CN117305285A - 磷酸烯醇丙酮酸羧激酶突变体及其应用 - Google Patents
磷酸烯醇丙酮酸羧激酶突变体及其应用 Download PDFInfo
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- CN117305285A CN117305285A CN202210716210.5A CN202210716210A CN117305285A CN 117305285 A CN117305285 A CN 117305285A CN 202210716210 A CN202210716210 A CN 202210716210A CN 117305285 A CN117305285 A CN 117305285A
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Abstract
本发明提供一种磷酸烯醇丙酮酸羧激酶突变体及其应用。本发明通过对具有核苷生产能力的微生物细胞内编码磷酸烯醇丙酮酸羧激酶的氨基酸序列进行改造,和/或将pckA基因的原有启动子变为强启动子和/或异位增加pckA的拷贝数,使得所述菌株产生核苷的能力与未修饰的菌株相比增强。本发明为核苷的发酵生产提供有效手段。
Description
技术领域
本发明涉及生物技术领域,具体地说,涉及磷酸烯醇丙酮酸羧激酶突变体及其应用。
背景技术
核苷是一类糖苷的总称。核苷是核酸和核苷酸的组成成分。核苷是由D-核糖或D-Z-脱氧核糖与嘧啶碱或嘌呤碱缩合而成。核苷一般为无色结晶,不溶于普通有机溶剂,易溶于热水,熔点为160~240℃。由D-核糖生成的核苷称核糖核苷,参与RNA组成,由D-α-脱氧核糖生成的核苷称脱氧核糖核苷,参与DNA组成。D-核糖与腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶缩合生成相应的腺嘌呤核糖核苷、鸟嘌呤核糖核苷、胞嘧啶核糖核苷、胸腺嘧啶核糖核苷和尿嘧啶核糖核苷,它们分别简称为腺苷(A)、鸟苷(G)、胞苷(C)、胸苷(T)和尿苷(U)。
鸟嘌呤核苷(鸟苷)和次黄嘌呤核苷(肌苷)在食品和医药行业有着广泛的作用。在食品领域,鸟苷和肌苷分别是鸟苷酸二钠和肌苷酸二钠的重要前体,而鸟苷酸二钠与肌苷酸二钠组合使用作为食品增鲜剂,广泛应用于鸡精、酱油等调味品中。在医药领域,鸟苷和肌苷可以作为多种抗病毒药物的医药中间体,如无环鸟苷、三氮唑核苷、三磷酸鸟苷钠等都需要鸟苷作为合成原料。肌苷是肌苷酸的重要前体,而肌苷酸可以作为合成腺苷酸(AMP)和鸟苷酸(GMP)的前体,适用于各种原因引起的白细胞减少症、血小板减少症、各种心脏疾患、急性及慢性肝炎、肝硬化等,此外还可治疗中心视网膜炎、视神经萎缩等。
腺苷即腺嘌呤核苷,化学名为6-氨基-9-β-D-呋喃核糖基-9-氢嘌呤,它是腺嘌呤核苷酸脱磷酸后的产物,属于重要的核苷酸衍生物。腺苷是一种遍布人体细胞的内源性核苷,可直接进入心肌经磷酸化生成腺苷酸,参与心肌能量代谢,同时还参与扩张冠脉血管,增加血流量。腺苷对心血管系统和肌体的许多其它系统及组织均有生理作用。腺苷除了可以用作治疗心脏的特效药物之外,还是用于合成三磷酸腺苷(ATP)、腺嘌呤、腺苷酸、阿糖腺苷的重要中间体,广泛应用于医药等行业。
目前,微生物发酵是生产核苷的主要方法,主要使用的微生物包括枯草芽孢杆菌、解淀粉芽孢杆菌或短小芽孢杆菌等。在生长菌株的选育与改造过程中,通过使用紫外诱变、硫酸二乙酯诱变育种,定向选育核苷高产菌株;或者根据细菌中核苷酸的代谢路径和调节机理,深入了解菌株遗传背景及菌株特性,通过代谢工程手段,有目的性地对菌株进行改造,以获得性状优良、能够高产核苷的生产菌株。但目前核苷菌种的发酵性能仍较差、核苷的转化率仍较低,难以满足大规模工业化生产的需求。
发明内容
本发明的目的是提供磷酸烯醇丙酮酸羧激酶突变体及其应用。
本发明的另一目的是提供能够高效率生产核苷的新微生物。
为了实现本发明目的,第一方面,本发明提供磷酸烯醇丙酮酸羧激酶突变体,所述突变体包含磷酸烯醇丙酮酸羧激酶第355位氨基酸由P到T的突变。
本发明中,来自枯草芽孢杆菌(Bacillus subtilis)的磷酸烯醇丙酮酸羧激酶在NCBI上的参考序列编号为BSU_30560;
来自解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的磷酸烯醇丙酮酸羧激酶在NCBI上的参考序列编号为BAMF_2839。
第二方面,本发明提供编码所述突变体的核酸分子或含有所述核酸分子的生物材料,所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第三方面,本发明提供所述核酸分子或所述生物材料的以下任一应用:
(1)用于核苷的发酵生产;
(2)用于提高核苷的发酵产量;
(3)用于构建产核苷的基因工程菌。
其中,所述核苷包括肌苷、鸟苷、腺苷,或它们对应的核苷衍生物,如次黄嘌呤、肌苷酸、鸟嘌呤、鸟苷酸、核黄素、二乙酰鸟苷酸等。
第四方面,本发明提供产核苷的菌株的构建方法,利用基因工程手段,在微生物基因组中引入突变,使其编码的磷酸烯醇丙酮酸羧激酶包含P355T突变位点。
所述微生物为具有核苷生产能力的微生物。
进一步地,所述方法包括进一步增强菌株中的pckA基因;
其中,解淀粉芽孢杆菌pckA基因在NCBI上的参考序列编号为BAMF_2839;枯草芽孢杆菌pckA基因在NCBI上的参考序列编号为BSU_30560。
所述增强的途径选自以下1)~6),或任选的组合:
1)通过导入具有所述基因的质粒而增强;
2)通过增加染色体上所述基因的拷贝数而增强;
3)通过改变染色体上所述基因的启动子序列而增强;
4)通过将强启动子与所述基因可操作地连接而增强;
5)通过导入增强子而增强;
6)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强。
优选地,增强方式为将pckA基因的原有启动子替换为强启动子;和/或
将pckA基因的拷贝数增加到二拷贝。
所述强启动子可选自P43、P41、Plaps等。
所述微生物优选所述微生物为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillus subtilis)、短小芽孢杆菌(Bacillus pumilus)或大肠杆菌(Escherichia coli)等,更优选解淀粉芽孢杆菌836,菌株836可参见CN112574934A;枯草芽孢杆菌A5,菌株A5可参见CN110257315B。
第五方面,本发明提供按照所述方法构建得到的产核苷的菌株。
第六方面,本发明提供一种生产核苷的方法,所述方法包括如下步骤:
1)培养所述菌株,以获得微生物的培养物;
2)从步骤1)中获得的所述培养物中收集所产生的核苷。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明提供的突变菌株B.a8348(pckAP355T点突变菌株)、B.a8349(pckA启动子强化菌株)、B.a8350(pckA二拷贝菌株)较出发菌相比,鸟苷产量由1.1g/L提高至3.0g/L、1.9g/L和2.3g/L。肌苷产量由0.6g/L分别提高至1.4g/L,1.4g/L和1.4g/L。腺苷生产菌株做了相应的改造后,其腺苷产量由出发菌的8.9g/L分别提高至10.0g/L、10.6g/L和10.1g/L,相应菌株的肌苷生产能力也有所提高,由1.1g/L分别提高至2.1g/L、1.6g/L和1.9g/L。
上述突变位点可应用于解淀粉芽孢杆菌及枯草芽孢杆菌,但不限于上述两种菌,如短小芽孢杆菌、大肠杆菌等宿主菌,用于产肌苷、鸟苷、腺苷等核苷或对应的核苷衍生物,如次黄嘌呤、肌苷酸、鸟嘌呤、鸟苷酸、核黄素、二乙酰鸟苷酸等。
具体实施方式
本发明旨在提供使用微生物生产嘌呤核苷的方法,以及创制在所述方法中使用的、能够高效率生产嘌呤核苷的新微生物。
研究发现,经过修饰枯草芽孢杆菌或解淀粉芽孢杆菌的磷酸烯醇丙酮酸羧激酶(由pckA基因编码,催化OAA生成PEP),使得PckA蛋白活性增强,使得微生物能够高效率、高速度地生成鸟嘌呤核苷、腺嘌呤核苷酸及次黄嘌呤核苷,并成功创制出能够高效生产核苷的新微生物。
通过强化pckA表达可提高微生物产核苷的能力。本发明首次在解淀粉芽孢杆菌或枯草芽孢杆菌中通过突变该基因使得氨基酸序列发生改变、替换原有启动子及增加拷贝数等,获得相应突变体可高效生产核苷,并成功创制出能够高效生产核苷的微生物。
本发明采用如下技术方案:
本发明提供一种解淀粉芽孢杆菌或枯草芽孢杆菌,其细胞内编码磷酸烯醇丙酮酸羧激酶的氨基酸序列发生变化,和/或将其起始密码子ATG前插入强启动子P43和/或异位增加pckA的拷贝数,使得所述菌株产生核苷的能力与未修饰的菌株相比增强。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
以下实施例中使用的引物序列如表1所示。
表1
引物名称 | 引物序列(5′-3′) |
pckA-1f | aacaaaataaggatcctctagatacagactgccgattacagtc |
pckA-1r | ccttcgtcagtttgctgatcgTagggagcacaccgaatgcatccgccg |
pckA-2f | cggcggatgcattcggtgtgctccctAcgatcagcaaactgacgaagg |
pckA-2r | gccaagcttgcatgcctgcagctatcgggtctatcctctcgat |
P43pckA-1f | acaaaataaggatcctctagacagcacaagacctgttgttacaga |
P43pckA-1r | gcgaaaacataccacctatcaaaaaaccttcctttgccggttt |
P43-F | tgataggtggtatgttttcg |
P43-R | gtgtacattcctctcttacc |
P43pckA-3f | taggtaagagaggaatgtacacatgaaattagttgatttaaccgca |
P43pckA-3r | gccaagcttgcatgcctgcagcaccgacgttggctgagcagtgcat |
pckA2nd-1f | acaaaataaggatcctctagagtcacctatgacagccagcatacc |
pckA2nd-1r | gcgaaaacataccacctatcacaggcggcttcctcacaaggag |
pckA2nd-2r | gttccgcttcatttcaatttattaaacgagaggtccgccggctttgg |
pckA2nd-3f | gccggcggacctctcgtttaataaattgaaatgaagcggaacatcaa |
pckA2nd-3r | gccaagcttgcatgcctgcagaatccgataacggatacggacctt |
A5pckA-1f | acaaaataaggatcctctagacaccattctttctgctccgcatt |
A5pckA-1r | cctgctccttcgttagtttgctgatcgTcggcaggactccgaaggca |
A5pckA-2f | tgccttcggagtcctgccgAcgatcagcaaactaacgaaggagcagg |
A5pckA-2r | gccaagcttgcatgcctgcagcaggcacagctgaagtgcagaag |
A5P43pckA-1f | acaaaataaggatcctctagacacagattttatccggatgcccc |
A5P43pckA-1r | agcgaaaacataccacctatcaatgaaaccttcctttatcgtttt |
A5P43pckA-3f | ataggtaagagaggaatgtacacatgaactcagttgatttgaccgc |
A5P43pckA-3r | gccagtgccaagcttgcatgcctgcaggaaaagggcgacatcgccttt |
A5pckA2nd-1f | acaaaataaggatcctctagagcgaatagaatcaattgcggtc |
A5pckA2nd-1r | caagcgaaaacataccacctatcagcatgaagaatggttccgcttttgatc |
A5pckA2nd-3r | cgtattgaacgaccaattccatttatacgagagggccgcctgcc |
A5pckA2nd-4f | atggaattggtcgttcaatacgtt |
A5pckA2nd-4r | gccaagcttgcatgcctgcagctttaaagcggaccttatggaat |
实施例1解淀粉芽孢杆菌中pckAP355T点突变菌株的构建
以B.a836菌株(参见CN112574934A)基因组为模板,使用pckA-1f/pckA-1r,pckA-2f/pckA-2r引物对,pfu高保真DNA聚合酶扩增获得pckA基因的上、下游同源臂。获得片段进行胶回收并融合,扩增获得pckAP355T全长片段,进行胶回收(对应的ORF框的核苷酸序列见SEQ ID No:1,氨基酸序列见SEQ ID No:2)。将pKSU质粒(pKSU质粒由南开大学王淑芳教授惠赠,参见A markerless gene replacement method for B.amyloliquefaciens LL3andits use in genome reduction and improvement of poly-γ-glutamic acidproduction[J],Applied Microbiology and Biotechnology,2014,98(21):8963-8973.Zhang W,Gao W,Feng J,et al DOI:10.1007/s00253-014-5824-2)使用XbaI/PstI进行双酶切并进行胶回收。使用组装试剂盒将酶切后的线性化质粒及pckAP355T片段进行组装,并转化至TransT1感受态中,后期进行鉴定筛选获得重组质粒pKSU-pckAP355T。转化至B.a836菌株中,用含2.5μg/mL氯霉素的LB平板在30℃下筛选转化子,将获得的转化子接到5mlLB液体培养基中,42℃200rpm培养12h并传一代,稀释涂布至含5μg/mL氯霉素的LB平板获得一次重组子;将一次重组子接到5ml LB液体培养基中,42℃200rpm培养12h并传一代,稀释涂布含0.8μM 5-FU的LB平板筛选二次重组子,筛选获得pckAP355T点突变菌株,命名为B.a8348。
实施例2解淀粉芽孢杆菌中pckA启动子强化菌株的构建
以B.a8348菌株基因组为模板,使用P43pckA-1f/P43pckA-1r,P43pckA-3f/P43pckA-3r引物对,pfu高保真DNA聚合酶扩增获得pckAP355T的上、下游同源臂。以PBE43质粒(PBE43质粒为全基因合成,参考文献Effects of overexpression of key enzyme geneson guanosine accumulation in Bacillus amyloliquefaciens)为模板,P43-F/P43-R为引物,扩增获得P43启动子片段。获得片段进行胶回收并融合,扩增获得全长片段P43-pckAP355T,进行胶回收,按照实施例1方法构建得到质粒pKSU-P43-pckAP355T,并将其转化至B.a8348菌株中,获得的菌株命名为B.a 8349。
实施例3解淀粉芽孢杆菌中pckA二拷贝菌株的构建
以B.a 8349菌株基因组为模板,使用pckA2nd-1f/pckA2nd-1r,P43-F/pckA2nd-2r,pckA2nd-3f/pckA2nd-3r引物对,pfu高保真DNA聚合酶扩增获得pckAP355T二拷贝方案的上游同源臂、P43-pckAP355T及下游同源臂。获得片段进行胶回收并融合,扩增获得P43-pckAP355T2nd全长片段,进行胶回收,按照实施例1方法构建得到质粒pKSU-P43-pckAP355T2nd,并将其转化至B.a8349菌株中,获得的菌株命名为B.a 8350。
实施例4枯草芽孢杆菌中pckAP355T点突变菌株的构建
为确定pckA的突变是否能提高腺苷产量,将pckAP355T点突变引入腺苷生产菌株B.subtilis A5(以B.subtilis 168为出发菌,构建得到的一株腺苷生产菌株,参见CN110257315B)中。以B.subtilis A5菌株基因组为模板,使用A5pckA-1f/A5pckA-1r,A5pckA-2f/A5pckA-2r引物对扩增出突变片段的左右同源臂,胶回收后进行融合获得A5-pckAP355T片段(对应的ORF框的核苷酸序列见SEQ ID No:3,氨基酸序列见SEQ ID No:4)。按照实施例1方法构建出重组质粒pKSU-A5pckAP355T。转化至B.subtilis A5菌株中,按照实施例1中筛选方法,筛选获得含有突变菌株,命名为B.subtilis A0001。
实施例5枯草芽孢杆菌中P43-pckAP355T启动子强化菌株的构建
以B.subtilis A0001菌株基因组为模板,分别使用A5P43pckA-1f/A5P43pckA-1r,A5P43pckA-3f/A5P43pckA-3r引物对扩增出本方案的左右同源臂,使用实施例2中扩增获得的P43启动子片段,将三个片段进行融合PCR,获得枯草芽孢杆菌中P43-pckAP355T全长片段;按照实施例1中构建质粒的方法构建得到pKSU-A5P43pckAP355T质粒,将其转化至B.subtilisA0001菌株中,按照相同的筛选方式筛选出P43启动子强化菌株,命名为B.subtilis A0002。
实施例6腺苷生产菌株中P43-pckAP355T二拷贝启动子强化菌株的构建
以B.subtilis A0002基因组为模板,A5pckA2nd-1f/A5pckA2nd-1r,P43-F/A5pckA2nd-3r,A5pckA2nd-4f/A5pckA2nd-4r引物对分别扩增出3个片段,并融合获得A5pckA2nd全长片段,按照实施例1方法构建出重组质粒pKSU-A5P43pckA2nd,并转化至B.subtilis A0002,筛选获得的二拷贝菌株为B.subtilisA0003。
实施例7突变菌株产苷性能验证
1、将甘油中保存的菌种37℃过夜培养划出单克隆。
2、挑单菌落接种至30mL种子培养基(葡萄糖20g/L,酵母粉5g/L,玉米浆干粉5g/L,磷酸二氢钾3g/L,硫酸镁0.5g/L,硫酸亚铁0.02g/L,硫酸锰0.01g/L,pH7.0~7.2。中,110rpm 37℃培养7~8h)。
3、按10%v/v接种量转接至30ml发酵培养基(葡萄糖120g/L,酵母粉3.5,磷酸二氢钾3g/L,硫酸铵25g/L,硫酸锰0.01g/L,硫酸镁5g/L,谷氨酸钠10g/L,玉米浆干粉15g/L,碳酸钙25g/L,pH7.0~7.2)中,摇床转速130rpm,解淀粉芽孢杆菌出发获得的突变菌株35℃培养70h(枯草芽孢杆菌出发获得的突变菌株发酵48h)。
4、使用液相色谱仪对发酵液中产苷进行检测(表2)。
表2 突变菌株摇瓶发酵产鸟苷及肌苷评估结果(三次重复均值)
菌株 | 鸟苷产量(g/L) | 肌苷产量(g/L) | 腺苷产量(g/L) | OD562 |
B.a 836 | 1.1 | 0.6 | 0.3 | 28.6 |
B.a 8348 | 3.0 | 1.4 | 1.4 | 28.3 |
B.a 8349 | 1.9 | 1.4 | 1.3 | 27.6 |
B.a 8350 | 2.3 | 1.4 | 1.6 | 26.9 |
B.subtilis A5 | 0 | 1.1 | 8.9 | 24.8 |
B.subtilis A0001 | 0 | 2.1 | 10.0 | 25.1 |
B.subtilis A0002 | 0 | 1.6 | 10.6 | 24.8 |
B.subtilis A0003 | 0 | 1.9 | 10.1 | 24.6 |
以上实验结果表明pckA基因发生P355T点突变或通过插入强启动子或二拷贝进行强化,能够提高鸟苷生产菌产鸟苷及肌苷的能力,同时将其构建至腺苷生产菌株中,同样能提高腺苷生产菌株产肌苷及腺苷的能力。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 磷酸烯醇丙酮酸羧激酶突变体及其应用
<130> KHP221115363.4
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1584
<212> DNA
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 1
atgaaattag ttgatttaac cgcagatctt caagcgttac tcgcaagtcc gaatgtgcat 60
cataatttat cagctccaaa acttactgaa aaagtgatca gccgaaatga aggaacgctg 120
acatcaacag gcgccgtacg cgccactacc ggcgcatata cgggccgatc acctaaagat 180
aaatttatcg ttcgtgaaaa aagcacagaa aatcaaattg attggggcgc ggtgaaccag 240
cccatttctg aagaagcgtt tgaaaagctg tacgccaaag tcgtcagcta cttaaaacag 300
agagatgaac tgtttgtttt tgaaggattc gcaggcgcag atgaaaaata cagactgccg 360
attacagtcg taaatgaatt tgcgtggcat aatttattcg cgcgccagct gtttatcaga 420
ccggagacga atgaaaaaga tccgcaagca cagccgttta ccattctttc agctccgcat 480
tttaaagctg atccggaaac ggacggcacg cattctgaaa catttattat tgtatccttc 540
gaaaagcgcg tcattttaat cggcggcacc gagtacgccg gagaaatgaa aaaatcaata 600
ttctcgatta tgaactttct gctcccgcag caggatattc tgccgatgca ctgctcagcc 660
aacgtcggtg aaaaaggcga tgtcgccctg tttttcggtc tttccggcac tgggaaaacg 720
acgctttcag ccgatgcgga ccggaaactg atcggcgatg acgagcacgg ctggtctgat 780
tccggcgttt ttaacattga aggcggatgt tacgctaaat gtatcaatct gagcgaagaa 840
aaagaaccgc agatttttca cgcgatccgc ttcgggtctg ttcttgagaa tgttgtgctt 900
gatgagaaaa cgggtgaggc ggattacgac aattccttct acacggaaaa cacaagagcc 960
gcatatccga ttgagatgat cggcaatatc gtgcagccta gcattgccgg ccacccgcag 1020
gcgatcgtct ttttaacggc ggatgcattc ggtgtgctcc ctccgatcag caaactgacg 1080
aaggagcagg cgatgtatca tttcttaagc gggtacacca gcaaacttgc cggaaccgaa 1140
cgcggtgtga catctccgca aacgactttt tccacgtgct tcggttcacc gttcctgccg 1200
cttccggctc acgtatatgc cgaaatgctc gggaagaaaa tcgacgaaca cggcgttcaa 1260
gtctttctgg tcaacacagg ctggaccggc ggagggtacg gagtcggaga gcggatgaaa 1320
ctttcgtata caagagcgat ggtcaaagcc gcaattgacg gcacgcttga tcagacagac 1380
atgacagcag acagcatttt cggtctgcac agccccgttc atgttccggg cgttccggat 1440
gaggttcttc agcctgaaaa cacatggagc gataaaaaag catacaacga aaaagcgctg 1500
tttttagcca atgagttcaa agaaaacttt aaaaagtttt cccatgcaga ctccattgcc 1560
aaagccggcg gacctctcgt ttaa 1584
<210> 2
<211> 527
<212> PRT
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 2
Met Lys Leu Val Asp Leu Thr Ala Asp Leu Gln Ala Leu Leu Ala Ser
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Pro Asn Val His His Asn Leu Ser Ala Pro Lys Leu Thr Glu Lys Val
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Thr Thr Gly Ala Tyr Thr Gly Arg Ser Pro Lys Asp Lys Phe Ile Val
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Arg Glu Lys Ser Thr Glu Asn Gln Ile Asp Trp Gly Ala Val Asn Gln
65 70 75 80
Pro Ile Ser Glu Glu Ala Phe Glu Lys Leu Tyr Ala Lys Val Val Ser
85 90 95
Tyr Leu Lys Gln Arg Asp Glu Leu Phe Val Phe Glu Gly Phe Ala Gly
100 105 110
Ala Asp Glu Lys Tyr Arg Leu Pro Ile Thr Val Val Asn Glu Phe Ala
115 120 125
Trp His Asn Leu Phe Ala Arg Gln Leu Phe Ile Arg Pro Glu Thr Asn
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Glu Lys Asp Pro Gln Ala Gln Pro Phe Thr Ile Leu Ser Ala Pro His
145 150 155 160
Phe Lys Ala Asp Pro Glu Thr Asp Gly Thr His Ser Glu Thr Phe Ile
165 170 175
Ile Val Ser Phe Glu Lys Arg Val Ile Leu Ile Gly Gly Thr Glu Tyr
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Ala Gly Glu Met Lys Lys Ser Ile Phe Ser Ile Met Asn Phe Leu Leu
195 200 205
Pro Gln Gln Asp Ile Leu Pro Met His Cys Ser Ala Asn Val Gly Glu
210 215 220
Lys Gly Asp Val Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr
225 230 235 240
Thr Leu Ser Ala Asp Ala Asp Arg Lys Leu Ile Gly Asp Asp Glu His
245 250 255
Gly Trp Ser Asp Ser Gly Val Phe Asn Ile Glu Gly Gly Cys Tyr Ala
260 265 270
Lys Cys Ile Asn Leu Ser Glu Glu Lys Glu Pro Gln Ile Phe His Ala
275 280 285
Ile Arg Phe Gly Ser Val Leu Glu Asn Val Val Leu Asp Glu Lys Thr
290 295 300
Gly Glu Ala Asp Tyr Asp Asn Ser Phe Tyr Thr Glu Asn Thr Arg Ala
305 310 315 320
Ala Tyr Pro Ile Glu Met Ile Gly Asn Ile Val Gln Pro Ser Ile Ala
325 330 335
Gly His Pro Gln Ala Ile Val Phe Leu Thr Ala Asp Ala Phe Gly Val
340 345 350
Leu Pro Thr Ile Ser Lys Leu Thr Lys Glu Gln Ala Met Tyr His Phe
355 360 365
Leu Ser Gly Tyr Thr Ser Lys Leu Ala Gly Thr Glu Arg Gly Val Thr
370 375 380
Ser Pro Gln Thr Thr Phe Ser Thr Cys Phe Gly Ser Pro Phe Leu Pro
385 390 395 400
Leu Pro Ala His Val Tyr Ala Glu Met Leu Gly Lys Lys Ile Asp Glu
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His Gly Val Gln Val Phe Leu Val Asn Thr Gly Trp Thr Gly Gly Gly
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Tyr Gly Val Gly Glu Arg Met Lys Leu Ser Tyr Thr Arg Ala Met Val
435 440 445
Lys Ala Ala Ile Asp Gly Thr Leu Asp Gln Thr Asp Met Thr Ala Asp
450 455 460
Ser Ile Phe Gly Leu His Ser Pro Val His Val Pro Gly Val Pro Asp
465 470 475 480
Glu Val Leu Gln Pro Glu Asn Thr Trp Ser Asp Lys Lys Ala Tyr Asn
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Glu Lys Ala Leu Phe Leu Ala Asn Glu Phe Lys Glu Asn Phe Lys Lys
500 505 510
Phe Ser His Ala Asp Ser Ile Ala Lys Ala Gly Gly Pro Leu Val
515 520 525
<210> 3
<211> 1584
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 3
atgaactcag ttgatttgac cgctgattta caagccttat taacatgtcc aaatgtgcgt 60
cataatttat cagcagcaca gctaacagaa aaagtcctct cccgaaacga aggcatttta 120
acatccacag gtgctgttcg cgcgacaaca ggcgcttaca caggacgctc acctaaagat 180
aaattcatcg tggaggaaga aagcacgaaa aataagatcg attggggccc ggtgaatcag 240
ccgatttcag aagaagcgtt tgagcggctg tacacgaaag ttgtcagcta tttaaaggag 300
cgagatgaac tgtttgtttt cgaaggattt gccggagcag acgagaaata caggctgccg 360
atcactgtcg taaatgagtt cgcatggcac aatttatttg cgcggcagct gtttatccgt 420
ccggaaggaa atgataagaa aacagttgag cagccgttca ccattctttc tgctccgcat 480
ttcaaagcgg atccaaaaac agacggcact cattccgaaa cgtttattat tgtctctttc 540
gaaaagcgga caattttaat cggcggaact gagtatgccg gtgaaatgaa gaagtccatt 600
ttctccatta tgaatttcct gctgcctgaa agagatattt tatctatgca ctgctccgcc 660
aatgtcggtg aaaaaggcga tgtcgccctt ttcttcggac tgtcaggaac aggaaagacc 720
accctgtcgg cagatgctga ccgcaagctg atcggtgacg atgaacatgg ctggtctgat 780
acaggcgtct ttaatattga aggcggatgc tacgctaagt gtattcattt aagcgaggaa 840
aaggagccgc aaatctttaa cgcgatccgc ttcgggtctg ttctcgaaaa tgtcgttgtg 900
gatgaagata cacgcgaagc caattatgat gattccttct atactgaaaa cacgcgggca 960
gcttacccga ttcatatgat taataacatc gtgactccaa gcatggccgg ccatccgtca 1020
gccattgtat ttttgacggc tgatgccttc ggagtcctgc cgacgatcag caaactaacg 1080
aaggagcagg cgatgtacca ttttttgagc ggttacacga gtaagcttgc cggaaccgaa 1140
cgtggtgtca cgtctcctga aacgacgttt tctacatgct tcggctcacc gttcctgccg 1200
cttcctgctc acgtctatgc tgaaatgctc ggcaaaaaga tcgatgaaca cggcgcagac 1260
gttttcttag tcaataccgg atggaccggg ggcggctacg gcacaggcga acgaatgaag 1320
ctttcttaca ctagagcaat ggtcaaagca gcgattgaag gcaaattaga ggatgctgaa 1380
atgataactg acgatatttt cggcctgcac attccggccc atgttcctgg cgttcctgat 1440
catatccttc agcctgaaaa cacgtggacc aacaaggaag aatacaaaga aaaagcagtc 1500
taccttgcaa atgaattcaa agagaacttt aaaaagttcg cacataccga tgccatcgcc 1560
caggcaggcg gccctctcgt ataa 1584
<210> 4
<211> 527
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 4
Met Asn Ser Val Asp Leu Thr Ala Asp Leu Gln Ala Leu Leu Thr Cys
1 5 10 15
Pro Asn Val Arg His Asn Leu Ser Ala Ala Gln Leu Thr Glu Lys Val
20 25 30
Leu Ser Arg Asn Glu Gly Ile Leu Thr Ser Thr Gly Ala Val Arg Ala
35 40 45
Thr Thr Gly Ala Tyr Thr Gly Arg Ser Pro Lys Asp Lys Phe Ile Val
50 55 60
Glu Glu Glu Ser Thr Lys Asn Lys Ile Asp Trp Gly Pro Val Asn Gln
65 70 75 80
Pro Ile Ser Glu Glu Ala Phe Glu Arg Leu Tyr Thr Lys Val Val Ser
85 90 95
Tyr Leu Lys Glu Arg Asp Glu Leu Phe Val Phe Glu Gly Phe Ala Gly
100 105 110
Ala Asp Glu Lys Tyr Arg Leu Pro Ile Thr Val Val Asn Glu Phe Ala
115 120 125
Trp His Asn Leu Phe Ala Arg Gln Leu Phe Ile Arg Pro Glu Gly Asn
130 135 140
Asp Lys Lys Thr Val Glu Gln Pro Phe Thr Ile Leu Ser Ala Pro His
145 150 155 160
Phe Lys Ala Asp Pro Lys Thr Asp Gly Thr His Ser Glu Thr Phe Ile
165 170 175
Ile Val Ser Phe Glu Lys Arg Thr Ile Leu Ile Gly Gly Thr Glu Tyr
180 185 190
Ala Gly Glu Met Lys Lys Ser Ile Phe Ser Ile Met Asn Phe Leu Leu
195 200 205
Pro Glu Arg Asp Ile Leu Ser Met His Cys Ser Ala Asn Val Gly Glu
210 215 220
Lys Gly Asp Val Ala Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr
225 230 235 240
Thr Leu Ser Ala Asp Ala Asp Arg Lys Leu Ile Gly Asp Asp Glu His
245 250 255
Gly Trp Ser Asp Thr Gly Val Phe Asn Ile Glu Gly Gly Cys Tyr Ala
260 265 270
Lys Cys Ile His Leu Ser Glu Glu Lys Glu Pro Gln Ile Phe Asn Ala
275 280 285
Ile Arg Phe Gly Ser Val Leu Glu Asn Val Val Val Asp Glu Asp Thr
290 295 300
Arg Glu Ala Asn Tyr Asp Asp Ser Phe Tyr Thr Glu Asn Thr Arg Ala
305 310 315 320
Ala Tyr Pro Ile His Met Ile Asn Asn Ile Val Thr Pro Ser Met Ala
325 330 335
Gly His Pro Ser Ala Ile Val Phe Leu Thr Ala Asp Ala Phe Gly Val
340 345 350
Leu Pro Thr Ile Ser Lys Leu Thr Lys Glu Gln Ala Met Tyr His Phe
355 360 365
Leu Ser Gly Tyr Thr Ser Lys Leu Ala Gly Thr Glu Arg Gly Val Thr
370 375 380
Ser Pro Glu Thr Thr Phe Ser Thr Cys Phe Gly Ser Pro Phe Leu Pro
385 390 395 400
Leu Pro Ala His Val Tyr Ala Glu Met Leu Gly Lys Lys Ile Asp Glu
405 410 415
His Gly Ala Asp Val Phe Leu Val Asn Thr Gly Trp Thr Gly Gly Gly
420 425 430
Tyr Gly Thr Gly Glu Arg Met Lys Leu Ser Tyr Thr Arg Ala Met Val
435 440 445
Lys Ala Ala Ile Glu Gly Lys Leu Glu Asp Ala Glu Met Ile Thr Asp
450 455 460
Asp Ile Phe Gly Leu His Ile Pro Ala His Val Pro Gly Val Pro Asp
465 470 475 480
His Ile Leu Gln Pro Glu Asn Thr Trp Thr Asn Lys Glu Glu Tyr Lys
485 490 495
Glu Lys Ala Val Tyr Leu Ala Asn Glu Phe Lys Glu Asn Phe Lys Lys
500 505 510
Phe Ala His Thr Asp Ala Ile Ala Gln Ala Gly Gly Pro Leu Val
515 520 525
Claims (10)
1.磷酸烯醇丙酮酸羧激酶突变体,其特征在于,所述突变体包含磷酸烯醇丙酮酸羧激酶第355位氨基酸由P到T的突变;
其中,来自枯草芽孢杆菌(Bacillus subtilis)的磷酸烯醇丙酮酸羧激酶在NCBI上的参考序列编号为BSU_30560;
来自解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的磷酸烯醇丙酮酸羧激酶在NCBI上的参考序列编号为BAMF_2839。
2.编码权利要求1所述突变体的核酸分子或含有所述核酸分子的生物材料;
所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体或工程菌。
3.权利要求2所述核酸分子或所述生物材料的以下任一应用:
(1)用于核苷的发酵生产;
(2)用于提高核苷的发酵产量;
(3)用于构建产核苷的基因工程菌;
其中,所述核苷包括肌苷、鸟苷、腺苷,或它们对应的核苷衍生物。
4.核苷生产菌株的构建方法,其特征在于,利用基因工程手段,在微生物基因组中引入突变,使其编码的磷酸烯醇丙酮酸羧激酶包含P355T突变位点;
其中,来自枯草芽孢杆菌(Bacillus subtilis)的磷酸烯醇丙酮酸羧激酶在NCBI上的参考序列编号为BSU_30560;
来自解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的磷酸烯醇丙酮酸羧激酶在NCBI上的参考序列编号为BAMF_2839。
5.根据权利要求4所述的方法,其特征在于,所述方法包括进一步增强菌株中的pckA基因;
所述增强的途径选自以下1)~6),或任选的组合:
1)通过导入具有所述基因的质粒而增强;
2)通过增加染色体上所述基因的拷贝数而增强;
3)通过改变染色体上所述基因的启动子序列而增强;
4)通过将强启动子与所述基因可操作地连接而增强;
5)通过导入增强子而增强;
6)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强。
6.根据权利要求5所述的方法,其特征在于,将pckA基因的原有启动子替换为强启动子;
所述强启动子选自P43、P41、Plaps。
7.根据权利要求6所述的方法,其特征在于,将pckA基因的拷贝数增加到二拷贝。
8.根据权利要求4-7任一项所述的方法,其特征在于,所述微生物为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillus subtilis)、短小芽孢杆菌(Bacillus pumilus)或大肠杆菌(Escherichia coli)。
9.按照权利要求4-8任一项所述方法构建得到的产核苷的菌株。
10.一种生产核苷的方法,其特征在于,所述方法包括如下步骤:
1)培养权利要求9所述菌株,以获得微生物的培养物;
2)从步骤1)中获得的所述培养物中收集所产生的核苷;
其中,所述核苷包括肌苷、鸟苷、腺苷,或它们对应的核苷衍生物。
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