CN117165501A - 产核苷及其衍生物的重组菌及其构建方法与应用 - Google Patents
产核苷及其衍生物的重组菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了产核苷及其衍生物的重组菌及其构建方法与应用。本发明通过使微生物中的全局转录调控因子CcpA活性减弱，使微生物能够高效地生成鸟嘌呤、次黄嘌呤或腺嘌呤核苷。解淀粉芽孢杆菌突变菌株8344、8345、8346和8347与出发菌相比，鸟苷产量由1.1g/L提高至3.2g/L、1.8g/L、1.9g/L和2.8g/L。肌苷产量由0.5g/L提高至1.5g/L、1.0g/L、1.0g/L和1.3g/L。枯草芽孢杆菌突变菌株A0004、A0005、A0006和A0007与出发菌相比，腺苷产量由8.9g/L提高至10.8g/L、10.6g/L、11.3g/L和11.9g/L，应用前景广阔。
Description
技术领域
本发明属于微生物工程技术领域，具体地说，涉及一种产核苷及其衍生物的重组菌及其构建方法与应用。
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，不能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一种产核苷及其衍生物的重组菌及其构建方法与应用。
为了实现本发明目的，第一方面，本发明提供一种修饰的微生物，所述微生物相比于未修饰的微生物，其全局转录调控因子CcpA的活性降低或丧失，且所述微生物相比于未修饰的微生物具有增强的核苷生产能力。
本发明中，所述全局转录调控因子CcpA在NCBI上的参考序列编号为BAMF_2774(解淀粉芽孢杆菌)或BSU_29740(枯草芽孢杆菌)。
进一步地，微生物中的全局转录调控因子CcpA的活性降低或丧失可以由选自以下1)-5)，或任选的组合实现的：
1)通过改变所述全局转录调控因子CcpA的编码基因的启动子序列而降低或丧失；
2)通过改变所述全局转录调控因子CcpA的编码基因的核糖体结合位点而降低或丧失；
3)通过改变所述全局转录调控因子CcpA的氨基酸序列而降低或丧失；
4)通过改变编码所述全局转录调控因子CcpA的核苷酸序列而降低或丧失；
5)通过敲除所述全局转录调控因子CcpA的编码序列而丧失。
可以采用诱变、定点突变或同源重组的方法来降低所述全局转录调控因子CcpA的编码基因的表达或敲除内源的所述全局转录调控因子CcpA的编码基因。
优选地，全局转录调控因子CcpA活性降低或丧失是通过如下A～D的至少一种方式来实现的：
A、全局转录调控因子CcpA的第95位氨基酸由I突变为M；
B、全局转录调控因子CcpA的第150位氨基酸由E突变为K；
C、全局转录调控因子CcpA的第205位氨基酸由L突变为P；
D、全局转录调控因子CcpA的编码基因的起始密码子由gtg或atg突变为ttg。
本发明中，所述微生物可以是芽孢杆菌属(Bacillus)或埃希氏菌属(Escherichia)菌种，如解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillus subtilis)、短小芽孢杆菌(Bacillus pumilus)、大肠杆菌(Escherichia coli)等。
第二方面，本发明提供一种产核苷及其衍生物的重组菌的构建方法，利用基因工程手段，失活或弱化具有核苷生产能力的微生物中的全局转录调控因子CcpA的编码基因。
所述弱化可以选自以下1)-5)，或任选的组合实现的：
1)通过改变所述全局转录调控因子CcpA的编码基因的启动子序列而降低或丧失；
2)通过改变所述全局转录调控因子CcpA的编码基因的核糖体结合位点而降低或丧失；
3)通过改变所述全局转录调控因子CcpA的氨基酸序列而降低或丧失；
4)通过改变编码所述全局转录调控因子CcpA的核苷酸序列而降低或丧失；
5)通过敲除所述全局转录调控因子CcpA的编码序列而丧失；
弱化的方法可以选自诱变、定点突变、同源重组中的至少一种。
优选地，弱化是通过如下A～D的至少一种方式来实现的：
A、全局转录调控因子CcpA的第95位氨基酸由I突变为M；
B、全局转录调控因子CcpA的第150位氨基酸由E突变为K；
C、全局转录调控因子CcpA的第205位氨基酸由L突变为P；
D、全局转录调控因子CcpA的编码基因的起始密码子由gtg或atg突变为ttg；
第三方面，本发明提供按照所述方法构建得到的重组菌。
第四方面，本发明提供所述修饰的微生物或所述重组菌在核苷及其衍生物的发酵生产或提高核苷及其衍生物的发酵产量中的应用。
所述核苷及其衍生物包括但不限于肌苷、鸟苷、腺苷、次黄嘌呤、肌苷酸、鸟嘌呤、腺嘌呤、鸟苷酸、核黄素、二乙酰鸟苷酸、腺苷酸、阿糖腺苷等。
第五方面，本发明提供一种生产核苷的方法，所述方法包括如下步骤：
a)培养所述修饰的微生物或所述重组菌，获得培养物；
b)从步骤a)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：
本发明通过使微生物中的全局转录调控因子CcpA活性减弱，使得微生物能够高效率、高速度地生成鸟嘌呤核苷、次黄嘌呤核苷或腺嘌呤核苷，突变菌株B.a 8344(含ccpAI95M点突变)、B.a 8345(含ccpAE150K点突变)、B.a 8346(含ccpAL205P点突变)和B.a 8347(ccpA起始密码子弱化)与出发菌相比，鸟苷产量由1.1g/L提高至3.2g/L、1.8g/L、1.9g/L和2.8g/L。肌苷产量由0.5g/L分别提高至1.5g/L、1.0g/L、1.0g/L和1.3g/L。突变菌株B.subtilisA0004(含ccpAI95M突变)、B.subtilis A0005(含ccpAE150K突变)、B.subtilis A0006(含ccpAL205P突变)和B.subtilis A0007(ccpA起始密码子弱化)与出发菌相比，腺苷产量由8.9g/L提高至10.8g/L、10.6g/L、11.3g/L和11.9g/L。本发明为大规模生产核苷提供有效手段，应用前景广阔。
具体实施方式
本发明提供一种利用微生物生产嘌呤核苷的方法，并成功创制了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全局转录调控因子CcpA(由ccpA基因编码)，使得CcpA蛋白活性减弱，使得微生物能够高效率、高速度地生成鸟嘌呤核苷或次黄嘌呤核苷，并且成功创制出能够高效生产核苷的新微生物，从而完成本发明。
CcpA是碳分解代谢物抑制(CCR)和碳分解代谢物活化(CCA)的全局转录调控因子，可确保在不同条件下实现最佳能量使用。与P-Ser-HPr或P-Ser-Crh相互作用，导致在分解代谢物反应元件(cre)处形成与DNA结合的复合物。与DNA结合可以激活或抑制许多不同的基因和操纵子。
目前尚无有关弱化CcpA表达可提高微生物产核苷能力的相关报道。本发明首次在解淀粉芽孢杆菌中通过突变该基因使得氨基酸序列发生改变或弱化其起始密码子，获得相应突变体可高效生产核苷，并成功构建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，其细胞内编码全局转录调控因子CcpA的氨基酸序列发生变化(如CcpA蛋白的第95位氨基酸由I突变为M和/或第150位氨基酸由E突变为K和/或第205位氨基酸由L突变为P)，和/或ccpA起始密码子由gtg或atg突变为ttg，使得所述菌株产生核苷的能力与未修饰的菌株相比增强。
上述突变位点可应用于解淀粉芽孢杆菌、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、腺嘌呤、腺苷酸、阿糖腺苷等。
本发明提供全局转录调控因子CcpA突变体的序列，解淀粉芽孢杆菌中分别如SEQID NO:2、4、6和8所示，其编码核酸序列分别如SEQ ID NO:1、3、5和7所示，野生型全局转录调控因子CcpA的氨基酸序列如SEQ ID NO:9所示；枯草芽孢杆菌中突变体序列分别如SEQID NO:10、12、14和16所示，其编码核酸序列分别如SEQ ID NO:11、13、15和17所示，野生型全局转录调控因子CcpA的氨基酸序列如SEQ ID NO:18所示。
本发明提供通过基因修饰使得ccpA活性减弱，弱化方式包括氨基酸突变、启动子弱化、RBS弱化、起始密码子弱化，包括但不限于上述4种弱化方式。
本发明提供弱化后的ccpA应用于生产鸟苷及鸟苷相关衍生物，如鸟嘌呤、鸟苷酸、核黄素、二乙酰鸟苷酸等。
本发明提供弱化的ccpA应用于生产肌苷，及肌苷相关衍生物，如肌苷酸、鸟苷酸、腺苷酸等。
本发明提供弱化后的ccpA应用于生产腺苷及腺苷相关衍生物，如腺嘌呤、腺苷酸、阿糖腺苷等。
本发明提供将弱化的ccpA应用于枯草芽孢杆菌、解淀粉芽孢杆菌、短小芽孢杆菌、大肠杆菌等微生物中。
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。
以下实施例中涉及的引物名称及引物序列如表1所示：
表1引物名称及序列信息
| 引物名称 | 引物序列(5′-3′) |
| ccpA95-1r | ggtctgaattgcttaacataatatt |
| ccpA95-2f | aatattatgttaagcaattcagacc |
| ccpA150-1f | acaaaataaggatcctctagagcataatacagtgttccgctc |
| ccpA150-1r | gccctgttctttaactgacgcagc |
| ccpA150-2f | gctgcgtcagttaaagaacagggc |
| ccpA150-2r | gccaagcttgcatgcctgcagctgataaacaccgctgcggca |
| ccpA205-1r | cccgcttcttcaggcgcccgtttgt |
| ccpA205-2f | acaaacgggcgcctgaagaagcggg |
| ccpA-ttg-1f | acaaaataaggatcctctagagcgattgattacattgacgtc |
| ccpA-ttg-1r | tatatcgtaatgttgctcaatgaaaccactcct |
| ccpA-ttg-2f | aggagtggtttcattgagcaacattacgatata |
| ccpA-ttg-2r | gccaagcttgcatgcctgcagcgccgtcggtttccggtcaag |
| BS0004-1f | acaaaataaggatcctctagacgtaatggctgaggttcaccctg |
| BS0004-1r | gctctttctccatgttttggtcagagttgctcaaCataatatt |
| BS0004-2f | atattatGttgagcaactctgaccaaaacatggagaaagagctgca |
| BS0004-2r | gccaagcttgcatgcctgcagaacaggtgtaagataatcaaaac |
| BS0005-1r | ttcctcctgctcttTtacagaagcggca |
| BS0005-2f | tgccgcttctgtaAaagagcaggaggaa |
| BS0006-1r | aggttcgcttcttcaGgcgcacgtttg |
| BS0006-2f | acaaacgtgcgcCtgaagaagcgaacct |
| BS0007-1f | acaaaataaggatcctctagacagaacatatgaaacagcaacg |
| BS0007-1r | tagatcgtaatattgctcaAcctaaaaccactccttt |
| BS0007-2f | aggagtggttttaggTtgagcaatattacgatctacgat |
| BS0007-2r | gccaagcttgcatgcctgcagacgaacgcgatgtctgtatgtcctt |
实施例1解淀粉芽孢杆菌中ccpAI95M点突变菌株的构建
以B.a 836菌株(参见CN112574934A)基因组为模板，使用ccpA150-1f/ccpA95-1r，ccpA95-2f/ccpA150-2r引物对，pfu高保真DNA聚合酶扩增获得ccpA的上、下游同源臂。获得片段进行胶回收并融合，扩增获得ccpAI95M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 LL3and its 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ccpAI95M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-ccpAI95M。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ccpA点突变菌株，命名为B.a 8344。
实施例2解淀粉芽孢杆菌ccpAE150K点突变菌株的构建
以B.a 836菌株(参见CN112574934A)基因组为模板，使用ccpA150-1f/ccpA150-1r，ccpA150-2f/ccpA150-2r引物对，pfu高保真DNA聚合酶扩增获得ccpA的上、下游同源臂。获得片段进行胶回收并融合，扩增获得全长片段ccpAE150K，进行胶回收(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中的构建方式构建获得质粒pKSU-ccpAE150K，并将其转化至B.a 836菌株中，获得的菌株命名为B.a 8345。
实施例3解淀粉芽孢杆菌ccpAL205P点突变菌株的构建
以B.a 836菌株(参见CN112574934A)基因组为模板，使用ccpA150-1f/ccpA205-1r，ccpA205-2f/ccpA150-2r引物对，pfu高保真DNA聚合酶扩增获得ccpA的上、下游同源臂。获得片段进行胶回收并融合，扩增获得ccpAL205P全长片段，进行胶回收(对应的ORF框的核苷酸序列见SEQ ID NO:5，氨基酸序列见SEQID NO:6)，按照实施例1中的构建方式构建获得质粒pKSU-ccpAL205P，并将其转化至B.a 836菌株中，获得的菌株命名为B.a 8346。
实施例4解淀粉芽孢杆菌ccpA起始密码子弱化菌株的构建
分别使用引物对ccpA-ttg-1f/ccpA-ttg-1r，ccpA-ttg-2f/ccpA-ttg-2r，B.a8341菌株基因组为模板，扩增获得左同源臂ccpA-ttg-L、ccpA-ttg-R片段并融合。获得ccpA-ttg全长片段(对应的ORF框的核苷酸序列见SEQ ID NO:7，氨基酸序列见SEQ ID NO:8)。按照实施例1中质粒的构建方法构建获得pKSU-ccpA-ttg质粒，将其转化至B.a 8341菌株中，筛选获得ccpA起始密码子弱化菌株，命名为B.a 8347。
实施例5枯草芽孢杆菌中ccpAI95M点突变菌株的构建
为确定ccpA的突变是否能提高腺苷产量，我们将ccpAI95M点突变引入腺苷生产菌株B.subtilis A5(菌株B.subtilis A5参见CN110257315B)中。以B.subtilis A5菌株基因组为模板，使用BS0004-1f/1r，BS0004-2f/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0004-ccpAI95M片段(对应的ORF框的核苷酸序列见SEQ ID NO:10，氨基酸序列见SEQ ID NO:11)。按照实施例1中的方法构建出重组质粒pKSU-0004-ccpAI95M。转化至B.subtilis A5菌株中，按照实施例1中的筛选方法，筛选获得含有突变菌株，命名为B.subtilis A0004。
实施例6枯草芽孢杆菌中ccpAE150K点突变菌株的构建
按照实施例5中的方法构建获得pKSU-0005-ccpAE150K质粒，使用的引物对为BS0005-1f/1r，BS0005-2f/2r。对应的ORF框的核苷酸和氨基酸序列分别见SEQ ID NO:12和SEQ ID NO:13，构建获得的菌株命名为B.subtilis A0005。
实施例7枯草芽孢杆菌中ccpAL205P点突变菌株的构建
按照实施例5中方法构建获得pKSU-0006-ccpAL205P质粒，使用的引物对为BS0006-1f/1r，BS0006-2f/2r。对应的ORF框的核苷酸和氨基酸序列分别见SEQ ID NO:14和SEQ IDNO:15，构建获得的菌株命名为B.subtilis A0006。
实施例8 ccpA起始密码子弱化菌株的构建
按照实施例5中方法构建获得pKSU-0007-ccpAttg质粒，使用的引物对为BS0007-1f/1r，BS0007-2f/2r。对应的ORF框的核苷酸和氨基酸序列分别见SEQ ID NO:16和SEQ IDNO:17，构建获得的菌株命名为B.subtilis A0007。
实施例9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，摇床转速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3.5g/L，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，35℃培养70h。
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)
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> 产核苷及其衍生物的重组菌及其构建方法与应用
<130> KHP221113209.1
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gtgagcaaca ttacgatata cgatgtggcc agagaagcaa atgtcagcat ggcgactgtt 60
tcccgcgtcg taaacggcaa tccgaatgta aaaccgacga ccagaaaaaa agtattggaa 120
gccattgacc gcctcggcta ccgcccgaac gcggtggcaa gaggactggc aagcaaaaaa 180
acgactacgg tcggcgtcat catccctgat atctcaagta ttttttacgc ggaacttgcg 240
cggggaattg aagatatcgc gacgatgtac aaatataata ttatgttaag caattcagac 300
caaaatctcg agaaggaact gcacctgtta aatacgatgc tcggtaaaca agtagacggt 360
atcgtattta tgggcggcaa cattacggac gagcatgtcg aagaatttaa gcgctctccc 420
gttccgatcg tattggctgc gtcagttgaa gaacagggcg agacgccttc tgtcgccatt 480
gattatgaac aggcgattta tgatgccgtc aagcttttca ttgataaggg gcatcgcgat 540
atcgcatttg tctccggacc gatggcagag ccgatcaacc gttcgaaaaa actgcaaggg 600
tacaaacggg cgcttgaaga agcgggcatt gcgtttaacg agcagttcgt ggcggaaggc 660
gattacacct atgattccgg gatggaggcg cttcaaagct tgatgggcct tgaccggaaa 720
ccgacggcga tcctttctgc gacggatgaa atggctctcg gcattattca tgcggcccag 780
gatcagggta tgtcgattcc tgacgatctt gacattatcg gttttgacaa tacgagattg 840
agcctgatgg tccgccctca gctttcaaca gtcgtgcagc ctacgtacga tatcggcgcc 900
gtggcgatga gacttttgac aaaactgatg aacaaagagc cggttgaaga gcatatcgtt 960
gaattgcctc accgcatcga attaagacaa tcaaccaagt cataa 1005
<210> 2
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Val Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Asp Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ala Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Met Leu
85 90 95
Ser Asn Ser Asp Gln Asn Leu Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Glu Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Gly Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Phe Ile Asp Lys
165 170 175
Gly His Arg Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Gly Ile Ala Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Met Glu Ala Leu Gln Ser Leu Met Gly Leu Asp Arg Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Met Ser Ile Pro Asp Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Gln Ser Thr Lys Ser
325 330
<210> 3
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtgagcaaca ttacgatata cgatgtggcc agagaagcaa atgtcagcat ggcgactgtt 60
tcccgcgtcg taaacggcaa tccgaatgta aaaccgacga ccagaaaaaa agtattggaa 120
gccattgacc gcctcggcta ccgcccgaac gcggtggcaa gaggactggc aagcaaaaaa 180
acgactacgg tcggcgtcat catccctgat atctcaagta ttttttacgc ggaacttgcg 240
cggggaattg aagatatcgc gacgatgtac aaatataata ttattttaag caattcagac 300
caaaatctcg agaaggaact gcacctgtta aatacgatgc tcggtaaaca agtagacggt 360
atcgtattta tgggcggcaa cattacggac gagcatgtcg aagaatttaa gcgctctccc 420
gttccgatcg tattggctgc gtcagttaaa gaacagggcg agacgccttc tgtcgccatt 480
gattatgaac aggcgattta tgatgccgtc aagcttttca ttgataaggg gcatcgcgat 540
atcgcatttg tctccggacc gatggcagag ccgatcaacc gttcgaaaaa actgcaaggg 600
tacaaacggg cgcttgaaga agcgggcatt gcgtttaacg agcagttcgt ggcggaaggc 660
gattacacct atgattccgg gatggaggcg cttcaaagct tgatgggcct tgaccggaaa 720
ccgacggcga tcctttctgc gacggatgaa atggctctcg gcattattca tgcggcccag 780
gatcagggta tgtcgattcc tgacgatctt gacattatcg gttttgacaa tacgagattg 840
agcctgatgg tccgccctca gctttcaaca gtcgtgcagc ctacgtacga tatcggcgcc 900
gtggcgatga gacttttgac aaaactgatg aacaaagagc cggttgaaga gcatatcgtt 960
gaattgcctc accgcatcga attaagacaa tcaaccaagt cataa 1005
<210> 4
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Val Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Asp Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ala Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Leu Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Glu Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Lys Glu Gln Gly Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Phe Ile Asp Lys
165 170 175
Gly His Arg Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Gly Ile Ala Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Met Glu Ala Leu Gln Ser Leu Met Gly Leu Asp Arg Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Met Ser Ile Pro Asp Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Gln Ser Thr Lys Ser
325 330
<210> 5
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtgagcaaca ttacgatata cgatgtggcc agagaagcaa atgtcagcat ggcgactgtt 60
tcccgcgtcg taaacggcaa tccgaatgta aaaccgacga ccagaaaaaa agtattggaa 120
gccattgacc gcctcggcta ccgcccgaac gcggtggcaa gaggactggc aagcaaaaaa 180
acgactacgg tcggcgtcat catccctgat atctcaagta ttttttacgc ggaacttgcg 240
cggggaattg aagatatcgc gacgatgtac aaatataata ttattttaag caattcagac 300
caaaatctcg agaaggaact gcacctgtta aatacgatgc tcggtaaaca agtagacggt 360
atcgtattta tgggcggcaa cattacggac gagcatgtcg aagaatttaa gcgctctccc 420
gttccgatcg tattggctgc gtcagttgaa gaacagggcg agacgccttc tgtcgccatt 480
gattatgaac aggcgattta tgatgccgtc aagcttttca ttgataaggg gcatcgcgat 540
atcgcatttg tctccggacc gatggcagag ccgatcaacc gttcgaaaaa actgcaaggg 600
tacaaacggg cgcctgaaga agcgggcatt gcgtttaacg agcagttcgt ggcggaaggc 660
gattacacct atgattccgg gatggaggcg cttcaaagct tgatgggcct tgaccggaaa 720
ccgacggcga tcctttctgc gacggatgaa atggctctcg gcattattca tgcggcccag 780
gatcagggta tgtcgattcc tgacgatctt gacattatcg gttttgacaa tacgagattg 840
agcctgatgg tccgccctca gctttcaaca gtcgtgcagc ctacgtacga tatcggcgcc 900
gtggcgatga gacttttgac aaaactgatg aacaaagagc cggttgaaga gcatatcgtt 960
gaattgcctc accgcatcga attaagacaa tcaaccaagt cataa 1005
<210> 6
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Val Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Asp Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ala Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Leu Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Glu Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Gly Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Phe Ile Asp Lys
165 170 175
Gly His Arg Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Pro Glu Glu Ala
195 200 205
Gly Ile Ala Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Met Glu Ala Leu Gln Ser Leu Met Gly Leu Asp Arg Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Met Ser Ile Pro Asp Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Gln Ser Thr Lys Ser
325 330
<210> 7
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttgagcaaca ttacgatata cgatgtggcc agagaagcaa atgtcagcat ggcgactgtt 60
tcccgcgtcg taaacggcaa tccgaatgta aaaccgacga ccagaaaaaa agtattggaa 120
gccattgacc gcctcggcta ccgcccgaac gcggtggcaa gaggactggc aagcaaaaaa 180
acgactacgg tcggcgtcat catccctgat atctcaagta ttttttacgc ggaacttgcg 240
cggggaattg aagatatcgc gacgatgtac aaatataata ttattttaag caattcagac 300
caaaatctcg agaaggaact gcacctgtta aatacgatgc tcggtaaaca agtagacggt 360
atcgtattta tgggcggcaa cattacggac gagcatgtcg aagaatttaa gcgctctccc 420
gttccgatcg tattggctgc gtcagttaaa gaacagggcg agacgccttc tgtcgccatt 480
gattatgaac aggcgattta tgatgccgtc aagcttttca ttgataaggg gcatcgcgat 540
atcgcatttg tctccggacc gatggcagag ccgatcaacc gttcgaaaaa actgcaaggg 600
tacaaacggg cgcttgaaga agcgggcatt gcgtttaacg agcagttcgt ggcggaaggc 660
gattacacct atgattccgg gatggaggcg cttcaaagct tgatgggcct tgaccggaaa 720
ccgacggcga tcctttctgc gacggatgaa atggctctcg gcattattca tgcggcccag 780
gatcagggta tgtcgattcc tgacgatctt gacattatcg gttttgacaa tacgagattg 840
agcctgatgg tccgccctca gctttcaaca gtcgtgcagc ctacgtacga tatcggcgcc 900
gtggcgatga gacttttgac aaaactgatg aacaaagagc cggttgaaga gcatatcgtt 960
gaattgcctc accgcatcga attaagacaa tcaaccaagt cataa 1005
<210> 8
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Leu Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Asp Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ala Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Leu Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Glu Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Lys Glu Gln Gly Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Phe Ile Asp Lys
165 170 175
Gly His Arg Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Gly Ile Ala Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Met Glu Ala Leu Gln Ser Leu Met Gly Leu Asp Arg Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Met Ser Ile Pro Asp Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Gln Ser Thr Lys Ser
325 330
<210> 9
<211> 334
<212> PRT
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 9
Val Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Asp Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ala Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Leu Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Glu Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Gly Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Phe Ile Asp Lys
165 170 175
Gly His Arg Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Gly Ile Ala Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Met Glu Ala Leu Gln Ser Leu Met Gly Leu Asp Arg Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Met Ser Ile Pro Asp Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Gln Ser Thr Lys Ser
325 330
<210> 10
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgagcaata ttacgatcta cgatgtagcg agagaagcta atgtaagcat ggcaaccgtt 60
tcccgtgtcg tgaacggcaa cccgaatgta aaaccgacaa cgaggaaaaa agtcttggaa 120
gccattgaac gtctcggtta ccgtccaaac gcggtggcaa gagggctggc aagtaaaaaa 180
acaacaactg taggtgtcat cattcccgat atctcaagca ttttctattc agagcttgcg 240
cgcggaattg aagatatcgc gacaatgtat aaatacaata ttatgttgag caactctgac 300
caaaacatgg agaaagagct gcacttgtta aacacaatgc tcggcaaaca agtggacggc 360
atcgtgttta tgggcggaaa cattacggac gagcatgtgg cggaatttaa gcgttctcca 420
gtgccgattg tacttgccgc ttctgtagaa gagcaggagg aaacaccgtc agtcgctatc 480
gattacgaac aggcgattta tgatgccgtg aagcttttgg ttgataaagg acatacagac 540
atcgcgttcg tttccggacc aatggcagaa ccgatcaacc gttcgaaaaa actccaaggc 600
tacaaacgtg cgcttgaaga agcgaacctt ccgtttaatg aacaatttgt agctgaaggg 660
gattacacat atgattccgg actcgaagca ctgcagcatc tgatgagcct ggataaaaaa 720
ccgacagcca ttctttctgc aactgatgaa atggcactcg gcattatcca tgccgctcag 780
gatcagggct tatccattcc ggaggatctc gacattatcg gttttgataa tacaagatta 840
agcctcatgg ttcgtcctca gctttcaaca gttgttcagc cgacatatga tatcggcgcc 900
gttgcgatga gactgctgac gaagctcatg aataaagagc cggttgaaga gcatatcgtc 960
gaactgccgc accgtataga gcttagaaag tcaaccaagt cataa 1005
<210> 11
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Glu Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ser Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Met Leu
85 90 95
Ser Asn Ser Asp Gln Asn Met Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Ala Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Glu Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Leu Val Asp Lys
165 170 175
Gly His Thr Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Asn Leu Pro Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Leu Glu Ala Leu Gln His Leu Met Ser Leu Asp Lys Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Leu Ser Ile Pro Glu Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Lys Ser Thr Lys Ser
325 330
<210> 12
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgagcaata ttacgatcta cgatgtagcg agagaagcta atgtaagcat ggcaaccgtt 60
tcccgtgtcg tgaacggcaa cccgaatgta aaaccgacaa cgaggaaaaa agtcttggaa 120
gccattgaac gtctcggtta ccgtccaaac gcggtggcaa gagggctggc aagtaaaaaa 180
acaacaactg taggtgtcat cattcccgat atctcaagca ttttctattc agagcttgcg 240
cgcggaattg aagatatcgc gacaatgtat aaatacaata ttattttgag caactctgac 300
caaaacatgg agaaagagct gcacttgtta aacacaatgc tcggcaaaca agtggacggc 360
atcgtgttta tgggcggaaa cattacggac gagcatgtgg cggaatttaa gcgttctcca 420
gtgccgattg tacttgccgc ttctgtaaaa gagcaggagg aaacaccgtc agtcgctatc 480
gattacgaac aggcgattta tgatgccgtg aagcttttgg ttgataaagg acatacagac 540
atcgcgttcg tttccggacc aatggcagaa ccgatcaacc gttcgaaaaa actccaaggc 600
tacaaacgtg cgcttgaaga agcgaacctt ccgtttaatg aacaatttgt agctgaaggg 660
gattacacat atgattccgg actcgaagca ctgcagcatc tgatgagcct ggataaaaaa 720
ccgacagcca ttctttctgc aactgatgaa atggcactcg gcattatcca tgccgctcag 780
gatcagggct tatccattcc ggaggatctc gacattatcg gttttgataa tacaagatta 840
agcctcatgg ttcgtcctca gctttcaaca gttgttcagc cgacatatga tatcggcgcc 900
gttgcgatga gactgctgac gaagctcatg aataaagagc cggttgaaga gcatatcgtc 960
gaactgccgc accgtataga gcttagaaag tcaaccaagt cataa 1005
<210> 13
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Glu Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ser Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Met Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Ala Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Lys Glu Gln Glu Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Leu Val Asp Lys
165 170 175
Gly His Thr Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Asn Leu Pro Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Leu Glu Ala Leu Gln His Leu Met Ser Leu Asp Lys Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Leu Ser Ile Pro Glu Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Lys Ser Thr Lys Ser
325 330
<210> 14
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgagcaata ttacgatcta cgatgtagcg agagaagcta atgtaagcat ggcaaccgtt 60
tcccgtgtcg tgaacggcaa cccgaatgta aaaccgacaa cgaggaaaaa agtcttggaa 120
gccattgaac gtctcggtta ccgtccaaac gcggtggcaa gagggctggc aagtaaaaaa 180
acaacaactg taggtgtcat cattcccgat atctcaagca ttttctattc agagcttgcg 240
cgcggaattg aagatatcgc gacaatgtat aaatacaata ttattttgag caactctgac 300
caaaacatgg agaaagagct gcacttgtta aacacaatgc tcggcaaaca agtggacggc 360
atcgtgttta tgggcggaaa cattacggac gagcatgtgg cggaatttaa gcgttctcca 420
gtgccgattg tacttgccgc ttctgtagaa gagcaggagg aaacaccgtc agtcgctatc 480
gattacgaac aggcgattta tgatgccgtg aagcttttgg ttgataaagg acatacagac 540
atcgcgttcg tttccggacc aatggcagaa ccgatcaacc gttcgaaaaa actccaaggc 600
tacaaacgtg cgcctgaaga agcgaacctt ccgtttaatg aacaatttgt agctgaaggg 660
gattacacat atgattccgg actcgaagca ctgcagcatc tgatgagcct ggataaaaaa 720
ccgacagcca ttctttctgc aactgatgaa atggcactcg gcattatcca tgccgctcag 780
gatcagggct tatccattcc ggaggatctc gacattatcg gttttgataa tacaagatta 840
agcctcatgg ttcgtcctca gctttcaaca gttgttcagc cgacatatga tatcggcgcc 900
gttgcgatga gactgctgac gaagctcatg aataaagagc cggttgaaga gcatatcgtc 960
gaactgccgc accgtataga gcttagaaag tcaaccaagt cataa 1005
<210> 15
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Glu Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ser Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Met Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Ala Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Glu Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Leu Val Asp Lys
165 170 175
Gly His Thr Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Pro Glu Glu Ala
195 200 205
Asn Leu Pro Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Leu Glu Ala Leu Gln His Leu Met Ser Leu Asp Lys Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Leu Ser Ile Pro Glu Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Lys Ser Thr Lys Ser
325 330
<210> 16
<211> 1005
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttgagcaata ttacgatcta cgatgtagcg agagaagcta atgtaagcat ggcaaccgtt 60
tcccgtgtcg tgaacggcaa cccgaatgta aaaccgacaa cgaggaaaaa agtcttggaa 120
gccattgaac gtctcggtta ccgtccaaac gcggtggcaa gagggctggc aagtaaaaaa 180
acaacaactg taggtgtcat cattcccgat atctcaagca ttttctattc agagcttgcg 240
cgcggaattg aagatatcgc gacaatgtat aaatacaata ttattttgag caactctgac 300
caaaacatgg agaaagagct gcacttgtta aacacaatgc tcggcaaaca agtggacggc 360
atcgtgttta tgggcggaaa cattacggac gagcatgtgg cggaatttaa gcgttctcca 420
gtgccgattg tacttgccgc ttctgtagaa gagcaggagg aaacaccgtc agtcgctatc 480
gattacgaac aggcgattta tgatgccgtg aagcttttgg ttgataaagg acatacagac 540
atcgcgttcg tttccggacc aatggcagaa ccgatcaacc gttcgaaaaa actccaaggc 600
tacaaacgtg cgcttgaaga agcgaacctt ccgtttaatg aacaatttgt agctgaaggg 660
gattacacat atgattccgg actcgaagca ctgcagcatc tgatgagcct ggataaaaaa 720
ccgacagcca ttctttctgc aactgatgaa atggcactcg gcattatcca tgccgctcag 780
gatcagggct tatccattcc ggaggatctc gacattatcg gttttgataa tacaagatta 840
agcctcatgg ttcgtcctca gctttcaaca gttgttcagc cgacatatga tatcggcgcc 900
gttgcgatga gactgctgac gaagctcatg aataaagagc cggttgaaga gcatatcgtc 960
gaactgccgc accgtataga gcttagaaag tcaaccaagt cataa 1005
<210> 17
<211> 334
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Leu Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Glu Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ser Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Met Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Ala Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Glu Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Leu Val Asp Lys
165 170 175
Gly His Thr Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Asn Leu Pro Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Leu Glu Ala Leu Gln His Leu Met Ser Leu Asp Lys Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Leu Ser Ile Pro Glu Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Lys Ser Thr Lys Ser
325 330
<210> 18
<211> 334
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 18
Met Ser Asn Ile Thr Ile Tyr Asp Val Ala Arg Glu Ala Asn Val Ser
1 5 10 15
Met Ala Thr Val Ser Arg Val Val Asn Gly Asn Pro Asn Val Lys Pro
20 25 30
Thr Thr Arg Lys Lys Val Leu Glu Ala Ile Glu Arg Leu Gly Tyr Arg
35 40 45
Pro Asn Ala Val Ala Arg Gly Leu Ala Ser Lys Lys Thr Thr Thr Val
50 55 60
Gly Val Ile Ile Pro Asp Ile Ser Ser Ile Phe Tyr Ser Glu Leu Ala
65 70 75 80
Arg Gly Ile Glu Asp Ile Ala Thr Met Tyr Lys Tyr Asn Ile Ile Leu
85 90 95
Ser Asn Ser Asp Gln Asn Met Glu Lys Glu Leu His Leu Leu Asn Thr
100 105 110
Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Gly Gly Asn Ile
115 120 125
Thr Asp Glu His Val Ala Glu Phe Lys Arg Ser Pro Val Pro Ile Val
130 135 140
Leu Ala Ala Ser Val Glu Glu Gln Glu Glu Thr Pro Ser Val Ala Ile
145 150 155 160
Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Val Lys Leu Leu Val Asp Lys
165 170 175
Gly His Thr Asp Ile Ala Phe Val Ser Gly Pro Met Ala Glu Pro Ile
180 185 190
Asn Arg Ser Lys Lys Leu Gln Gly Tyr Lys Arg Ala Leu Glu Glu Ala
195 200 205
Asn Leu Pro Phe Asn Glu Gln Phe Val Ala Glu Gly Asp Tyr Thr Tyr
210 215 220
Asp Ser Gly Leu Glu Ala Leu Gln His Leu Met Ser Leu Asp Lys Lys
225 230 235 240
Pro Thr Ala Ile Leu Ser Ala Thr Asp Glu Met Ala Leu Gly Ile Ile
245 250 255
His Ala Ala Gln Asp Gln Gly Leu Ser Ile Pro Glu Asp Leu Asp Ile
260 265 270
Ile Gly Phe Asp Asn Thr Arg Leu Ser Leu Met Val Arg Pro Gln Leu
275 280 285
Ser Thr Val Val Gln Pro Thr Tyr Asp Ile Gly Ala Val Ala Met Arg
290 295 300
Leu Leu Thr Lys Leu Met Asn Lys Glu Pro Val Glu Glu His Ile Val
305 310 315 320
Glu Leu Pro His Arg Ile Glu Leu Arg Lys Ser Thr Lys Ser
325 330
Claims (10)
1.一种修饰的微生物，其特征在于，所述微生物相比于未修饰的微生物，其全局转录调控因子CcpA的活性降低或丧失，且所述微生物相比于未修饰的微生物具有增强的核苷生产能力；
其中，所述全局转录调控因子CcpA在NCBI上的参考序列编号为BAMF_2774或BSU_29740。
2.根据权利要求1所述的微生物，其特征在于，微生物中的全局转录调控因子CcpA的活性降低或丧失是由选自以下1)-5)，或任选的组合实现的：
1)通过改变所述全局转录调控因子CcpA的编码基因的启动子序列而降低或丧失；
2)通过改变所述全局转录调控因子CcpA的编码基因的核糖体结合位点而降低或丧失；
3)通过改变所述全局转录调控因子CcpA的氨基酸序列而降低或丧失；
4)通过改变编码所述全局转录调控因子CcpA的核苷酸序列而降低或丧失；
5)通过敲除所述全局转录调控因子CcpA的编码序列而丧失。
3.根据权利要求2所述的微生物，其特征在于，采用诱变、定点突变或同源重组的方法来降低所述全局转录调控因子CcpA的编码基因的表达或敲除内源的所述全局转录调控因子CcpA的编码基因。
4.根据权利要求1所述的微生物，其特征在于，全局转录调控因子CcpA活性降低或丧失是通过如下A～D的至少一种方式来实现的：
A、全局转录调控因子CcpA的第95位氨基酸由I突变为M；
B、全局转录调控因子CcpA的第150位氨基酸由E突变为K；
C、全局转录调控因子CcpA的第205位氨基酸由L突变为P；
D、全局转录调控因子CcpA的编码基因的起始密码子由gtg或atg突变为ttg。
5.根据权利要求1-4任一项所述的微生物，其特征在于，所述微生物为芽孢杆菌属(Bacillus)或埃希氏菌属(Escherichia)菌种；
优选解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillussubtilis)、短小芽孢杆菌(Bacillus pumilus)、大肠杆菌(Escherichia coli)。
6.产核苷及其衍生物的重组菌的构建方法，其特征在于，利用基因工程手段，失活或弱化具有核苷生产能力的微生物中的全局转录调控因子CcpA的编码基因；
所述弱化选自以下1)-5)，或任选的组合实现的：
1)通过改变所述全局转录调控因子CcpA的编码基因的启动子序列而降低或丧失；
2)通过改变所述全局转录调控因子CcpA的编码基因的核糖体结合位点而降低或丧失；
3)通过改变所述全局转录调控因子CcpA的氨基酸序列而降低或丧失；
4)通过改变编码所述全局转录调控因子CcpA的核苷酸序列而降低或丧失；
5)通过敲除所述全局转录调控因子CcpA的编码序列而丧失；
其中，所述全局转录调控因子CcpA在NCBI上的参考序列编号为BAMF_2774或BSU_29740。
7.根据权利要求6所述的方法，其特征在于，弱化的方法选自诱变、定点突变、同源重组中的至少一种；
优选地，弱化是通过如下A～D的至少一种方式来实现的：
A、全局转录调控因子CcpA的第95位氨基酸由I突变为M；
B、全局转录调控因子CcpA的第150位氨基酸由E突变为K；
C、全局转录调控因子CcpA的第205位氨基酸由L突变为P；
D、全局转录调控因子CcpA的编码基因的起始密码子由gtg或atg突变为ttg；
所述微生物为芽孢杆菌属(Bacillus)或埃希氏菌属(Escherichia)菌种；
优选解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、枯草芽孢杆菌(Bacillussubtilis)、短小芽孢杆菌(Bacillus pumilus)、大肠杆菌(Escherichia coli)。
8.按照权利要求6或7所述方法构建得到的重组菌。
9.权利要求1-5任一项所述微生物或权利要求8所述重组菌在核苷及其衍生物的发酵生产或提高核苷及其衍生物的发酵产量中的应用；
所述核苷及其衍生物选自肌苷、鸟苷、腺苷、次黄嘌呤、肌苷酸、鸟嘌呤、腺嘌呤、鸟苷酸、核黄素、二乙酰鸟苷酸、腺苷酸、阿糖腺苷。
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：
a)培养权利要求1-5任一项所述微生物或权利要求8所述重组菌，获得培养物；
b)从步骤a)中获得的所述培养物中收集所产生的核苷；
所述核苷及其衍生物选自肌苷、鸟苷、腺苷、次黄嘌呤、肌苷酸、鸟嘌呤、腺嘌呤、鸟苷酸、核黄素、二乙酰鸟苷酸、腺苷酸、阿糖腺苷。
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