CN114891712B - 一种提高n-乙酰神经氨酸产量的重组大肠杆菌 - Google Patents

一种提高n-乙酰神经氨酸产量的重组大肠杆菌 Download PDF

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CN114891712B
CN114891712B CN202210690425.4A CN202210690425A CN114891712B CN 114891712 B CN114891712 B CN 114891712B CN 202210690425 A CN202210690425 A CN 202210690425A CN 114891712 B CN114891712 B CN 114891712B
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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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Abstract

本发明公开了一种提高N‑乙酰神经氨酸产量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。本发明通过构建具有N‑乙酰神经氨酸合成能力的重组菌株BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::PgprE‑NeuC,ΔpoxB，并在基因组上重组表达由低强度组成型启动子PgrpE调控的glmM，由中等强度组成型启动子PssrA调控的glmU和glmS突变体glmSA，并用低强度组成型启动子PgrpE调控脑膜炎奈瑟菌来源的NemNeuB的表达，获得了N‑乙酰神经氨酸产量提高的重组菌株，使产量达到11.28g/L，为工业化生产N‑乙酰神经氨酸奠定了基础。

Description

一种提高N-乙酰神经氨酸产量的重组大肠杆菌
技术领域
本发明涉及一种提高N-乙酰神经氨酸产量的重组大肠杆菌，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
本发明所要解决的技术问题是提供一种提高N-乙酰神经氨酸产量的重组大肠杆菌菌株及其构建方法。
本发明提供了一种重组大肠杆菌，所述重组大肠杆菌敲除了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(GeneID: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)，并且表达由低等强度组成型启动子调控的内源酶glmM(Gene ID:947692)、由中等强度组成型启动子调控的glmU(Gene ID:948246)和葡萄糖胺合酶突变体glmSA，并引入(Neisseria meningitidis)来源的N-乙酰神经氨酸合酶NemNeuB和脑膜炎奈瑟菌(Neisseria meningitidis)来源的UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC。
在一种实施方式中，所述组成型启动子调控的内源酶glmM，glmU和葡萄糖胺合酶突变体glmSA和N-乙酰神经氨酸合酶NemNeuB均在基因组上整合表达。
在一种实施方式中，glmM，glmU和葡萄糖胺合酶突变体glmSA的整合位点为大肠杆菌motA基因(Gene ID:947564)所在位置。
在一种实施方式中，所述NemNeuB整合在ΔpoxB基因所在位置。
在一种实施方式中，所述低等强度组成型启动子为PgrpE，所述中等强度组成型启动子为PssrA。
在一种实施方式中，所述N-乙酰神经氨酸合酶NemNeuB通过启动子Ptrc调控表达；所述UDP-N-乙酰氨基葡糖-2-差向异构酶NeuC通过启动子PgrpE调控表达。
在一种实施方式中，以大肠杆菌E.coli.BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ,ΔpoxB为出发菌株，在ΔmanXYZ处整合PgrpE调控的内源酶NeuC基因，并在ΔpoxB处整合PgrpE调控的NemNeuB。
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、SEQ ID NO.16或SEQ ID NO.18所示；编码NeuB基因的核苷酸序列分别如SEQ ID NO.4、SEQID NO.17或SEQ ID NO.19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，利用不同强度的启动子组合表达所述glmM,glmU-glmSA；所述不同强度的启动子选自PssrA、PgrpE、P224。
在一种实施方式中，采用SEQ ID NO.10所示的启动子PgrpE强化表达所述glmM,采用SEQ ID NO.8所示启动子PssrA强化表达所述glmU和突变体glmSA。
在一种实施方式中，采用SEQ ID NO.10所示的启动子PgrpE强化表达所述N-乙酰神经氨酸合酶NemNeuB。
本发明还提供了一种提高重组大肠杆菌N-乙酰神经氨酸合成能力的方法，所述方法是以所述重组大肠杆菌E.coli.BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::PgprE-NeuC,ΔpoxB为出发菌株，以不同强度的组成型启动子组合强化glmM,glmU和glmS突变体glmSA，并以不同强度的启动子优化脑膜炎奈瑟菌(Neisseria meningitidis)来源的NeuB在基因组上的表达水平，所述启动子选自SEQ ID NO.7～15任一所示的启动子。
在一种实施方式中，用启动子PgrpE调控glmM基因，并用PssrA调控的glmU和glmS突变体glmSA的表达，
在一种实施方式中，用启动子PgrpE调控N-乙酰神经氨酸合酶NemNeuB、NeuC的表达。
本发明还提供了所述重组大肠杆菌在发酵生产N-乙酰神经氨酸中的应用。
在一种实施方式中，所述发酵以甘油为碳源，甘油的浓度可选自20～40g/L。
在一种实施方式中，所述发酵是在含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，所述方法将基因改造后的重组大肠杆菌接种于LB培养基中，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，所述发酵在35～37℃发酵至少24h。
本发明提供了所述大肠杆菌在生产含N-乙酰神经氨酸的产品中的应用。
在一种实施方式中，所述产品包括但不限于药物或保健品
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，通过采用不同强度的组成型大肠杆菌启动子强化glmM,glmU和glmSA提高N-乙酰神经氨酸合成途径的代谢通量，并以不同强度的组成型启动子优化不同来源的N-乙酰神经氨酸合酶NeuB在基因组上的表达水平，使构建的重组大肠杆菌发酵至48h时N-乙酰神经氨酸产量提高至11.28g/L以上。
附图说明
图1为N-乙酰神经氨酸的代谢流程图。
具体实施方式
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。Neisseriameningitidis来源的N-乙酰神经氨酸合酶NemNeuB的核苷酸序列如SEQ ID NO.17所示。Moritella viscosa来源的N-乙酰神经氨酸合酶编码基因MovNeuB的核苷酸序列如SEQIDNO.4所示。Ecoli来源的N-乙酰神经氨酸合酶编码基因EcoNeuB的核苷酸序列如SEQ IDNO.19所示。
启动子Ptrc、PssrA、Pdnakj、PgrpE、P566、P224、P333、Ptac、PalsAR的核苷酸序列分别为SEQ IDNO.7～15；
重组大肠杆菌种子培养及发酵：
种子液培养基：10g/L胰蛋白胨，10g/L氯化钠，5g/L酵母粉。
发酵培养基配方为：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
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，在37℃220rpm条件下反应48h。
样品检测方法：N-乙酰神经氨酸的检测采用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重组菌株Ecoli.BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::PgprE-NeuC,ΔpoxB::Ptrc-MovNeuB的构建
(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(GeneID: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(GeneID: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。
(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’-CTCCCGGACCAAAACGAAAAAAGACGCTTTTCAGCGTCTTTTTTTTTTTTTTTGGTAC CGAGgaatctgttagaggcgcaatagtgacag-3’,
合成核苷酸序列如SEQ ID NO.10所示的PgrpE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、PgrpE启动子片段、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PgrpE-NeuC。
(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’-CACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAaaagggtggcatttcccgtcataataa ggacat-3’
合成核苷酸序列如SEQ ID NO.14所示的Ptrc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、Ptrc启动子片段、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Ptrc-NeuB。
(4)基因的整合
将步骤(2)构建的融合片段PgrpE-NeuC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质粒的步骤(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，验证正确后命名为NBC-2。
将步骤(3)构建的融合片段融合片段Ptrc-NeuB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。
实施例2构建由不同强度组成型启动子组合调控的glmM,glmU和glmSA重组片段
以大肠杆菌BL21(DE3)基因组为模板，设计引物glm-L-F2和glm-L–R2，扩增重组P第一启动子-glmM-P第二启动子-glmU-glmSA整合位点的左侧同源臂基因片段；
glm-L-F2:5’-acttagttAGCTTGGCCgtaacgcccgcagtttcggatc-3’；
glm-L-R2:5’-cctcggcattttattggcttacgg-3’；
以大肠杆菌BL21(DE3)基因组为模板，设计引物glm-R-F2和glm-R-R2，扩增重组P第一启动子-glmM-P第二启动子-glmU-glmSA整合位点的右侧同源臂基因片段；
glm-R-F2：5’-GGTACCGAGgatttcgccatcaaccgataaagcagagc-3’；
glm-R-R2：5’-taaccttgaacagtgcccacaagcag-3’；
合成如SEQ ID NO.6的核苷酸序列所示的glmSA片段；
合成核苷酸序列如SEQ ID NO.8、9、12所示的启动子片段；
以大肠杆菌BL21(DE3)基因组为模板，设计引物glmM-F2和glmM R2，扩增重组整合glmM的基因片段；
glmM-F2:5’-ccctgaaactgatccccataataagcgaagttagcgagatgaatgcgaaaaaaacgGTTCACACAGGAAACCTATAATGatgagtaatcg-3’；
glmM-R2:5’-agatcccggtgcctaatgagtgag-3’；
以大肠杆菌BL21(DE3)基因组为模板，设计引物glmU-F2和glmU R2，扩增重组整合glmU的基因片段；
glmU-F2:5’-cattgaggctggtcatggcgctcataaatctggtatacttacctttacacattGTTCACACAGGAAACCTATAATGatgttgaataatgc-3’；
glmU-R2:5’-ggatttctgctacatcacgttgcgc-3’；
通过PCR扩增获得整合位点左侧同源臂、启动子片段(分别如SEQ ID NO.8、9、12所示所示)、glmM片段、glmU片段、glmSA片段和右侧同源臂。利用融合PCR技术将含有三种强度的启动子片段分别与glmM片段和glmU-SA片段融合，并根据启动子的不同将其调控的片段分别命名为glmM1～glmM3；glmU-SA1～glmU-SA3。其中，glmM1对应含有SEQ ID NO.10所示的PgrpE启动子的glmM融合片段，glmM2对应含有SEQ ID NO.8所示的PssrA启动子的glmM融合片段，glmM3对应含有SEQ ID NO.12所示的P224启动子的glmM融合片段。glmU-SA命名方式与之类似，glmU-SA1对应含有SEQ ID NO.10所示的PgrpE启动子的glmU-SA融合片段，glmU-SA2对应含有SEQ ID NO.8所示的PssrA启动子的glmU-SA融合片段，glmU-SA3对应含有SEQ IDNO.12所示的P224启动子的glmU-SA融合片段。
表1各启动子序列
实施例3由不同强度组成型启动子组合调控glmM,glmU和glmSA的重组菌株的构建
以实施例1构建的重组大肠杆菌宿主NBC-3(E.coli.BL21(DE3)ΔnanATEK,ΔnagABE,ΔmanXYZ::PgprE-NeuC,ΔpoxB::Ptrc-MovNeuB)作为出发菌株，将实施例2构建的含有不同强度启动子组合调控的glmM1～glmM3和glmU-SA1～glmU-SA3的DNA片段经过Gibson组装试剂盒与CRISPER/Cas9系统中的含有N23识别序列(tcgtcgattatctgcgcctgatt)的靶向切割质粒pTarget载体连接，并将glmM和glmU-SA根据不同强度的要求进行9种不同强度的组合。质粒构建好测序后，制作含有pCas9的E.coli.BL21(DE3)ΔnanATEK,ΔnagABE::glmSA,ΔmanXYZ::PgprE-NeuC,ΔpoxB::Ptrc-MovNeuB大肠杆菌宿主感受态细胞，实现glmM1～glmM3以及glmU-SA1～glmU-SA3在宿主菌中motA处的重组整合。转化后将转化子涂布于相应抗性平板上，挑取单菌落进行菌落PCR验证阳性转化子并在再次进行序列测序。最终将含有glmM1～glmM3和glmU-SA1～glmU-SA3片段的重组大肠杆菌，分别命名为NBC-5-1～NBC-5-9(其中，NBC-5-1对应glmM1-glmU-SA1的组合，NBC-5-2对应glmM1-glmU-SA2，NBC-5-3对应glmM1-glmU-SA3，NBC-5-4对应glmM2-glmU-SA1，NBC-5-5对应glmM2-glmU-SA2，NBC-5-6对应glmM2-glmU-SA3，NBC-5-7对应glmM3-glmU-SA1，NBC-5-8对应glmM3-glmU-SA2，NBC-5-9对应glmM3-glmU-SA3)。
分别将重组大肠杆菌NBC-5-1～NBC-5-9接种于LB液体培养基中过夜培养12-14h，再将种子液以2％的接种量转接于TMM发酵培养基中于37℃，220rpm发酵培养48h。经测定，发酵液中N-乙酰神经氨酸(NeuAc)的关键前体物质N-乙酰基-D-氨基甘露糖(ManNAc)的产量分别为2.79g/L，9.24g/L，8.5g/L，2.75g/L，1.91g/L，2.72g/L，2.75g/L，2.72g/L，8.3g/L。将N-乙酰基-D-氨基甘露糖(ManNAc)产量为9.24g/L的大肠杆菌工程菌(即PgrpE-glmM-PssrA-glmU-glmSA)命名为NBC-5。
实施例4构建基因组整合的NeuB重组片段
根据不同来源的N-乙酰神经氨酸合酶编码基因NeuB，合成其编码基因NeuB的核苷酸序列(如SEQ ID NO.4、18、20所示)；
以脑膜炎奈瑟菌(Neisseria meningitidis)来源的NemNeuB(氨基酸序列如SEQID NO.17所示)为例：以大肠杆菌BL21(DE3)为模板，设计引物NemNeuB-L-F1和NemNeuB-L-R1，扩增重组整合NemNeuB左臂基因片段；
NemNeuB-L-F1：5’-gaggcgttaatcagcacgtttctcgct-3’；
NemNeuB-L-R1：5’-aaagggtggcatttcccgtcataataaggacat-3’；
合成如SEQ ID NO.9～13和SEQ ID NO.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NemNeuB-R-F1和NemNeuB-R-R1，扩增重组整合NemNeuB右臂基因片段；
NemNeuB-R-F1:5’-GCCGAAGCGGGTTTTTACGTAAAACAGGTGAAACTGACggttctccatctcctgaatgtgataacggtaacaagtt-3’；
NemNeuB-R-R1:5’-aatatgcactggtcagcgtgcgtaactc-3’；
将扩增得到的NemNeuB左臂基因片段、启动子片段(分别如SEQ ID NO.8～12和SEQID NO.15所示)、NemNeuB基因片段(核苷酸序列如SEQ ID NO.17)和NemNeuB右臂片段通过融合PCR技术构建成重组整合NemNeuB基因片段，根据启动子的不同将其分别命名为NemNeuB1～NemNeuB6；其中，NemNeuB1对应含有SEQ ID NO.8所示的PssrA启动子的NemNeuB融合片段，NemNeuB2对应含有SEQ ID NO.10所示的PgrpE启动子的NemNeuB融合片段，NemNeuB3对应含有SEQ ID NO.12所示的P224启动子的NemNeuB融合片段，NemNeuB4对应含有SEQ ID NO.9所示的Pdnakj启动子的NemNeuB融合片段，NemNeuB5对应含有SEQ ID NO.11所示的P566启动子的NemNeuB融合片段,NemNeuB6对应含有SEQ ID NO.15所示的PalsAR启动子的NemNeuB融合片段。
按照上述相同策略，构建Moritella viscosa来源的MovNeuB(核苷酸序列如SEQID NO.4所示)和E.coli来源的EcoNeuB(核苷酸序列如SEQ ID NO.19所示)的重组整合片段，根据启动子的不同分别将重组片段命名为MovNeuB1～MovNeuB5，EcoNeuB1～EcoNeuB6。其中MovNeuB1～MovNeuB5分别对应含有SEQ ID NO.13～SEQ ID NO.15、SEQ ID NO.7所示启动子以及MovNeuB的融合片段；EcoNeuB1～EcoNeuB6分别对应含有SEQ ID NO.7～10、13、15、所示启动子的EcoNeuB融合片段。
表2各启动子序列
实施例5重组整合NemNeuB基因的大肠杆菌的构建
将实施例4中得到的由不同强度启动子调控的NemNeuB片段(NemNeuB1～NemNeuB6)分别经过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载体连接后，采用化学转化法，将构建好的质粒转化到含有Cas9质粒的NBC-5大肠工程菌感受态细胞中，使不同启动子-NemNeuB片段替换原Ptrc-MovNeuB。取测序正确的含有不同强度启动子调控的NemNeuB的DNA片段的阳性转化子分别命名为NBC8-1～NBC8-6。
消除基因编辑系统中的工具质粒后于发酵培养基中37℃，220rpm培养48h后利用高效液相色谱测定上清液中产物的浓度，NBC8-1～NBC8-6中检测到的N-乙酰神经氨酸(NeuAc)产量分别为8.76g/L，11.28g/L，5.2g/L，0.945g/L，9.87g/L，2.575g/L。结果显示在脑膜炎奈瑟菌(Neisseria meningitidis)来源的NemNeuB受到低强度启动子PgrpE调控的菌株中N-乙酰神经氨酸(NeuAc)产量相对最高，为11.28g/L，将该菌株命名为NBC-8。
对比例1
将实施例5的大肠重组菌株中脑膜炎奈瑟菌(Neisseria meningitidis)来源的NemNeuB序列(SEQ ID NO.17)分别替换成Moritella viscosa来源的MovNeuB(SEQ IDNO.4)和Ecoli来源的EcoNeuB(SEQ ID NO.19)序列，取测序正确的含有不同强度启动子调控的EcoNeuB，MovNeuB的DNA片段的阳性转化子分别命名为NBC6-1～NBC6-6(分别融合了Ptac、Pssra、Pdnakj、PgrpE、PalsaR、P333启动子)，NBC7-1～NBC7-5(分别融合了Ptac、Pssra、Pdnakj、P566、P333、Ptrc启动子)。检测发酵上清液发现NBC6-1～NBC6-6检测到的N-乙酰神经氨酸(NeuAc)产量分别为4.1g/L，3.5g/L，0.76g/L，2.81g/L，8.2g/L，6.85g/L。NBC7-1～NBC7-5中检测到的N-乙酰神经氨酸(NeuAc)产量分别为0.87g/L，0.765g/L，0.785g/L，0.905g/L，0.8g/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> 一种提高N-乙酰神经氨酸产量的重组大肠杆菌
<130> BAA220674A
<160> 19
<170> PatentIn version 3.3
<210> 1
<211> 377
<212> PRT
<213> Neisseria meningitidis
<400> 1
Met Lys Arg Ile Leu Cys Ile Thr Gly Thr Arg Ala Asp Phe Gly Lys
1 5 10 15
Leu Lys Pro Leu Leu Ala Tyr Ile Glu Asn His Pro Asp Leu Glu Leu
20 25 30
His Leu Ile Val Thr Gly Met His Met Met Lys Thr Tyr Gly Arg Thr
35 40 45
Tyr Lys Glu Val Thr Arg Glu Asn Tyr Gln His Thr Tyr Leu Phe Ser
50 55 60
Asn Gln Ile Gln Gly Glu Pro Met Gly Ala Val Leu Gly Asn Thr Ile
65 70 75 80
Thr Phe Ile Ser Arg Leu Ser Asp Glu Ile Glu Pro Asp Met Val Met
85 90 95
Ile His Gly Asp Arg Leu Glu Ala Leu Ala Gly Ala Ala Val Gly Ala
100 105 110
Leu Ser Ser Arg Leu Val Cys His Ile Glu Gly Gly Glu Leu Ser Gly
115 120 125
Thr Val Asp Asp Ser Ile Arg His Ser Ile Ser Lys Leu Ser His Ile
130 135 140
His Leu Val Ala Asn Glu Gln Ala Val Thr Arg Leu Val Gln Met Gly
145 150 155 160
Glu Lys Arg Lys His Ile His Ile Ile Gly Ser Pro Asp Leu Asp Val
165 170 175
Met Ala Ser Ser Thr Leu Pro Ser Leu Glu Glu Val Lys Glu Tyr Tyr
180 185 190
Gly Leu Pro Tyr Glu Asn Tyr Gly Ile Ser Met Phe His Pro Val Thr
195 200 205
Thr Glu Ala His Leu Met Pro Gln Tyr Ala Ala Gln Tyr Phe Lys Ala
210 215 220
Leu Glu Leu Ser Gly Gln Asn Ile Ile Ser Ile Tyr Pro Asn Asn Asp
225 230 235 240
Thr Gly Thr Glu Ser Ile Leu Gln Glu Leu Leu Lys Tyr Gln Ser Asp
245 250 255
Lys Phe Ile Ala Phe Pro Ser Ile Arg Phe Glu Tyr Phe Leu Val Leu
260 265 270
Leu Lys His Ala Lys Phe Met Val Gly Asn Ser Ser Ala Gly Ile Arg
275 280 285
Glu Ala Pro Leu Tyr Gly Val Pro Ser Ile Asp Val Gly Thr Arg Gln
290 295 300
Ser Asn Arg His Met Gly Lys Ser Ile Ile His Thr Asp Tyr Glu Thr
305 310 315 320
Lys Asn Ile Phe Asp Ala Ile Gln Gln Ala Cys Ser Leu Gly Lys Phe
325 330 335
Glu Ala Asp Asp Thr Phe Asn Gly Gly Asp Thr Arg Thr Ser Thr Glu
340 345 350
Arg Phe Ala Glu Val Ile Asn Asn Pro Glu Thr Trp Asn Val Ser Ala
355 360 365
Gln Lys Arg Phe Ile Asp Leu Asn Leu
370 375
<210> 2
<211> 1134
<212> DNA
<213> 人工序列
<400> 2
atgaaaagaa ttttatgcat cacaggaaca cgcgcagatt ttggcaaact gaaaccgctg 60
cttgcgtata ttgaaaatca tccggatctg gaacttcatt taatcgttac aggaatgcat 120
atgatgaaaa catacggcag aacatacaaa gaagtgacac gcgaaaacta ccaacataca 180
tacctgtttt caaaccaaat tcagggcgaa ccgatgggag cagtgctggg caacacaatc 240
acatttatct ctagactttc agatgaaatc gaaccggata tggtcatgat ccatggagat 300
agacttgaag cattagcggg agcagcggtg ggcgcgttat caagccgcct ggtctgtcat 360
attgaaggcg gagaattaag cggcacagtc gatgattcta ttcgccattc aatcagcaaa 420
cttagccata tccatctggt tgctaacgaa caagccgtta caagacttgt gcagatggga 480
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
ggcacaagac aatctaatcg ccatatggga aaatcaatca tccatacaga ttacgaaaca 960
aaaaacattt ttgatgcaat ccaacaggcg tgctctctgg gcaaatttga agcagatgat 1020
acatttaacg gcggagatac aagaacatct acagaacgct ttgcagaagt cattaataac 1080
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> 349
<212> PRT
<213> Neisseria meningitidis
<400> 16
Met Gln Asn Asn Asn Glu Phe Lys Ile Gly Asn Arg Ser Val Gly Tyr
1 5 10 15
Asn His Glu Pro Leu Ile Ile Cys Glu Ile Gly Ile Asn His Glu Gly
20 25 30
Ser Leu Lys Thr Ala Phe Glu Met Val Asp Ala Ala Tyr Asn Ala Gly
35 40 45
Ala Glu Val Val Lys His Gln Thr His Ile Val Glu Asp Glu Met Ser
50 55 60
Asp Glu Ala Lys Gln Val Ile Pro Gly Asn Ala Asp Val Ser Ile Tyr
65 70 75 80
Glu Ile Met Glu Arg Cys Ala Leu Asn Glu Glu Asp Glu Ile Lys Leu
85 90 95
Lys Glu Tyr Val Glu Ser Lys Gly Met Ile Phe Ile Ser Thr Pro Phe
100 105 110
Ser Arg Ala Ala Ala Leu Arg Leu Gln Arg Met Asp Ile Pro Ala Tyr
115 120 125
Lys Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Ile Lys Leu Val
130 135 140
Ala Ser Phe Gly Lys Pro Ile Ile Leu Ser Thr Gly Met Asn Ser Ile
145 150 155 160
Glu Ser Ile Lys Lys Ser Val Glu Ile Ile Arg Glu Ala Gly Val Pro
165 170 175
Tyr Ala Leu Leu His Cys Thr Asn Ile Tyr Pro Thr Pro Tyr Glu Asp
180 185 190
Val Arg Leu Gly Gly Met Asn Asp Leu Ser Glu Ala Phe Pro Asp Ala
195 200 205
Ile Ile Gly Leu Ser Asp His Thr Leu Asp Asn Tyr Ala Cys Leu Gly
210 215 220
Ala Val Ala Leu Gly Gly Ser Ile Leu Glu Arg His Phe Thr Asp Arg
225 230 235 240
Met Asp Arg Pro Gly Pro Asp Ile Val Cys Ser Met Asn Pro Asp Thr
245 250 255
Phe Lys Glu Leu Lys Gln Gly Ala His Ala Leu Lys Leu Ala Arg Gly
260 265 270
Gly Lys Lys Asp Thr Ile Ile Ala Gly Glu Lys Pro Thr Lys Asp Phe
275 280 285
Ala Phe Ala Ser Val Val Ala Asp Lys Asp Ile Lys Lys Gly Glu Leu
290 295 300
Leu Ser Gly Asp Asn Leu Trp Val Lys Arg Pro Gly Asn Gly Asp Phe
305 310 315 320
Ser Val Asn Glu Tyr Glu Thr Leu Phe Gly Lys Val Ala Ala Cys Asn
325 330 335
Ile Arg Lys Gly Ala Gln Ile Lys Lys Thr Asp Ile Glu
340 345
<210> 17
<211> 1050
<212> DNA
<213> 人工序列
<400> 17
atgcaaaaca acaacgaatt taaaatcggc aacagatcag tcggatataa tcatgaaccg 60
cttattatct gcgaaattgg catcaaccat gaaggaagct taaaaacagc ctttgaaatg 120
gtcgatgcag cgtataatgc cggagcagaa gttgtgaaac atcaaacaca tatcgttgaa 180
gatgaaatgt ctgatgaagc caaacaggtg atcccgggca acgcagatgt ctcaatctac 240
gaaatcatgg aaagatgtgc gctgaacgaa gaagatgaaa tcaaactgaa agaatacgtt 300
gaaagcaaag gaatgatctt tatctctaca ccgttttcac gcgctgccgc acttagatta 360
cagcgcatgg atattccggc ctataaaatc ggctctggag aatgcaacaa ctacccgctg 420
atcaaactgg tggcaagctt tggcaaaccg atcatcctgt ctacaggaat gaactcaatc 480
gaaagcatca aaaaatcagt tgaaatcatc agagaagcgg gcgtgccgta tgctctgctt 540
cattgtacaa acatttatcc gacaccgtat gaagatgttc gcctgggcgg aatgaatgat 600
ctttcagaag cctttccgga tgcaattatc ggccttagcg atcatacatt agataactat 660
gcatgcctgg gagcggtggc tcttggcgga tctatcctgg aaagacattt tacagataga 720
atggatcgcc cgggcccgga tatcgtctgt tcaatgaatc cggatacatt taaagaactg 780
aaacaaggag cccatgcact gaaacttgcg agaggcggca agaaagatac aattatcgct 840
ggcgaaaaac cgacaaaaga ttttgcgttt gctagcgtcg ttgcggataa agatattaag 900
aaaggcgaac tgctgtctgg agataacctg tgggtcaaaa gaccgggcaa cggagatttt 960
agcgttaacg aatacgaaac actttttggc aaagtggcgg cttgcaatat ccgcaaagga 1020
gctcagatta agaaaacaga tatcgaataa 1050
<210> 18
<211> 346
<212> PRT
<213> Escherichia coli
<400> 18
Met Ser Asn Ile Tyr Ile Val Ala Glu Ile Gly Cys Asn His Asn Gly
1 5 10 15
Ser Val Asp Ile Ala Arg Glu Met Ile Leu Lys Ala Lys Glu Ala Gly
20 25 30
Val Asn Ala Val Lys Phe Gln Thr Phe Lys Ala Asp Lys Leu Ile Ser
35 40 45
Ala Ile Ala Pro Lys Ala Glu Tyr Gln Ile Lys Asn Thr Gly Glu Leu
50 55 60
Glu Ser Gln Leu Glu Met Thr Lys Lys Leu Glu Met Lys Tyr Asp Asp
65 70 75 80
Tyr Leu His Leu Met Glu Tyr Ala Val Ser Leu Asn Leu Asp Val Phe
85 90 95
Ser Thr Pro Phe Asp Glu Asp Ser Ile Asp Phe Leu Ala Ser Leu Lys
100 105 110
Gln Lys Ile Trp Lys Ile Pro Ser Gly Glu Leu Leu Asn Leu Pro Tyr
115 120 125
Leu Glu Lys Ile Ala Lys Leu Pro Ile Pro Asp Lys Lys Ile Ile Ile
130 135 140
Ser Thr Gly Met Ala Thr Ile Asp Glu Ile Lys Gln Ser Val Ser Ile
145 150 155 160
Phe Ile Asn Asn Lys Val Pro Val Gly Asn Ile Thr Ile Leu His Cys
165 170 175
Asn Thr Glu Tyr Pro Thr Pro Phe Glu Asp Val Asn Leu Asn Ala Ile
180 185 190
Asn Asp Leu Lys Lys His Phe Pro Lys Asn Asn Ile Gly Phe Ser Asp
195 200 205
His Ser Ser Gly Phe Tyr Ala Ala Ile Ala Ala Val Pro Tyr Gly Ile
210 215 220
Thr Phe Ile Glu Lys His Phe Thr Leu Asp Lys Ser Met Ser Gly Pro
225 230 235 240
Asp His Leu Ala Ser Ile Glu Pro Asp Glu Leu Lys His Leu Cys Ile
245 250 255
Gly Val Arg Cys Val Glu Lys Ser Leu Gly Ser Asn Ser Lys Val Val
260 265 270
Thr Ala Ser Glu Arg Lys Asn Lys Ile Val Ala Arg Lys Ser Ile Ile
275 280 285
Ala Lys Thr Glu Ile Lys Lys Gly Glu Val Phe Ser Glu Lys Asn Ile
290 295 300
Thr Thr Lys Arg Pro Gly Asn Gly Ile Ser Pro Met Glu Trp Tyr Asn
305 310 315 320
Leu Leu Gly Lys Ile Ala Glu Gln Asp Phe Ile Pro Asp Glu Leu Ile
325 330 335
Ile His Ser Glu Phe Lys Asn Gln Gly Glu
340 345
<210> 19
<211> 1041
<212> DNA
<213> 人工序列
<400> 19
atgtctaaca tctacatcgt ggcagaaatc ggctgcaatc ataacggatc agtcgatatc 60
gcgagagaaa tgattttaaa agctaaagaa gccggcgtga acgctgtcaa atttcaaaca 120
tttaaagccg ataaactgat cagcgcaatt gcgccgaaag cagaatacca aatcaaaaac 180
acaggagaat tagaatctca gctggaaatg acgaaaaaac tggaaatgaa atacgatgat 240
taccttcatc tgatggaata cgcagtcagc ctgaatcttg atgtttttag cacaccgttt 300
gatgaagatt ctattgattt tctggcgtca ctgaaacaaa aaatctggaa aattccgtca 360
ggcgaactgc ttaaccttcc gtacctggaa aaaatcgcta aacttccgat cccggataag 420
aaaattatca ttagcacagg catggccaca atcgatgaaa tcaaacagtc tgtctcaatc 480
tttatcaata acaaagtccc ggttggaaac atcacaatcc tgcattgtaa cacagaatat 540
ccgacaccgt ttgaagatgt taaccttaac gctatcaacg atctgaaaaa acattttccg 600
aaaaacaaca tcggcttttc tgatcattca agcggatttt atgcagcgat tgctgccgtt 660
ccgtatggca tcacatttat cgaaaaacat tttacactgg ataaaagcat gtctggaccg 720
gatcatcttg cttcaatcga accggatgaa ctgaaacatc tttgcattgg cgttagatgt 780
gtggaaaaat cactgggatc aaatagcaaa gttgtgacag ccagcgaaag aaaaaacaaa 840
atcgttgcac gcaaatctat catcgcgaaa acagaaatca aaaaaggaga agtgttttca 900
gagaaaaata tcacaacaaa aagaccgggc aacggaatta gcccgatgga atggtataat 960
ttactgggca aaatcgcgga acaagatttt atcccggatg aacttatcat ccatagcgaa 1020
tttaaaaacc agggagaata a 1041

Claims (5)

1.一种重组大肠杆菌，其特征在于，敲除了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、氨基葡萄糖-6-磷酸脱氨酶nagB、N-乙酰氨基葡萄糖特异性EIICBA组分nagE、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、唾液酸转运蛋白nanT、N-乙酰甘露糖胺-6-磷酸2-差向异构酶nanE、N-乙酰甘露糖胺激酶nanK、甘露糖特异性EIIAB组分相关基因manXYZ、丙酮酸氧化酶poxB基因，并表达由低等强度组成型启动子调控的内源酶glmM、由中等强度组成型启动子调控的glmU和葡萄糖胺合酶突变体glmSA；所述低等强度组成型启动子为PgrpE，所述中等强度组成型启动子为PssrA；所述glmM，glmU和葡萄糖胺合酶突变体glmSA的整合位点为大肠杆菌motA基因所在位置；并在ΔmanXYZ处整合PgrpE调控的UDP-N-乙酰氨基葡糖-2-差向异构酶基因NeuC，并在ΔpoxB处整合PgrpE调控的脑膜炎奈瑟菌(Neisseria meningitidis)来源的N-乙酰神经氨酸合酶基因NemNeuB；
基因nagA的核苷酸序列如Gene ID:945289所示，基因nagB的核苷酸序列如Gene ID:945290所示，基因nagE的核苷酸序列如Gene ID:945292所示；基因nanA的核苷酸序列如Gene ID:947742)所示；基因nanT的核苷酸序列如Gene ID:947740所示；基因nanE的核苷酸序列如Gene ID:947745所示；基因nanK的核苷酸序列如Gene ID:947757所示；基因poxB的核苷酸序列如Gene ID:946132所示；glmU的核苷酸序列如Gene ID:948246所示；glmM的核苷酸序列如Gene ID:947692所示；所述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；所述N-乙酰神经氨酸合酶NemNeuB的核苷酸序列如SEQ ID NO.17所示；所述启动子PssrA的核苷酸序列如SEQ ID NO.8所示；所述PgrpE的核苷酸序列如SEQ ID NO.10所示；编码NeuC基因的核苷酸序列如SEQ ID NO.2所示。
2.一种提高重组大肠杆菌N-乙酰神经氨酸合成能力的方法，其特征在于，在出发菌株的基础上，敲除了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、氨基葡萄糖-6-磷酸脱氨酶nagB、N-乙酰氨基葡萄糖特异性EIICBA组分nagE、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、唾液酸转运蛋白nanT、N-乙酰甘露糖胺-6-磷酸2-差向异构酶nanE、N-乙酰甘露糖胺激酶nanK、甘露糖特异性EIIAB组分相关基因manXYZ、丙酮酸氧化酶poxB基因，并表达由低等强度组成型启动子调控的内源酶glmM、由中等强度组成型启动子调控的glmU和葡萄糖胺合酶突变体glmSA；所述低等强度组成型启动子为PgrpE，所述中等强度组成型启动子为PssrA；所述glmM，glmU和葡萄糖胺合酶突变体glmSA的整合位点为大肠杆菌motA基因所在位置；并在ΔmanXYZ处整合PgrpE调控的UDP-N-乙酰氨基葡糖-2-差向异构酶基因NeuC，并在ΔpoxB处整合PgrpE调控的脑膜炎奈瑟菌(Neisseria meningitidis)来源的N-乙酰神经氨酸合酶基因NemNeuB；
基因nagA的核苷酸序列如Gene ID:945289所示，基因nagB的核苷酸序列如Gene ID:945290所示，基因nagE的核苷酸序列如Gene ID:945292所示；基因nanA的核苷酸序列如Gene ID:947742)所示；基因nanT的核苷酸序列如Gene ID:947740所示；基因nanE的核苷酸序列如Gene ID:947745所示；基因nanK的核苷酸序列如Gene ID:947757所示；基因poxB的核苷酸序列如Gene ID:946132所示；glmU的核苷酸序列如Gene ID:948246所示；glmM的核苷酸序列如Gene ID:947692所示；所述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；所述N-乙酰神经氨酸合酶NemNeuB的核苷酸序列如SEQ ID NO.17所示；所述启动子PssrA的核苷酸序列如SEQ ID NO.8所示；所述PgrpE的核苷酸序列如SEQ ID NO.10所示；编码NeuC基因的核苷酸序列如SEQ ID NO.2所示。
3.一种生产N-乙酰神经氨酸的方法，其特征在于，应用权利要求1所述的重组大肠杆菌发酵生产N-乙酰神经氨酸。
4.根据权利要求3所述的方法，其特征在于，所述发酵以甘油为碳源，在35～37℃发酵至少24h。
5.权利要求1所述的大肠杆菌，或权利要求3～4任一所述方法在生产含N-乙酰神经氨酸的产品中的应用。
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