Foundation
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Abstract

本发明涉及一种从插入源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物中生产2’‑岩藻糖基乳糖的方法，本发明的插入源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物比常规大肠杆菌更安全，同时可以高浓度、高产率、高生产率生产2’‑岩藻糖基乳糖。

Description

利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基 乳糖的生产方法
技术领域
本发明涉及一种2’-岩藻糖基乳糖(2'-fucosyllactose,2'-FL)的生产方法，更具体地，涉及一种从插入源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物中生产2’-岩藻糖基乳糖的方法。
背景技术
与其它哺乳类的乳汁相比，人的母乳中存在相当高浓度(5～15g/L)的200多种以上的具有独特结构的低聚糖(human milk oligosaccharides,HMO)。HMO由D-葡萄糖(Glc)、D-半乳糖、N-乙酰氨基葡萄糖(N-aetylglucosamine,GlcNAc)、L-岩藻糖(L-fucose,Fuc)和唾液酸(sialc acid)[Sia；N-acetyl neuraminic acid(Neu5Ac)]构成。
由于HMO的结构非常多样且复杂，因此具有其它残基和糖基化键的约200个异构体可能以相互不同的聚合度(DP 3-20)存在。然而，尽管结构复杂，HMO仍呈现几个共同的结构，其中，大部分HMO在还原端具有乳糖(Galβ1-4Glc)残基。乳糖的Gal可以以α-(2,3)-和α-(2,6)-键各自唾液化为3-唾液乳糖(3-sialyllactose)或6-唾液乳糖(6-sialyllactose)的形态，或者以α-(1,2)-和α-(1,3)-键各自岩藻糖基化(fucosylation)为2-岩藻糖基乳糖(2'-fucosyllactose,2'-FL)或3-岩藻糖基乳糖(3'-fucosyllactose,3-FL)的形态。
包括母乳中含量最高的三种低聚糖在内的137个被岩藻糖基化，其比率约为77％，而其余低聚糖大部分被唾液化(39个)，约为28％。其中，尤其，2-岩藻糖基乳糖和3-岩藻糖基乳糖是提供有助于肠内乳酸菌生长的益生元(prebiotic)效果、预防病原菌感染、调节免疫系统以及对大脑发育等婴儿的发育以及健康具有积极影响的各种生物学活性的主要HMO，因此专家们强调婴儿期的母乳喂养非常重要。
但是，已知约20％的女性由于合成岩藻糖基低聚糖(fucosyl-oligosaccharides)的岩藻糖基转移酶(fucosyltransferase)的突变，在体内无法正常合成它们。因此，实际上需要岩藻糖基乳糖的产业化生产。
另一方面，作为岩藻糖基乳糖的生产方法，有直接从母乳提取的方法和通过化学或者酶方法合成的方法。直接提取的方法存在母乳供求限制和生产率低的问题，化学合成方法存在基质昂贵、异构体选择性(stereo-selectivity)和生产产率低以及有毒有机溶剂的使用等问题。并且，酶合成方法存在用作岩藻糖的供体(dornor)的GDP-L-fucose非常昂贵，岩藻糖基转移酶的提炼费用高的问题。
由于上述问题，直接提取、化学或酶生产方法难以应用于岩藻糖基乳糖的大量生产，作为该问题的解决方案，提出了利用微生物来生产2’-岩藻糖基乳糖的解决方案。利用微生物来生产2’-岩藻糖基乳糖的现有技术大部分是利用重组大肠杆菌生产的技术。但是，用于实验的大部分大肠杆菌实际上并不是病原菌，但消费者普遍认为是有害菌。
并且，大肠杆菌的细胞膜成分有可能起到内毒素作用，因此在生产2’-岩藻糖基乳糖时，分离提炼费用高。因此，目前还难以将大肠杆菌用作生产食品以及医药品的原材料，即岩藻糖基乳糖的宿主细胞。
发明内容
技术课题
本发明的目的在于提供一种作为生产食品以及医药品的原材料，即岩藻糖基乳糖的宿主细胞，利用插入源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物来生产2’-岩藻糖基乳糖的方法。
技术课题
本发明提供一种重组棒状杆菌属(Corynebacterium SP.)微生物，其特征在于，其转化为表达出源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的具有对应于序列号5的氨基酸序列的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)，转化为表达出GDP-D-甘露糖-4,6-脱水酶(GDP-D-mannose-4,6-dehydratase)，转化为表达出GDP-L-岩藻糖合酶(GDP-L-fucose synthase)，转化为表达出乳糖渗透酶(lactose permease)，且保留磷酸甘露糖变位酶(phosphomannomutase)以及GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphate guanylyltransferase)。
优选地，本发明的重组棒状杆菌属(Corynebacterium SP.)微生物选自谷氨酸棒状杆菌(Corynebacterium glutamicum)、产氨棒状杆菌(Corynebacterium ammoniagenes)以及热产氨棒状杆菌(Corynebacterium thermoaminogenes)中的任意一个。
在本发明的重组棒状杆菌属(Corynebacterium SP.)微生物中，优选地，上述具有对应于序列号5的氨基酸序列的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)被序列号4所记载的核酸序列加密。
在本发明的重组棒状杆菌属(Corynebacterium SP.)微生物中，优选地，上述重组棒状杆菌属(Corynebacterium SP.)微生物转化为超表达出磷酸甘露糖变位酶(phosphomannomutase)，转化为超表达出GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphate guanylytransferase)。
另外，本发明提供一种2’-岩藻糖基乳糖的生产方法，其特征在于，在添加有乳糖的培养基中培养上述本发明的重组棒状杆菌属(Corynebacterium SP.)微生物。
在本发明的2’-岩藻糖基乳糖的生产方法中，优选地，上述培养基还包含葡萄糖。
发明效果
本发明的插入源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物比常规大肠杆菌更安全，同时可以高浓度、高产率、高生产率生产2’-岩藻糖基乳糖。
附图说明
图1是pFGW(Ps)质粒的示意图。
图2是pXIL质粒的示意图。
图3是pFGW(Hp)质粒的示意图。
图4是插入有源自幽门螺杆菌(Helicobacter pylori)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的培养结果(●：干燥细胞重量，■：乳糖，▼：葡萄糖，◆：2'-FL，◇：乳酸盐，○：醋酸盐)。
图5是插入有源自舞蹈土地杆菌(Pedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的培养结果(●：干燥细胞重量，■：乳糖，▼：葡萄糖，◆：2'-FL，◇：乳酸盐，○：醋酸盐)。
具体实施方式
本发明提供一种重组棒状杆菌属(Corynebacterium SP.)微生物，其特征在于，其转化为表达出源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的具有对应于序列号5的氨基酸序列的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)，转化为表达出GDP-D-甘露糖-4,6-脱水酶(GDP-D-mannose-4,6-dehydratase)，转化为表达出GDP-L-岩藻糖合酶(GDP-L-fucose synthase)，转化为表达出乳糖渗透酶(lactose permease)，且保留磷酸甘露糖变位酶(phosphomannomurase)以及GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphate guanylyltransferase)。
优选地，本发明的重组棒状杆菌属(Corynebacterium SP.)微生物选自谷氨酸棒状杆菌(Corynebacterium glutamicum)、产氨棒状杆菌(Corynebacterium ammoniagenes)以及热产氨棒状杆菌(Corynebacterium thermoaminogenes)中的任意一个。
另一方面，黄色短杆菌(Brevibacterium flavum)目前被分类为谷氨酸棒状杆菌(Corynebacterium glutamicum)，因此黄色短杆菌(Brevibacterium flavum)菌株也包含在本发明的范围内。
并且，根据UniProt数据库，解糖短杆菌(Brevibacterium saccharolyticum)是谷氨酸棒状杆菌(Corynebacterium glutamicum)的同义词，并且乳酸发酵短杆菌(Brevibacterium lactofermentum)也以谷氨酸棒状杆菌(Corynebacterium glutamicum)的别名命名，因此解糖短杆菌(Brevibacterium saccharolyticum)以及乳酸发酵短杆菌(Brevibacterium lactofermentum)也包含在本发明的范围内(W.LIEBL等，INTERNATIONALJOURNAL OF SYSTEMATIBCA CTERIOLOGY,Apr.1991,p.255-260；LOTHAR EGGELING等，JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol.92,No.3,201-213.2001；JillA.Haynes等，FEMS Microbiology Letters 61(1989)329-334)。
本发明人之前通过韩国授权专利第10-17312630000号(2017.04.24)获得过利用棒状杆菌的2’-岩藻糖基乳糖的生产方法的专利。在本发明中，将源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)插入棒状杆菌中，从而可以获得比常规的通过将源自幽门螺杆菌(Helicovacter pylori)的岩藻糖基转移酶(fucosyltransferase)插入棒状杆菌中而生产的2’-岩藻糖基乳糖显著大量的2'岩藻糖基乳糖。
另一方面，与常规使用的大肠杆菌不同，谷氨酸棒状杆菌(Corynebacteriumglutamicum)或产氨棒状杆菌(Corynebacterium ammoniagenes)不仅是被认定为GRAS(generally recognized as safe)的菌株，而且是不产生内毒素且广泛应用于作为食品添加剂的氨基酸以及核酸的产业化生产的菌株。因此，可以说谷氨酸棒状杆菌(Corynebacterium glutamicum)或产氨棒状杆菌(Corynebacterium ammoniagenes)是适合生产食品以及医药品的原材料的菌株，具有可以打消消费者对于安全性方面的担忧的优点。
另一方面，由于大肠杆菌和包含谷氨酸棒状杆菌(Corynebacteriumglutamicum)、产氨棒状杆菌(Corynebacterium ammoniagenes)以及热产氨棒状杆菌(Corynebacterium thermoaminogenes)的棒状杆菌属(Corynebacterium SP.)微生物的菌株本身的遗传特性不同，因此需要使用与应用于大肠杆菌的战略不同的战略。为了生产2’-岩藻糖基乳糖，大肠杆菌和棒状杆菌属(Corynebacterium SP.)微生物基本上都需要引入外来的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)，但是，棒状杆菌属(Corynebacterium SP.)微生物除此之外还需进一步引入GDP-D-甘露糖-4,6-脱水酶(GDP-D-mannose-4,6-degydratase,Gmd)、GDP-L-岩藻糖合酶(GDP-L-fucose synthase,WcaG)、乳糖渗透酶(lactose permease,LacY)。
此时，在本发明的重组棒状杆菌属(Corynebacterium SP.)微生物中，优选地，上述具有对应于序列号5的氨基酸序列的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)被序列号4所记载的核酸序列加密，并且作为加密上述GDP-D-甘露糖-4,6-脱水酶(GDP-D-mannose-4,6-dehydratase,Gmd)、GDP-L-岩藻糖合酶(GDP-L-fucose synthase,GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase,WcaG)以及乳糖渗透酶(lactose permease,LacY)的基因可以使用常见的基因，优选地使用源自大肠杆菌的基因。然而，乳糖渗透酶(lactose permease,LacY)是参与将存在于菌株外部的乳糖输送至菌株内部的酶，在本发明中，引入Lac操纵子的目的在于引入乳糖，因此仅引入lacY基因即可，无需非要引入lacA基因。
另一方面，重组棒状杆菌属(Corynebacterium SP.)微生物本身可以保留加密磷酸甘露糖变位酶(phosphomannomutase,ManB)、GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphate guanylyltransferase,ManC)的基因，并表达出来，因此无需非要超表达该酶，但是，为了大量生产，需要超表达该酶。因此，在本发明的重组棒状杆菌属(Corynebacterium SP.)微生物中，优选地，上述重组棒状杆菌属(Corynebacterium SP.)微生物转化为超表达出磷酸甘露糖变位酶(phosphomannomurase)，转化为超表达出GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphate guanylytransferase)。
并且，本发明提供一种2’-岩藻糖基乳糖的生产方法，其特征在于，在添加有乳糖的培养基中培养上述本发明的重组棒状杆菌属(Corynebacterium SP.)微生物。与使用现有菌株相比，根据以下本发明的实验，可以高浓度、高产率、高生产率从重组棒状杆菌属(Corynebacterium SP.)微生物中生产2’-岩藻糖基乳糖。
另一方面，在本发明的2’-岩藻糖基乳糖的生产方法中，优选地，上述培养基还包含高浓度的葡萄糖。通过将葡萄糖添加到培养基中，菌株的生长变得活跃，从而可以更高的生产率生产2’-岩藻糖基乳糖。
另一方面，优选地，本发明的2’-岩藻糖基乳糖的生产方法是附加供给葡萄糖或者乳糖的补料分批培养。这是因为通过补料分批培养持续供给葡萄糖或者乳糖时，可以进一步增大细胞的成长，并且以高纯度、高产率、高生产率生产岩藻糖基乳糖。关于补料分批培养的具体细节技术可以采用本领域公知技术，因此省略对其的记载。
下面，通过以下实施例更加详细说明本发明的内容。但是，本发明的保护范围并不仅仅限定于以下实施例，而甚至包括与其等同的技术思想的变形。
另一方面，以下仅利用谷氨酸棒状杆菌(Corynebacterium glutamicum)作为宿主细胞来确认了效果，但由于谷氨酸棒状杆菌(Corynebacterium glutamicum)和产氨棒状杆菌(Corynebacterium ammoniagenes)、热产氨棒状杆菌(Corynebacteriumthermoaminogenes)、黄色短杆菌(Brevibacterium flavum)以及乳酸发酵短杆菌(Brevibacterium lactofermentum)可以同样地应用转化系统，因此可以期待相同的效果。
[制造例1：重组质粒的制备]
为了制备质粒并生产2’-岩藻糖基乳糖(fucosyllactose,2'-FL)，分别利用了大肠杆菌(Escherichia coli)K-12MG1655和谷氨酸棒状杆菌(Corynebacteriumglutamicum)ATCC 13032。
为了组建pFGW(Ps)质粒，从大肠杆菌，即K-12MG1655的基因组(genomic)DNA通过利用两个DNA引物(primer)GW-F和GW-R的PCR反应放大gmd-wcaG基因簇(cluster)，并且从谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC 13032的基因组DNA通过利用两个DNA引物Sod-F和Sod-R的PCR反应放大Sod基因的启动子之后，利用两个DNA引物Sod-F和GW-R通过重叠(overlap)PCR反应合成了pSod-Gmd-WcaG DNA片段。
并且，从pXMJ19质粒(plasmid)通过利用两个DNA引物Ter-F和Ter-R的PCR反应放大转录终止序列之后，使用上述合成的pSod-Gmd-WcaG和转录终止序列作为模板通过利用两个DNA引物Sod-F和Ter-R的PCR反应合成pSod-Gmd-WcaG-ter序列之后，将其插入用限制酶BamHⅠ切割的pCES208质粒中，从而组建了pGW质粒。
并且，从谷氨酸棒状杆菌ATCC 13032的基因组DNA通过利用两个DNA引物Tuf-F1和Tuf-R1的PCR反应放大Tuf基因启动子，并且从源自合成的舞蹈拟土地杆菌(Pseudopedobacter saltans)DSM 12145的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)通过利用两个DNA引物FT(Ps)-F和FT(Ps)-R的PCR反应放大α-1,2-岩藻糖基转移酶之后，利用两个引物Tuf-F和FT(Ps)-R通过重叠(overlap)PCR反应合成了pTuf-FT(Ps)DNA片段。通过用限制酶NotⅠ处理上述组建的pGW质粒来插入pTuf-FT(Ps)，从而组建了pFGW(Ps)质粒。
另一方面，为了组建pXIL质粒，从大肠杆菌K-12MG1655的基因组DNA通过利用两个DNA引物ilvC-lacY-F和lacY pX-R的PCR反应放大lacY基因，并且从谷氨酸棒状杆菌ATCC13032的基因组DNA通过利用两个DNA引物pX-ilvC-F和ilvC-lacY-R的PCR反应放大ilvC基因的启动子之后，利用两个DNA引物pX-ilvC-F和lacY pX-R通过重叠(overlap)PCR反应合成pilvC-lacY DNA片段之后，将其插入由限制酶NotⅠ和EcoRⅠ处理的pX质粒(pXMJ19)中，从而组建了pXIL质粒。
另一方面，为了组建pFGW(Hp)载体，利用了上述组建的pGW质粒，从谷氨酸棒状杆菌ATCC 13032的基因组DNA通过利用两个DNA引物Tuf-F1和Tuf-R2的PCR反应放大Tuf基因启动子，并且从源自通过密码子优化(codon optimization)谷氨酸棒状杆菌而合成的幽门螺杆菌(Helicobacter pylori)ATCC 700392的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)通过利用两个DNA引物FT(Hp)-F和FT(Hp)-R的PCR反应放大α-1,2-岩藻糖基转移酶之后，利用两个引物Tuf-F1和FT(Hp)-R通过重叠(overlap)PCR反应合成了pTuf-FT(Ps)DNA片段。通过用限制酶NotⅠ处理上述组建的pGW质粒来插入pTuf-FT(Hp)，从而组建了pFGW(Hp)质粒。
本制造例中使用的菌株(strain)、引物(primer)、质粒(plasmid)、核酸以及氨基酸序列示于下表1至4中。
[表1]
菌株
E.Coli K-12 MG1655 F-,lambda-,rph-1
C.glutamicum Wild-type strain,ATCC13032
[表2]
核酸以及氨基酸序列
gmd核酸序列 序列号1
wcaG核酸序列 序列号2
lacY核酸序列 序列号3
FT(Ps)核酸序列 序列号4
FT(Ps)氨基酸序列 序列号5
FT(Hp)核酸序列 序列号6
FT(Hp)氨基酸序列 序列号7
[表3]
引物
引物 序列(5'→3')
pX-ilvC-F GTCATATGATGGTCGCGGATCCGAATTCCCAGGCAAGCTCCGC
ilvC-lacY-R GTTTTTTAAATAGTACATAATCTCGCCTTTCGTAAAAATTTTGGT
ilvC-lacY-F TTACGAAAGGCGAGATTATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGG
lacY pX-R GCCTTTCGTTTTATTTGCTCGAGTGCGGCCGCTTAAGCGACTTCATTCACCTGACGAC
Tuf-F1 TGGAGCTCCACCGCGGTGGCTGGCCGTTACCCTGCGAA
Tuf-R1 CAAATATCATTGTATGTCCTCCTGGACTTCG
FT(ps)-F AGGACATACAATGATATTTGTAACCGGATATG
FT(ps)-R CGCTTCACTAGTTCTAGAGCTTAAATAATGTGTCGAAACAGATTC
Sod-F GCTCTAGAACTAGTGAAGCGCCTCATCAGCG
Sod-R TACACCGGTGATGAGAGCGACTTTTGACATGGTAAAAAATCCTTTCGTAGGTTTCCGCAC
GW-F ATGTCAAAAGTCGCTCTCATCACCGGTGTA
GW-R CAAGCTGAATTCTTACCCCCGAAAGCGGTC
ter-F GACCGCTTTCGGGGGTAAGAATTCAGCTTG
ter-R GGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGAAAAGGCCATCCGTCAGGAT
Tuf-R2 TGAAAGCCATTGTATGTCCTCCTGGACTTCGT
FT(Hp)-F GGACATACAATGGCTTTCAAGGTGGTCCAAAT
FT(Hp)-R GCTTCACTAGTTCTAGAGCTTAAGCATTGTATTTCTGGCTCTTCACTTCG
[表4]
质粒
Figure GDA0002273085460000101
[实施例1：插入有源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的培养]
在装有包含25μg/mL卡那霉素(kanamycin)和5μg/mL氯霉素的5mL的BHI(BrainHeart Infusion)培养基的实验中，将插入有pFGW(Ps，图1)和pXIL(图2)的谷氨酸棒状杆菌(C.glutamicum)ATCC 13032在30℃，250rpm下种菌培养12小时。图1是pFGW(Ps)质粒的示意图，图2是pXIL质粒的示意图。
分批培养利用了装有50mL的最低培养基((NH4)2SO4 20g/L，Urea 5g/L，KH2 PO41g/L，K2HPO4 1g/L，MgSO4·7H2O 0.25g/L，MOPS 42g/L，CaCl2 10mg/L，Biotin 0.2mg/L，Protocatechuic acid 30mg/L，FeSO4·7H2O 10mg/L，MnSO4·H2O 10mg/L，ZnSO4·7H2O1mg/L，CuSO4 0.2mg/L，NiCl2·6H2O 0.02mg/L，Glucose 40g/L，Lactose 10g/L，pH7.0)的250mL烧瓶，在30℃，250rpm下进行了90小时。
[比较例1：插入有源自幽门螺杆菌(Helicobacter pylori)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的培养]
使用了与实施例1中相同的培养方法，不同之处在于，将pFGW(Ps，图1)变为pFGW(Hp，图3)。图3是pFGW(Hp)质粒的示意图。
[实验例1：实施例1和比较例1重组菌株的2'-FL的生产比较]
对实施例1和比较例1中制造的重组菌株中的2'-FL的产量进行了比较(图4、图5、表5)。图4是插入有源自幽门螺杆菌(Helicobacter pylori)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的培养结果，图5是插入有源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的培养结果(●：干燥细胞重量，■：乳糖，▼：葡萄糖，◆：2'-FL，◇：乳酸盐，○：醋酸盐)。
[表5]
Figure GDA0002273085460000111
实验结果，可以确认，与插入有源自幽门螺杆菌(Helicobacter pylori)的岩藻糖基转移酶(fucosyltransferase)的重组菌株相比，插入有源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的岩藻糖基转移酶(fucosyltransferase)的重组菌株的2’-岩藻糖基乳糖生产率高约2倍。
<110> 高级蛋白质技术公司
<120> 利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法
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ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240
accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300
tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360
ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420
ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480
atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540
gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600
gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660
aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720
caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780
tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840
ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900
gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960
ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020
tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttctt taagcaactg 1080
gcgatgattt ttatgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140
gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200
agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc ttaa 1254
<210> 4
<211> 807
<212> DNA
<213> Unknown
<220>
<223> Pseudopedobacter saltans
<400> 4
atgatatttg taaccggata tggccagatg tgtaacaaca tccttcaatt tgggcatttc 60
tttgcttatg caaaaagaaa tggtttaaaa acggttggct tacgtttttg ctacaaatac 120
acttttttca agattagtaa cgaaaaaggc tataattggc cgacctatct ttatgcaaaa 180
tatggggcaa aaataggact tataaagtct gttgattttg acgaatcatt cgaaggtaca 240
aatgtagatt ctcttcaatt agacaaacaa accgtgttag ccaaaggctg gtattttaga 300
gactaccagg gatttcttaa ttaccgtaat gagcttaaag cacttttcga ctttaaagag 360
catattaaga aaccggtaga acagtttttt tcaacgttat caaaagacac catcaaagta 420
ggcctgcata taagacgtgg tgattataag acctggcacc agggtaaata cttttttagc 480
gacgaagaat acggtcaaat cgtaaattct tttgctaaaa gtttagataa accggtagaa 540
ttaattattg ttagcaatga tcccaaacta aacagcaaaa gttttgaaaa tttaacatcc 600
tgtaaagtat caatgttaaa tggcaatcct gccgaagatc tttaccttct ttctaaatgt 660
gattatatta ttggccctcc cagcactttt tctttaatgg cagcttttta cgaagaccgc 720
cctttatatt ggatatttga taaagaaaaa cagcttttag cagaaaactt tgacaagttc 780
gagaatctgt ttcgacacat tatttaa 807
<210> 5
<211> 268
<212> PRT
<213> Unknown
<220>
<223> Pseudopedobacter saltans
<400> 5
Met Ile Phe Val Thr Gly Tyr Gly Gln Met Cys Asn Asn Ile Leu Gln
1 5 10 15
Phe Gly His Phe Phe Ala Tyr Ala Lys Arg Asn Gly Leu Lys Thr Val
20 25 30
Gly Leu Arg Phe Cys Tyr Lys Tyr Thr Phe Phe Lys Ile Ser Asn Glu
35 40 45
Lys Gly Tyr Asn Trp Pro Thr Tyr Leu Tyr Ala Lys Tyr Gly Ala Lys
50 55 60
Ile Gly Leu Ile Lys Ser Val Asp Phe Asp Glu Ser Phe Glu Gly Thr
65 70 75 80
Asn Val Asp Ser Leu Gln Leu Asp Lys Gln Thr Val Leu Ala Lys Gly
85 90 95
Trp Tyr Phe Arg Asp Tyr Gln Gly Phe Leu Asn Tyr Arg Asn Glu Leu
100 105 110
Lys Ala Leu Phe Asp Phe Lys Glu His Ile Lys Lys Pro Val Glu Gln
115 120 125
Phe Phe Ser Thr Leu Ser Lys Asp Thr Ile Lys Val Gly Leu His Ile
130 135 140
Arg Arg Gly Asp Tyr Lys Thr Trp His Gln Gly Lys Tyr Phe Phe Ser
145 150 155 160
Asp Glu Glu Tyr Gly Gln Ile Val Asn Ser Phe Ala Lys Ser Leu Asp
165 170 175
Lys Pro Val Glu Leu Ile Ile Val Ser Asn Asp Pro Lys Leu Asn Ser
180 185 190
Lys Ser Phe Glu Asn Leu Thr Ser Cys Lys Val Ser Met Leu Asn Gly
195 200 205
Asn Pro Ala Glu Asp Leu Tyr Leu Leu Ser Lys Cys Asp Tyr Ile Ile
210 215 220
Gly Pro Pro Ser Thr Phe Ser Leu Met Ala Ala Phe Tyr Glu Asp Arg
225 230 235 240
Pro Leu Tyr Trp Ile Phe Asp Lys Glu Lys Gln Leu Leu Ala Glu Asn
245 250 255
Phe Asp Lys Phe Glu Asn Leu Phe Arg His Ile Ile
260 265
<210> 6
<211> 903
<212> DNA
<213> Helicobacter pylori
<400> 6
atggctttca aggtggtcca aatttgtggt ggacttggca atcagatgtt tcagtacgca 60
tttgcgaaaa gcctgcaaaa acattcgaat acgcctgtac tcctcgacat cacctcattt 120
gattggagcg atcggaaaat gcagctcgag cttttcccca ttgatctccc ctatgcgtct 180
gccaaggaga ttgccattgc gaaaatgcaa caccttccga aattggtacg tgacgcgctg 240
aaatgcatgg gattcgaccg tgtgtctcag gaaatcgtct ttgagtacga acctaagctg 300
ctgaagccat cgcgtttgac gtattttttc ggatactttc aagacccacg gtattttgac 360
gcaattagcc cacttattaa gcagacgttt actttgccac ccccaccaga aaacaacaag 420
aataacaaca agaaggagga agagtatcaa tgcaaactta gcttgatttt ggcagctaaa 480
aattcggtat ttgttcatat ccgccggggt gactatgtcg gaatcggatg ccaacttgga 540
atcgactacc agaaaaaggc tcttgagtac atggctaagc gtgtccccaa tatggaactc 600
ttcgtctttt gcgaggatct ggagttcacc cagaacctcg atctcggcta tccgttcatg 660
gacatgacca cccgcgacaa agaggaagaa gcatactggg atatgctgtt gatgcagtct 720
tgtcagcacg cgatcattgc aaattccact tactcatggt gggcggccta ccttattgaa 780
aatcctgaga aaattatcat cggaccaaag cactggctct ttggacacga aaacattctc 840
tgtaaggagt gggtgaagat cgaaagccat ttcgaagtga agagccagaa atacaatgct 900
taa 903
<210> 7
<211> 300
<212> PRT
<213> Helicobacter pylori
<400> 7
Met Ala Phe Lys Val Val Gln Ile Cys Gly Gly Leu Gly Asn Gln Met
1 5 10 15
Phe Gln Tyr Ala Phe Ala Lys Ser Leu Gln Lys His Ser Asn Thr Pro
20 25 30
Val Leu Leu Asp Ile Thr Ser Phe Asp Trp Ser Asp Arg Lys Met Gln
35 40 45
Leu Glu Leu Phe Pro Ile Asp Leu Pro Tyr Ala Ser Ala Lys Glu Ile
50 55 60
Ala Ile Ala Lys Met Gln His Leu Pro Lys Leu Val Arg Asp Ala Leu
65 70 75 80
Lys Cys Met Gly Phe Asp Arg Val Ser Gln Glu Ile Val Phe Glu Tyr
85 90 95
Glu Pro Lys Leu Leu Lys Pro Ser Arg Leu Thr Tyr Phe Phe Gly Tyr
100 105 110
Phe Gln Asp Pro Arg Tyr Phe Asp Ala Ile Ser Pro Leu Ile Lys Gln
115 120 125
Thr Phe Thr Leu Pro Pro Pro Pro Glu Asn Asn Lys Asn Asn Asn Lys
130 135 140
Lys Glu Glu Glu Tyr Gln Cys Lys Leu Ser Leu Ile Leu Ala Ala Lys
145 150 155 160
Asn Ser Val Phe Val His Ile Arg Arg Gly Asp Tyr Val Gly Ile Gly
165 170 175
Cys Gln Leu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Met Ala
180 185 190
Lys Arg Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Glu
195 200 205
Phe Thr Gln Asn Leu Asp Leu Gly Tyr Pro Phe Met Asp Met Thr Thr
210 215 220
Arg Asp Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser
225 230 235 240
Cys Gln His Ala Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp Ala Ala
245 250 255
Tyr Leu Ile Glu Asn Pro Glu Lys Ile Ile Ile Gly Pro Lys His Trp
260 265 270
Leu Phe Gly His Glu Asn Ile Leu Cys Lys Glu Trp Val Lys Ile Glu
275 280 285
Ser His Phe Glu Val Lys Ser Gln Lys Tyr Asn Ala
290 295 300

Claims (6)

1.一种重组棒状杆菌属(Corynebacterium SP.)微生物，其特征在于，
其转化为表达出源自舞蹈拟土地杆菌(Pseudopedobacter saltans)的氨基酸序列为序列号5的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)，
转化为表达出GDP-D-甘露糖-4,6-脱水酶(GDP-D-mannose-4,6-dehydratase)，
转化为表达出GDP-L-岩藻糖合酶(GDP-L-fucose synthase)，
转化为表达出乳糖渗透酶(lactose permease)，
且保留磷酸甘露糖变位酶(phosphomannomutase)以及GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphate guanylyltransferase)。
2.根据权利要求1所述的重组棒状杆菌属(Corynebacterium SP.)微生物，其特征在于，
上述重组棒状杆菌属(Corynebacterium SP.)微生物是选自谷氨酸棒状杆菌(Corynebacterium glutamicum)、产氨棒状杆菌(Corynebacterium ammoniagenes)以及热产氨棒状杆菌(Corynebacterium thermoaminogenes)中的任意一个。
3.根据权利要求1所述的重组棒状杆菌属(Corynebacterium SP.)微生物，其特征在于，
上述的氨基酸序列为序列号5的α-1,2-岩藻糖基转移酶(α-1,2-fucosyltransferase)被序列号4所记载的核酸序列编码。
4.根据权利要求1所述的重组棒状杆菌属(Corynebacterium SP.)微生物，其特征在于，
上述重组棒状杆菌属(Corynebacterium SP.)微生物转化为超表达出磷酸甘露糖变位酶(phosphomannomutase)，
转化为超表达出GTP-甘露糖-1-磷酸鸟嘌呤基转移酶(GTP-mannose-1-phosphateguanylytransferase)。
5.一种2’-岩藻糖基乳糖的生产方法，其特征在于，
在添加有乳糖的培养基中培养权利要求1所述的重组棒状杆菌属(CorynebacteriumSP.)微生物。
6.根据权利要求5所述的2’-岩藻糖基乳糖的生产方法，其特征在于，
上述培养基还包含葡萄糖。
CN201980002419.1A 2018-04-04 2019-02-14 利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法 Active CN110637091B (zh)

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