CN114874967A - 一种产n-乙酰神经氨酸的重组大肠杆菌及其构建方法 - Google Patents

一种产n-乙酰神经氨酸的重组大肠杆菌及其构建方法 Download PDF

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CN114874967A
CN114874967A CN202210690512.XA CN202210690512A CN114874967A CN 114874967 A CN114874967 A CN 114874967A CN 202210690512 A CN202210690512 A CN 202210690512A CN 114874967 A CN114874967 A CN 114874967A
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刘延峰
刘龙
刘畅
吕雪芹
田荣臻
李江华
堵国成
陈坚
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Jiangnan University
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Abstract

本发明公开了一种产N‑乙酰神经氨酸的重组大肠杆菌及其构建方法，属于代谢工程和基因工程技术领域。本发明构建的重组大肠杆菌(Escherichia coli)是由大肠杆菌BL21(DE3)为出发菌株，通过在基因组上敲除nanATEK基因,nagABE基因、manXYZ基因、poxB基因，并引入外源的UDP‑N‑乙酰氨基葡糖‑2‑差向异构酶编码基因NeuC和N‑乙酰神经氨酸合酶编码基因NeuB，并且重组表达由诱导型启动子T7调控的内源酶glmM，glmU和glmS突变体glmSA而得到的一株产N‑乙酰神经氨酸的重组菌株，其产量达到3.85g/L，为工业化生产N‑乙酰神经氨酸奠定了基础。

Description

一种产N-乙酰神经氨酸的重组大肠杆菌及其构建方法
技术领域
本发明涉及一种产N-乙酰神经氨酸的重组大肠杆菌及其构建方法，属于代谢工程和基因工程技术领域。
背景技术
N-乙酰神经氨酸(N-acetylneuraminic acid)广泛存在于自然界中，它具有非常重要的应用价值，在医用和保健方面N-乙酰神经氨酸可用于抗菌排毒，并且它作为抗流感病毒药物扎那米韦的前体可用于研发唾液酸酶抑制剂类抗流感药物，在食品方面，N-乙酰神经氨酸是婴幼儿大脑生长发育的条件性必须营养因子，可促进记忆力和智力发育，添加到食品中也可提高肠道的抗菌能力。
大肠杆菌(Escherichia coli)是一种被大规模应用于代谢工程改造生产化学品的模式工业微生物，其在医药、化工、农业等方面都具有非常广泛的应用。近年来随着合成生物学的研究发展，大肠杆菌的基因编辑工具多样、操作简单且发展成熟。此外，大肠杆菌具有易于培养、遗传背景清晰、生长迅速、表达量高且遗传操作简便易于代谢工程改造等优点。
在底盘细胞的开发与改造中，质粒游离表达外源基因稳定性较差，易增加细胞的代谢负担从而降低目标产物产量与生产效率，难以满足大规模工业化生产，通过将外源基因整合到大肠杆菌基因组中可以实现稳定表达。目前在N-乙酰神经氨酸高产菌株中代谢途径主要是以葡萄糖为底物，以N-乙酰氨基葡萄糖为前体外源表达AGE酶，易导致代谢流强度不够，因此解除限速步骤中的反馈抑制作用，强化N-乙酰神经氨酸合成途径的代谢流，寻找适合大肠杆菌中N-乙酰神经氨酸生产的外源酶、发酵碳源以及高效的合成途径十分重要。
发明内容
本发明提供了一种重组大肠杆菌，所述重组大肠杆菌敲除基因组中的葡萄糖胺转运相关的磷酸转移酶系统相关的基因N-乙酰葡糖胺-6-磷酸脱乙酰酶nagA(Gene ID:945289)、氨基葡萄糖-6-磷酸脱氨酶nagB(Gene ID:945290)、N-乙酰氨基葡萄糖特异性EIICBA组分nagE(Gene ID:945292)、N-乙酰神经氨酸转运载体相关基因N-乙酰神经氨酸裂解酶nanA(Gene ID:947742)、唾液酸转运蛋白nanT(Gene ID:947740)、N-乙酰甘露糖胺-6-磷酸2-差向异构酶nanE(Gene ID:947745)、N-乙酰甘露糖胺激酶nanK(Gene ID:947757)、甘露糖特异性EIIAB组分相关基因manXYZ(Gene ID:946334,946332)丙酮酸氧化酶poxB基因(Gene ID:946132),表达来源于脑膜炎奈瑟菌(Neisseria meningitidis)的UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC和粘放线菌(Moritella viscosa)来源的N-乙酰神经氨酸合酶NeuB，并重组表达由诱导型启动子T7调控的内源酶glmM(Gene ID:947692)，glmU(GeneID:948246)和葡萄糖胺合酶突变体glmSA。
在一种实施方式中，所述UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC、N-乙酰神经氨酸合酶NeuB以及由诱导型启动子T7调控的内源酶glmM，glmU和glmS突变体glmSA均重组于基因组上整合表达。
在一种实施方式中，所述glmS突变体glmSA整合于△nagABE所在位置。
在一种实施方式中，所述NeuC整合于敲除了甘露糖特异性EIIAB组分相关基因△manXYZ的所在位置。
在一种实施方式中，所述NeuB整合于敲出了丙酮酸氧化酶基因△poxB的所在位置。
在一种实施方式中，所述UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC的氨基酸序列如SEQ ID NO.1所示；编码NeuC基因的核苷酸序列如SEQ ID NO.2所示。
在一种实施方式中，所述N-乙酰神经氨酸合酶NeuB的氨基酸序列如SEQ ID NO.3所示；编码NeuB基因的核苷酸序列如SEQ ID NO.4所示。
在一种实施方式中，所述葡萄糖胺合酶突变体glmSA的氨基酸序列如SEQ ID NO.5所示；编码glmSA基因的核苷酸序列如SEQ ID NO.6所示。
在一种实施方式中，所述glmM的Gene ID为947692，glmU的Gene ID为948246。
在一种实施方式中，利用不同强度的启动子表达所述UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC；所述不同强度的启动子包括Ptac、PssrA、Pdnakj、PgrpE、P566、P224、P333、Ptrc、PalsAR。
在一种实施方式中，所述启动子Ptac、PssrA、Pdnakj、PgrpE、P566、P224、P333、Ptrc、PalsAR的核苷酸序列分别如SEQ ID NO.7～15所示。
在一种实施方式中，采用SEQ ID NO.10强化表达所述UDP-N-乙酰氨基葡糖-2-差向异构酶编码基因NeuC。
在一种实施方式中，采用SEQ ID NO.14强化表达所述N-乙酰神经氨酸合酶编码基因NeuB。
在一种实施方式中，以大肠杆菌Escherichia coli BL21(DE3)为出发菌株。
本发明还提供了一种生产N-乙酰神经氨酸的方法，所述方法是以所述重组大肠杆菌为发酵菌株，以甘油为底物发酵生产N-乙酰神经氨酸。
在一种实施方式中，所述发酵碳源甘油的工作浓度为20～40g/L。
在一种实施方式中，用于发酵的发酵培养基含有30g/L甘油，6g/L尿素，3.8mg/L七水硫酸锌，0.33g/L一水硫酸锰，5g/L七水硫酸铁，0.1g/L五水硫酸铜，0.1g/L六水氯化钴，4.8g/L酵母粉，2.4g/L胰蛋白胨，5.336g/L磷酸二氢钾，3.284g/L三水合磷酸氢二钾，2.84g/L一水柠檬酸，2g/L七水硫酸镁，4g/L硫酸铵，PH调至7的培养基中进行的。
在一种实施方式中，所述基因改造后的重组大肠杆菌接种于LB培养基中，35～37℃培养12～14h获得种子液，再以1～2％的接种量转接至发酵培养基中发酵。
在一种实施方式中，当OD600达到0.6时加入0.5mM IPTG，在30℃条件下反应48h。
在一种实施方式中，所述应用是用于制备药物或保健品。
本发明提供了所述大肠杆菌在生产N-乙酰神经氨酸中的应用。
有益效果：本发明提供的重组大肠杆菌可以实现N-乙酰神经氨酸在胞外积累，通过敲除N-乙酰神经氨酸分解代谢途径的相关基因，以9个不同强度的组成型启动子优化UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC以及N-乙酰神经氨酸合酶NeuB在基因组上的表达水平，并且引入glmS的突变体解除限速反应中glmS受到的反馈抑制，强化glmM,glmU和glmSA提高N-乙酰神经氨酸合成途径的代谢通量，并使大肠杆菌发酵48h的N-乙酰神经氨酸产量达到3.85g/L以上。
具体实施方式
Neisseria meningitidis来源的UDP-N-乙酰氨基葡萄糖-2-差向异构酶NeuC的核苷酸序列如SEQ ID NO.2所示。N-乙酰神经氨酸合酶NeuB的核苷酸序列如SEQ ID NO.4所示。葡萄糖胺合酶突变体glmSA的基因的核苷酸序列如SEQ ID NO.6所示。
启动子Ptac、PssrA、Pdnakj、PgrpE、P566、P224、P333、Ptrc、PalsAR、PT7的核苷酸序列分别为SEQID NO.7～16所示；
重组大肠杆菌种子培养及发酵：
种子培养基：10g/L胰蛋白胨，10g/L氯化钠，5g/L酵母粉。
发酵培养基：6g/L尿素，3.8mg/L七水硫酸锌，0.33g/L一水硫酸锰，5g/L七水硫酸铁，0.1g/L五水硫酸铜，0.1g/L六水氯化钴，4.8g/L酵母粉，2.4g/L胰蛋白胨，5.336g/L磷酸二氢钾，3.284g/L三水合磷酸氢二钾，2.84g/L一水柠檬酸，2g/L七水硫酸镁，4g/L硫酸铵，pH调至7
培养条件：将重组大肠杆菌接种于LB培养基中，37℃220rpm培养12～14h获得种子液，再以1—2％的接种量转接至发酵培养基中发酵，当OD600达到0.6时加入0.5mM IPTG，在30℃220rpm条件下反应48h。
样品检测方法：N-乙酰神经氨酸及N-乙酰基-D-氨基甘露糖采用Agilent液相色谱进行检测，色谱柱为Aminex HPX-87H column(300×7.8mm)，紫外210nm检测吸收峰，流动相为10mM硫酸，流速为0.5mL/min，N-乙酰神经氨酸的出峰时间约为9.8分钟。
实施例1 nagABE、nanATEK、manXYZ和poxB敲除菌株的构建
通过CRISPER/Cas9基因编辑技术实现在大肠杆菌基因组上葡萄糖胺转运相关的磷酸转移酶系统相关的基因N-乙酰葡糖胺-6-磷酸脱乙酰酶nagA(Gene ID:945289)、氨基葡萄糖-6-磷酸脱氨酶nagB(Gene ID:945290)、PTS系统N-乙酰氨基葡萄糖特异性EIICBA组分nagE(Gene ID:945292)、N-乙酰神经氨酸转运载体相关基因N-乙酰神经氨酸裂解酶nanA(Gene ID:947742)、唾液酸转运蛋白nanT(Gene ID:947740)、N-乙酰甘露糖胺-6-磷酸2-差向异构酶nanE(Gene ID:947745)、N-乙酰甘露糖胺激酶nanK(Gene ID:947757)、PTS系统甘露糖特异性EIIAB组分相关基因manXYZ(Gene ID:946334,946332)丙酮酸氧化酶poxB基因(Gene ID:946132)的敲除。其中nagABE的N20序列为attgccctgagcaaggagcc，nanATEK的N20序列为gctttggtatgaaaattgta，manXYZ的N20序列为acgaagccgaggtagaagaa，poxB的N20序列为ggtgaaaatagcgtcatcgg。首先将含有Cas9切割蛋白的pCas9质粒用化学转化法转化到大肠杆菌宿主菌中，以大肠杆菌BL21(DE3)为模板，PCR扩增敲除位点的上游、下游片段各1000bp左右，通过PCR扩增获得片段后，再经过Gibson组装试剂盒与CRISPER/Cas9系统中的靶向切割质粒pTarget载体连接。质粒构建好测序后，制作含有pCas9的大肠杆菌感受态细胞，实现目的基因的敲除。转化后将转化子涂布于抗性平板上，挑取单菌落进行菌落PCR验证阳性转化子并测序，依次构建在基因组上敲除nagABE、nanATEK、manXYZ和poxB的重组大肠杆菌，将该菌株命名为NBC-1。
实施例2构建基因组重组整合NeuC片段
根据Neisseria meningitidis来源的UDP-N-乙酰氨基葡萄糖-2-差向异构酶NeuC(氨基酸序列如SEQ ID NO.1所示)，合成其编码基因NeuC的核苷酸序列(如SEQ ID NO.2所示)；将NeuC的整合位点选择在PTS系统甘露糖特异性EIIAB组分相关基因manXYZ的所在位置进行敲入。
以大肠杆菌BL21(DE3)为模板，设计引物NeuC-L-F1和NeuC-L-R1，扩增重组整合NeuC左臂基因片段；
NeuC-L-F1：5’-catcaataccgtttccggcaaaggc-3’,
NeuC-L-R1：5’-CTCCCGGACCAAAACGAAAAAAGACGCTTTTCAGCGTCTTTTTTTTTTTTTTTGGTACCGAGgaatctgttagaggcgcaatagtgacag-3’,
合成如SEQ ID NO.7～15的核苷酸序列所示的启动子片段；
以大肠杆菌BL21(DE3)为模板，设计引物NeuC-R-F1和NeuC-R-R1，扩增重组整合NeuC右臂基因片段；
NeuC-R-F1:5’-CGGGGGCTTTctcatgcgtttcccaggtggaagccctatttcttttatg-3’；
NeuC-R-R1:5’-gtagagttcactcctgccgatccg-3’；
将扩增得到的NeuC左臂基因片段、启动子片段(分别如SEQ ID NO.7～15所示)、NeuC基因片段和NeuC右臂片段通过融合PCR技术构建成重组整合NeuC基因片段，根据启动子的不同将其分别命名为NeuC1～NeuC9；其中，NeuC1对应含有SEQ ID NO.7所示的Ptac启动子的NeuC融合片段，NeuC2对应含有SEQ ID NO.8所示的PssrA启动子的NeuC融合片段，以此类推，NeuC10对应含有SEQ ID NO.15所示的PalsAR启动子的NeuC融合片段。
表1各启动子序列
Figure BDA0003699392210000051
实施例3构建基因组重组整合NeuB片段
根据Moritella viscosa来源的N-乙酰神经氨酸合酶编码基因NeuB(氨基酸序列如SEQ ID NO.3所示)，合成其编码基因NeuB的核苷酸序列(如SEQ ID NO.4所示)；NeuB的整合位点选择在丙酮酸氧化酶poxB基因(Gene ID:946132)所在位置进行敲入整合。
以大肠杆菌BL21(DE3)为模板，设计引物NeuB-L-F1和NeuB-L-R1，扩增重组整合NeuB左臂基因片段；
NeuB-L-F1：5’-gaggcgttaatcagcacgtttctcgct-3’,
NeuB-L-R1：5’-CACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAaaagggtggcatttcccgtcataataaggacat-3’；
合成如SEQ ID NO.7～15的核苷酸序列所示的启动子片段；
以大肠杆菌BL21(DE3)为模板，设计引物NeuB-R-F1和NeuB-R-R1，扩增重组整合NeuB右臂基因片段；
NeuB-R-F1：5’-GCCGAAGCGGGTTTTTACGTAAAACAGGTGAAACTGACggttctccatctcctgaatgtgataacggtaacaagtt-3’,
NeuB-R-R1：5’-aatatgcactggtcagcgtgcgtaactc-3’；
将扩增得到的NeuB左臂基因片段、启动子片段(分别如SEQ ID NO.7～15所示)、NeuB基因片段和NeuB右臂片段通过融合PCR技术构建成重组整合NeuB基因片段。
实施例4构建基因组重组整合glmM，glmU-SA片段
以大肠杆菌BL21(DE3)基因组为模板，设计引物glm-L-F1和glm-L-R1，扩增重组PT7-glmM-PT7-glmU-glmSA整合位点的左侧同源臂基因片段；
glm-L-F1:5’-GAGctatcccaacaactcttccttaacatccatcg-3’，
glm-L-R1:5’-gttAGCTTGGCCgatggcgctacgtcgtcatatgagc-3’，
以大肠杆菌BL21(DE3)基因组为模板，设计引物glm-R-F1和glm-R-R1，扩增重组PT7-glmM-PT7-glmU-glmSA整合位点的右侧同源臂基因片段，以及如SEQ ID NO.16所示的T7启动子片段；
glm-R-F1：5’-GTACCGAGttactgatacaccggcagtaaattaaagctcg-3’，
glm-R-R1：5’-ggtaatagatctaacggcaaagatccgaaaaccatcgtg-3’，
合成如SEQ ID NO.6的核苷酸序列所示的glmSA片段；
以大肠杆菌BL21(DE3)基因组为模板，设计引物glmM-F1和glmM R1，扩增重组整合glmM(Gene ID：947692)的基因片段；
glmM-F1:5’-gcgttgcgcTAATACGACTCACTATAGGGTTCACACAGGAAACCTATAATGatgagtaatcgtaaatatttcggtaccgatgggattcg-3’，
glmM-R1:5’-cgttgccagttatctaacgctttaaacggc-3’；
以大肠杆菌BL21(DE3)基因组为模板，设计引物glmU-F1和glmU R1，扩增重组整合glmU(Gene ID：948246)的基因片段；
glmU-F1:5’-GGGTTCACACAGGAAACCTATAATGatgttgaataatgctatgagcgtagtgatcc-3’，
glmU-R1:5’-ggatttctgctacatcacgttgcgc-3’；
通过PCR扩增获得整合位点左侧同源臂、glmM片段、glmU片段和glmSA片段和右侧同源臂后，再经过Gibson组装试剂盒与CRISPER/Cas9系统中的含有N20识别序列的靶向切割质粒pTarget载体连接，得到PT7-glmM-PT7-glmU-glmSA。
实施例5重组整合NeuC基因的大肠杆菌的构建
将实施例2得到的由SEQ ID NO.10所示PgrpE调控的NeuC融合片段经过Gibson组装试剂盒与CRISPER/Cas9系统中的含有N20识别序列(agccctttctttttatagtt)的靶向切割质粒pTarget载体连接后，采用化学转化法，将构建好的质粒转化到含有Cas9质粒的实施例1构建的大肠杆菌NBC-1感受态细胞中，获得将PgprE-NeuC整合在ΔmanXYZ处的重组菌株。取测序正确的阳性转化子，消除抗性后于发酵培养基中发酵48h后利用高效液相色谱测定产物的浓度，结果显示在NeuC受到低强度启动子PgrpE调控的菌株中N-乙酰神经氨酸(NeuAc)的前体物质N-乙酰基-D-氨基甘露糖(ManNAc)产量达0.48g/L，将该菌株命名为NBC-2。
实施例6重组整合NeuB基因和glmM，glmU-SA片段的大肠杆菌的构建
实施例3得到由SEQ ID NO.14所示Ptrc启动子调控的NeuB融合片段经过Gibson组装试剂盒与CRISPER/Cas9系统中的含有N20识别序列(ggtgaaaatagcgtcatcgg)的靶向切割质粒pTarget载体连接后，采用化学转化法，将构建好的质粒转化到含有Cas9质粒的实施例5构建的大肠杆菌NBC-2感受态细胞中，获得将Ptrc-NeuB整合在ΔpoxB处的重组菌株。取转化子进行菌落PCR验证，整合成功后的菌株命名为NBC-3。消除NBC-2中的pTarget质粒后将NBC-2按照大肠杆菌超级感受态细胞制备方法制作成感受态细胞，将实施例4构建的分别由T7诱导调控的glmM，glmU-SA质粒PT7-glmM-PT7-glmU-glmSA转化至NBC-3，验证后获得将PT7-glmM-PT7-glmU-glmSA整合在ΔnagABE处的阳性转化子，将该菌株命名为NBC-4。
实施例7重组菌株发酵生产N-乙酰神经氨酸
取实施例6构建的单菌落接种于种子培养基中于37℃，220rpm下培养10-14h得到种子液，以2％的接种量转接入发酵培养基中，以工作浓度为30g/L的甘油为碳源，于37℃，220rpm培养至OD600达到0.6加入终浓度0.5mM的IPTG诱导剂，发酵48h后，取发酵上清液检测产量，最终N-乙酰神经氨酸产量达到3.85g/L，实现了构建高产N-乙酰神经氨酸的重组工程菌。
对比例1
具体实施方式同实施例5，区别在于，将实施例5中的由PgrpE(SEQ ID NO.10)调控的NeuC融合片段替换为由SEQ ID NO.7～9,11～15所示的不同强度的启动子调控的NeuC融合片段，检测发现，构建的由SEQ ID NO.7～9,11～14所示启动子调控NeuC融合片段的重组大肠杆菌发酵48h后发酵上清液中N-乙酰基-D-氨基甘露糖(ManNAc)无积累，由SEQ IDNO.15调控NeuC融合片段的大肠杆菌重组菌株中N-乙酰基-D-氨基甘露糖(ManNAc)产量为0.1g/L。
对比例2
将实施例7中的重组菌株发酵碳源替换为工作浓度30g/L的葡萄糖，并按照实施例7的发酵方法进行发酵，并测定发酵上清液中的N-乙酰神经氨酸，结果显示，N-乙酰神经氨酸产量为1.5g/L。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种产N-乙酰神经氨酸的重组大肠杆菌及其构建方法
<130> BAA220600A
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 377
<212> PRT
<213> Neisseria meningitidis
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Asn Gln Ile Gln Gly Glu Pro Met Gly Ala Val Leu Gly Asn Thr Ile
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Thr Phe Ile Ser Arg Leu Ser Asp Glu Ile Glu Pro Asp Met Val Met
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Ile His Gly Asp Arg Leu Glu Ala Leu Ala Gly Ala Ala Val Gly Ala
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tacctgtttt caaaccaaat tcagggcgaa ccgatgggag cagtgctggg caacacaatc 240
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agacttgaag cattagcggg agcagcggtg ggcgcgttat caagccgcct ggtctgtcat 360
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gaaaaacgca aacatatcca tattatcggc tcaccggatt tagatgtgat ggcttcttca 540
acactgccga gccttgaaga agtcaaagaa tattatggac tgccgtacga aaactacggc 600
atctcaatgt ttcatccggt tacaacagaa gctcatctta tgccgcaata tgctgcccag 660
tattttaaag ccctggaact ttcaggacag aacattatca gcatttatcc gaataacgat 720
acaggcacag aaagcatcct tcaagaactg ctgaaatacc agagcgataa atttatcgct 780
tttccgtcta tcagatttga atattttctg gttcttctga aacatgccaa atttatggtg 840
ggaaatagct ctgctggcat tcgcgaagcc ccgctgtatg gagtcccgag catcgatgtt 900
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ccggaaacat ggaatgtttc agcgcagaaa agatttatcg atttaaacct gtaa 1134
<210> 3
<211> 347
<212> PRT
<213> Moritella viscosa
<400> 3
Met Thr Asn Pro Val Phe Glu Ile Ser Gly Arg Lys Val Gly Leu Asp
1 5 10 15
Tyr Ala Pro Leu Val Ile Ala Glu Ile Gly Ile Asn His Glu Gly Ser
20 25 30
Leu Lys Thr Ala Phe Glu Met Val Asp Ala Ala Ile Glu Gly Gly Ala
35 40 45
Glu Ile Ile Lys His Gln Thr His Val Ile Glu Asp Glu Met Ser Ser
50 55 60
Glu Ala Lys Lys Val Ile Pro Gly Asn Ala Asp Val Ser Ile Tyr Glu
65 70 75 80
Ile Met Asp Arg Cys Ser Leu Asn Glu Glu Asp Glu Ile Lys Leu Lys
85 90 95
Lys Tyr Ile Glu Ser Lys Gly Ala Ile Phe Ile Ser Thr Pro Phe Ser
100 105 110
Arg Ala Ala Ala Leu Arg Leu Glu Arg Met Gly Val Ser Ala Tyr Lys
115 120 125
Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Leu Asp Leu Ile Ala
130 135 140
Ser Tyr Gly Lys Pro Val Ile Leu Ser Thr Gly Met Asn Asp Ile Pro
145 150 155 160
Ser Ile Arg Lys Ser Val Glu Ile Phe Arg Lys Tyr Lys Thr Pro Leu
165 170 175
Cys Leu Leu His Thr Thr Asn Leu Tyr Pro Thr Pro Asp His Leu Ile
180 185 190
Arg Ile Gly Ala Met Glu Glu Met Gln Arg Glu Phe Ser Asp Val Val
195 200 205
Val Gly Leu Ser Asp His Ser Ile Asp Asn Leu Ala Cys Leu Gly Ala
210 215 220
Val Ala Ala Gly Ala Ser Val Leu Glu Arg His Phe Thr Asp Asn Lys
225 230 235 240
Ala Arg Ser Gly Pro Asp Ile Cys Cys Ser Met Asp Gly Ala Glu Cys
245 250 255
Ala Glu Leu Ile Ser Gln Ser Lys Arg Met Ala Gln Met Arg Gly Gly
260 265 270
Ser Lys Gly Ala Val Lys Glu Glu Gln Val Thr Ile Asp Phe Ala Tyr
275 280 285
Ala Ser Val Val Thr Ile Lys Glu Ile Lys Ala Gly Glu Ala Phe Thr
290 295 300
Lys Asp Asn Leu Trp Val Lys Arg Pro Gly Thr Gly Asp Phe Leu Ala
305 310 315 320
Asp Asp Tyr Glu Met Leu Leu Gly Lys Lys Ala Ser Gln Asn Ile Asp
325 330 335
Phe Asp Val Gln Leu Lys Lys Glu Phe Ile Lys
340 345
<210> 4
<211> 1044
<212> DNA
<213> 人工序列
<400> 4
atgacaaatc cggtctttga aatttctggc agaaaagttg gacttgatta tgccccgtta 60
gtgatcgcag aaattggcat caaccatgaa ggatcactga aaacagcctt tgaaatggtg 120
gatgcagcga ttgaaggcgg agcagaaatc atcaaacatc aaacacatgt cattgaagat 180
gaaatgtcaa gcgaagcaaa gaaagttatc ccgggcaatg ctgatgtgag catctacgaa 240
atcatggata gatgctctct gaacgaagaa gatgaaatca aactgaaaaa atacatcgaa 300
tcaaaaggcg ctatctttat ctcaacaccg tttagccgcg ctgccgcact gagacttgaa 360
cgcatgggag ttagcgccta taaaattggc tctggagaat gcaataacta tccgctgctt 420
gatcttattg cgtcttatgg caaaccggtc atcttatcaa caggaatgaa tgatattccg 480
tctatcagaa aatcagttga aatctttcgc aaatacaaaa caccgctttg tttactgcat 540
acaacaaacc tgtatccgac accggatcat cttattagaa tcggcgcaat ggaagaaatg 600
caacgcgaat ttagcgatgt tgtggtcgga ctgagcgatc attctatcga taacctggct 660
tgtctgggag ctgtggctgc tggagcttct gtcctggaaa gacattttac agataacaaa 720
gctcgctcag gcccggatat ttgctgtagc atggatggag cggaatgtgc tgaacttatc 780
tctcaatcaa aaagaatggc ccagatgcgc ggcggatcaa aaggcgcagt caaagaagaa 840
caggttacaa ttgattttgc ctatgcaagc gttgtgacaa ttaaagaaat caaagccgga 900
gaagcattta caaaagataa tctgtgggtt aaacgcccgg gcacaggaga ttttcttgcg 960
gatgattatg aaatgctttt aggcaagaaa gcaagccaaa acattgattt tgatgtgcag 1020
ctgaagaaag aatttatcaa ataa 1044
<210> 5
<211> 609
<212> PRT
<213> 人工序列
<400> 5
Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Lys Ile
1 5 10 15
Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala
20 25 30
Gly Leu Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg
35 40 45
Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu
50 55 60
His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu
65 70 75 80
Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val
85 90 95
Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu
100 105 110
Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile
115 120 125
Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu
130 135 140
Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val
145 150 155 160
Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly
165 170 175
Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser
180 185 190
Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu
195 200 205
Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp
210 215 220
Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln
225 230 235 240
Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu
245 250 255
Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile
260 265 270
Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu
275 280 285
Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser
290 295 300
Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly
305 310 315 320
Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser
325 330 335
Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu
340 345 350
Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr
355 360 365
Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg
370 375 380
Glu Ser Val Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val
385 390 395 400
Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu
405 410 415
Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His
420 425 430
Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met
435 440 445
Leu Pro Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp
450 455 460
Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala
465 470 475 480
Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu
485 490 495
Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp
500 505 510
Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Gly Leu Leu Glu
515 520 525
Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu
530 535 540
Tyr Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met
545 550 555 560
His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe
565 570 575
Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys
580 585 590
Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val
595 600 605
Glu
<210> 6
<211> 1830
<212> DNA
<213> 人工序列
<400> 6
atgtgtggaa ttgttggcgc gatcgcgcaa cgtgatgtag caaaaatcct tcttgaaggt 60
ttacgtcgtc tggaataccg cggatatgac tctgccggtc tggccgttgt tgatgcagaa 120
ggtcatatga cccgcctgcg tcgcctcggt aaagtccaga tgctggcaca ggcagcggaa 180
gaacatcctc tgcatggcgg cactggtatt gctcacactc gctgggcgac ccacggtgaa 240
ccttcagaag tgaatgcgca tccgcatgtt tctgaacaca ttgtggtggt gcataacggc 300
atcatcgaaa accatgaacc gctgcgtgaa gagctaaaag cgcgtggcta taccttcgtt 360
tctgaaaccg acaccgaagt gattgcccat ctggtgaact gggagctgaa acaaggcggg 420
actctgcgtg aggccgttct gcgtgctatc ccgcagctgc gtggtgcgta cggtacagtg 480
atcatggact cccgtcaccc ggataccctg ctggcggcac gttctggtag tccgctggtg 540
attggcctgg ggatgggcga aaactttatc gcttctgacc agctggcgct gttgccggtg 600
acccgtcgct ttatcttcct tgaagagggc gatattgcgg aaatcactcg ccgttcggta 660
aacatcttcg ataaaactgg cgcggaagta aaacgtcagg atatcgaatc caatctgcaa 720
tatgacgcgg gcgataaagg catttaccgt cactacatgc agaaagagat ctacgaacag 780
ccgaacgcga tcaaaaacac ccttaccgga cgcatcagcc acggtcaggt tgatttaagc 840
gagctgggac cgaacgccga cgaactgctg tcgaaggttg agcatattca gatcctcgcc 900
tgtggtactt cttataactc cggtatggtt tcccgctact ggtttgaatc gctagcaggt 960
attccgtgcg acgtcgaaat cgcctctgaa ttccgctatc gcaaatctgc cgtgcgtcgt 1020
aacagcctga tgatcacctt gtcacagtct ggcgaaaccg cggataccct ggctggcctg 1080
cgtctgtcga aagagctggg ttaccttggt tcactggcaa tctgtaacgt tccgggttct 1140
tctctggtgc gcgaatccgt tctggcgcta atgaccaacg cgggtacaga aatcggcgtg 1200
gcatccacta aagcattcac cactcagtta actgtgctgt tgatgctggt ggcgaagctg 1260
tctcgcctga aaggtctgga tgcctccatt gaacatgaca tcgtgcatgg tctgcaggcg 1320
ctgccgagcc gtattgagca gatgctgcct caggacaaac gcattgaagc gctggcagaa 1380
gatttctctg acaaacatca cgcgctgttc ctgggccgtg gcgatcagta cccaatcgcg 1440
ctggaaggcg cattgaagtt gaaagagatc tcttacattc acgctgaagc ctacgctgct 1500
ggcgaactga aacacggtcc gctggcgcta attgatgccg atatgccggt tattgttgtt 1560
gcaccgaaca acggattgct ggaaaaactg aaatccaaca ttgaagaagt tcgcgcgcgt 1620
ggcggtcagt tgtatgtctt cgccgatcag gatgcgggtt ttgtaagtag cgataacatg 1680
cacatcatcg agatgccgca tgtggaagag gtgattgcac cgatcttcta caccgttccg 1740
ctgcagctgc tggcttacca tgtcgcgctg atcaaaggca ccgacgttga ccagccgcgt 1800
aacctggcaa aatcggttac ggttgagtaa 1830
<210> 7
<211> 28
<212> DNA
<213> 人工序列
<400> 7
ttgacaatta atcatcggct cgtataat 28
<210> 8
<211> 146
<212> DNA
<213> 人工序列
<400> 8
attggctatc acatccgaca caaatgttgc catcccattg cttaatcgaa taaaaatcag 60
gctacatggg tgctaaatct ttaacgataa cgccattgag gctggtcatg gcgctcataa 120
atctggtata cttaccttta cacatt 146
<210> 9
<211> 160
<212> DNA
<213> 人工序列
<400> 9
gcacaaaaaa tttttgcatc tcccccttga tgacgtggtt tacgacccca tttagtagtc 60
aaccgcagtg agtgagtctg caaaaaaatg aaattgggca gttgaaacca gacgtttcgc 120
ccctattaca gactcacaac cacatgatga ccgaatatat 160
<210> 10
<211> 88
<212> DNA
<213> 人工序列
<400> 10
gattgatgac aatgtgagtg cttcccttga aaccctgaaa ctgatcccca taataagcga 60
agttagcgag atgaatgcga aaaaaacg 88
<210> 11
<211> 60
<212> DNA
<213> 人工序列
<400> 11
aaaaaacggc ctctcgaaat agagggttga cactcttttg agaatatgtt atattatcag 60
<210> 12
<211> 80
<212> DNA
<213> 人工序列
<400> 12
ttgaggaatc atagaatttt taatttaaat tttatttgac aaaaatgggc tcgtgttgta 60
taatctaagc tagtgtattt 80
<210> 13
<211> 82
<212> DNA
<213> 人工序列
<400> 13
ttgaggaatc atagaatttt gacttaaaaa tttcagttgc ttaatcccta caattcttga 60
tataatattc tcatagtttg aa 82
<210> 14
<211> 30
<212> DNA
<213> 人工序列
<400> 14
ttgacaatta atcatccggc tcgtataatg 30
<210> 15
<211> 191
<212> DNA
<213> 人工序列
<400> 15
agcaacatct atcatctaaa aaaccagaaa aacaaataac atcatgtttt taaactaatt 60
aaatgaaata aaattttaag ccactcgcca ttgttcacaa taaaataaac tttataaatt 120
ttattttttt gtgaagtcgc cagcatcttt tctgttcttg ctgtggtgat atagtggcgt 180
cttcaattca a 191
<210> 16
<211> 19
<212> DNA
<213> 人工序列
<400> 16
taatacgact cactatagg 19

Claims (10)

1.一种产N-乙酰神经氨酸的重组大肠杆菌，其特征在于，沉默或缺失了大肠杆菌基因组中的N-乙酰葡糖胺-6-磷酸脱乙酰酶基因nagA、氨基葡萄糖-6-磷酸脱氨酶基因nagB、N-乙酰氨基葡萄糖特异性EIICBA组分基因nagE、N-乙酰神经氨酸裂解酶基因nanA、唾液酸转运蛋白基因nanT、N-乙酰甘露糖胺-6-磷酸2-差向异构酶基因nanE、N-乙酰甘露糖胺激酶基因nanK、甘露糖特异性EIIAB组分相关基因manXYZ、丙酮酸氧化酶poxB基因，并表达来源于脑膜炎奈瑟菌(Neisseria meningitidis)的UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC和来源于粘放线菌(Moritella viscosa)的N-乙酰神经氨酸合酶NeuB，并表达内源酶glmM、glmU和葡萄糖胺合酶突变体glmSA。
2.根据权利要求1所述的重组大肠杆菌，其特征在于，所述葡萄糖胺合酶突变体glmSA整合于原nagABE所在位置；所述NeuC整合于原甘露糖特异性EIIAB组分相关基因manXYZ所在位置；所述NeuB整合于原丙酮酸氧化酶poxB基因所在位置。
3.根据权利要求1或2所述的重组大肠杆菌，其特征在于，所述葡萄糖胺合酶突变体glmSA的氨基酸序列如SEQ ID NO.5所示。
4.根据权利要求1～3任一所述的重组大肠杆菌，其特征在于，利用SEQ ID NO.7～15任一所示启动子表达所述UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC。
5.根据权利要求1～4任一所述的重组大肠杆菌，其特征在于，利用SEQ ID NO.10所示启动子表达所述N-乙酰神经氨酸合酶NeuB。
6.根据权利要求1～5任一所述的重组大肠杆菌，其特征在于，所述glmM，glmU和glmS突变体glmSA由诱导型启动子T7调控表达。
7.一种生产N-乙酰神经氨酸的方法，其特征在于，以权利要求1～6任一所述重组大肠杆菌为发酵菌株，以甘油为底物，在30～37℃发酵培养至少24h。
8.根据权利要求7所述的方法，其特征在于，将重组大肠杆菌接种于LB培养基中，培养12～14h获得种子液，再以1～2％的接种量转接至发酵培养基中发酵。
9.根据权利要求7或8所述的方法，其特征在于，当OD600达到0.6后加入诱导剂IPTG，于28～30℃培养24～48h。
10.权利要求1～6任一所述的大肠杆菌在生产含N-乙酰神经氨酸的产品中的应用。
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