CN112143751A - 高产核苷的枯草芽孢杆菌工程菌及其构建方法与应用 - Google Patents

高产核苷的枯草芽孢杆菌工程菌及其构建方法与应用 Download PDF

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CN112143751A
CN112143751A CN202011003905.6A CN202011003905A CN112143751A CN 112143751 A CN112143751 A CN 112143751A CN 202011003905 A CN202011003905 A CN 202011003905A CN 112143751 A CN112143751 A CN 112143751A
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孙莹莹
胡丹
白立宽
吴涛
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Abstract

本发明提供高产核苷的枯草芽孢杆菌工程菌及其构建方法与应用。本发明还提供发酵生产核苷的方法，包括：(1)增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA；和/或增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA；(2)将步骤(1)获得的基因增强菌株用于核苷的发酵生产。本发明提供的枯草芽孢杆菌工程菌为核苷高产菌株，能有效积累核苷，提高核苷的产量，为核苷的工业化生产奠定了基础。

Description

高产核苷的枯草芽孢杆菌工程菌及其构建方法与应用
技术领域
本发明涉及微生物领域和生物工程技术领域，具体地说，涉及高产核苷的枯草芽孢杆菌工程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)。
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。
鸟苷，化学名称：9-β-D-呋喃核糖鸟嘌呤。它可作为食品或医药原料的中间体，用于生产5’-鸟苷酸二钠、鸟嘌呤、利巴韦林、阿昔洛韦、泛昔洛韦等食品添加剂或医药原料。
肌苷，化学名称：9-β-D-核糖次黄嘌呤。是细胞代谢改善药，参与体内核酸代谢，在体内转变为肌苷酸及三磷酸腺苷，参与细胞的能量代谢和蛋白质合成，提高各种酶，特别是辅酶A与丙酮酸氧化酶的活性，从而使细胞在缺氧状态下继续进行代谢，活化肝脏功能，促进受损伤肝脏的恢复，可刺激体内产生抗体并促进肠道对铁的吸收。适用于各种慢性肝脏疾患、心脏疾患、白血球或血小板减少症、中心性视网膜炎等，并可预防接触锑剂引起的对肝脏心脏的副作用。
嘌呤核苷既可通过化学法合成，也可通过微生物发酵法合成，并且微生物发酵法由于其诸多的优势(条件温和、环境污染小)，已成为主流生产方法。但发酵法的缺点是成本高、转化率低。因此，亟需通过代谢工程的手段，改善菌种性能。
发明内容
本发明的目的是提供一种发酵生产核苷的方法或提高核苷发酵产量的方法。
本发明的另一目的是提供高产核苷的枯草芽孢杆菌工程菌及其构建方法与应用。
本发明构思如下：核苷合成对氮同化的需求远高于氨基酸合成，腺嘌呤环涉及5个氮同化，其中2个来自谷胺酰胺。发明人在研发中意外地发现，通过增强核苷合成代谢过程中的氮供应可以明显地促进嘌呤核苷的合成，且强化氮供应的方式不止一种，组合强化可起到叠加的效果。
尽管gltA不是核苷合成的关键基因，却是谷氨酸合酶合成的关键基因，该酶催化谷氨酸合成；而glnA又是谷氨酰胺合成酶的关键基因，催化谷氨酸向谷氨酰胺合成。本发明通过gltA点突变、gltA和glnA强启动子替换达到gltA和glnA基因强化目的。
gltA强化条件下，促进谷氨酸合成，glnA强化条件下，促进谷氨酰胺合成，谷氨酰胺是核苷合成的直接前体，进一步满足核苷合成氮同化需求。
为了实现本发明目的，第一方面，本发明提供一种发酵生产核苷的方法或提高核苷发酵产量的方法，包括以下步骤：
(1)增强枯草芽孢杆菌(Bacillus subtilis)染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA；和/或
增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA；
(2)将步骤(1)获得的基因增强菌株用于核苷的发酵生产。
增强的途径可选自以下1)～3)，或任选的组合：
1)通过点突变而增强；
2)通过改变染色体上所述基因的启动子序列而增强；
3)通过将强启动子与所述基因可操作地连接而增强。
优选地，通过点突变增强谷氨酸合酶基因gltA，使其编码的谷氨酸合酶的第1181位丙氨酸突变为缬氨酸。
优选地，通过将强启动子与谷氨酸合酶基因gltA可操作地连接而增强。
优选地，通过将强启动子与谷氨酰胺合成酶基因glnA可操作地连接而增强；
本发明中，所述强启动子可以是来自枯草芽孢杆菌cdd基因(GenBank ID：937885)的P43启动子。
优选地，所述枯草芽孢杆菌为B.subtilis A5。菌株B.subtilis A5参见CN110257315A说明书实施例4。
本发明中，所述核苷选自腺苷、肌苷、鸟苷和黄苷中的至少一种。
第二方面，本发明提供高产核苷的枯草芽孢杆菌工程菌，所述工程菌是通过增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA和/或编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA，获得的基因增强菌株。
优选地，所述枯草芽孢杆菌为B.subtilis A5。
增强的途径可选自以下a)～c)，或任选的组合：
a)通过点突变增强谷氨酸合酶基因gltA，使其编码的谷氨酸合酶的第1181位丙氨酸突变为缬氨酸；
b)通过将强启动子与谷氨酸合酶基因gltA可操作地连接；
c)通过将强启动子与谷氨酰胺合成酶基因glnA可操作地连接。
第三方面，本发明提供高产核苷的枯草芽孢杆菌工程菌的构建方法，包括：利用基因工程手段增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA和/或编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA，获得基因增强菌株。
优选地，所述枯草芽孢杆菌为B.subtilis A5。
前述的方法，增强的途径可选自以下a)～c)，或任选的组合：
a)通过点突变增强谷氨酸合酶基因gltA，使其编码的谷氨酸合酶的第1181位丙氨酸突变为缬氨酸；
b)通过将强启动子与谷氨酸合酶基因gltA可操作地连接；
c)通过将强启动子与谷氨酰胺合成酶基因glnA可操作地连接。
更优选地，增强的途径为a)和c)组合。
第四方面，本发明提供所述的枯草芽孢杆菌工程菌或按照上述方法构建的枯草芽孢杆菌工程菌在发酵生产核苷中的应用。
第五方面，本发明提供谷氨酸合酶突变体，其氨基酸序列如SEQ ID NO:5所示。
第六方面，本发明提供编码所述氨酸合酶突变体的基因或含有该基因的生物材料在发酵生产核苷或提高核苷(如腺苷)发酵产量中的应用。
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、噬菌体载体、病毒载体、工程菌或转基因细胞系。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明提供的枯草芽孢杆菌工程菌为核苷高产菌株，能有效积累核苷，提高核苷的产量，为核苷的工业化生产奠定了基础。
(一)核苷在食品医药等领域应用较广，而目前产量较低，有较大提升空间。通过基因工程手段获得核苷高产菌株，可操作性强。
(二)核苷自身发酵周期长，本发明通过优化发酵条件缩短核苷发酵周期，并且提升产苷，降低了发酵成本。
(三)本发明对于其它路径相似的产品(如核黄素、组氨酸等)具有借鉴作用。
附图说明
图1为本发明较佳实施例中工程菌株B.subtilis A9、A10、A11、A12、A13(B.s A9、B.s A10、B.s A11、B.s A12、B.s A12)产核苷能力与对照B.subtilis A5(B.s A5)对比。
具体实施方式
本发明提供一种高产核苷的枯草芽孢杆菌，所述枯草芽孢杆菌为B.subtilis A5菌株中gltA、glnA中至少一个位点经修饰而强化。
其中，所述gltA基因的表达是通过点突变、起始密码子及强启动子替换来强化的；glnA基因的表达是起始密码子及强启动子替换来强化的。
本发明还提供上述枯草芽孢杆菌的构建方法，包括：
步骤A、分别制备gltA、glnA基因片段，与载体连接分别获得点突变、起始密码子及强启动子替换质粒；
步骤B、两个质粒分别转化B.subtilis A5菌株分别获得点突变、起始密码子及强启动子替换的枯草芽孢杆菌。
进一步地，上述构建方法中步骤A所用载体为pKSU。pKSU由南开大学生命科学学院惠赠。
进一步地，两个单一位点强化质粒分别为pKSU-gltA*，pKSU-P43-gltA，pKSU-P43-glnA，单一位点强化的枯草芽孢杆菌为B.subtilis A9、B.subtilis A10、B.subtilis A11菌株。
进一步地，上述构建方法中步骤B为两个位点强化质粒依次转化B.subtilis A5菌株，获得两个位点均强化的枯草芽孢杆菌B.subtilis A12和B.subtilis A13。
本发明还提供上述枯草芽孢杆菌在发酵生产核苷中的应用。
本发明还提供一种核苷的生产方法，将上述枯草芽孢杆菌接种于种子培养基进行扩繁，然后将扩繁后的培养物转入发酵培养基发酵。
其中，所述种子培养基(g/L)为：葡萄糖20，酵母粉5，玉米浆干粉5，磷酸二氢钾3，硫酸镁0.5，硫酸亚铁0.02，硫酸锰0.01，pH 7.0～7.2。
所述发酵培养基(g/L)为：葡萄糖60，酵母粉3.5，磷酸二氢钾3，硫酸铵25，硫酸锰0.01，硫酸镁5，谷氨酸钠10，玉米浆干粉15，碳酸钙25，pH 7.0～7.2。
发酵条件为：35.5℃发酵46h。
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试剂均为市售产品，均可以通过商业渠道购买获得。菌株B.subtilis A5购自(Bacillus Genetic Stock Center，http://www.bgsc.org/)。本发明使用的腺苷、鸟苷、肌苷标准品购自Sigma公司(http://www.sigmaaldrich.com/sigma-aldrich)，所用DNA聚合酶、DNA纯化试剂盒、限制性内切酶、去磷酸化酶、DNA连接酶等分子生物学试剂购自Thermo公司(http://www.thermoscientificbio.com/fermentas)，所用其他生化试剂购自生工生物工程(上海)股份有限公司(http://www.sangon.com/)。
以下实施例中使用的LB液体培养基(g/L)的配制：蛋白胨10，酵母提取物5，NaCl10，调节pH至7.2，0.15MPa条件下灭菌20min。
LB固体培养基/LB平板(g/L)的配制：在LB液体培养基中加入琼脂粉(终浓度18g/L)，121℃条件下灭菌20min。
以下实施例中使用的引物信息见表1。
表1引物序列
引物 序列5′→3′
gltA-1f caaaataaggatcctctagagtcgactcaaaagcaggtgtaggaacaatc
gltA-1r tcctttgcccgttcacttgtatgaagtacatcagtgcggc
gltA-2f gccgcactgatgtacttcatccaagtgaacgggcaaagga
gltA-2r ccagtgccaagcttgcatgcctgcagatatgcttctccgcttgtcgcacc
P43-gltA-1f caaaataaggatcctctagagtcgac cggatattcttctttaaacgctga
P43-gltA-1r tagcaagagggtgatgtttgtcaattcctctcccccgatcaatt
P43-gltA-2f aaggtaagggaggaatttacacatgacgtacaatcaaatg
P43-gltA-2r ccagtgccaagcttgcatgcctgcagcagcctgtttacgaatgacataca
P43-glnA-1f caaaataaggatcctctagagtcgacgacacataatataacatcacctat
P43-glnA-1r tagcaagagggtgatgtttgtcttggtaaaattcctcctcttaa
P43-glnA-2f aaggtaagggaggaatttacacatggcaaagtacactagagaag
P43-glnA-2r ccagtgccaagcttgcatgcctgcagcaagtacgatatcgcggcggcagt
P43-f gacaaacatcaccctcttgcta
P43-r gtgtaaattcctcccttacctttaatggtaccgctatcactt
实施例1工程菌株B.subtilis A9(gltAA1181V)的构建
用引物gltA-1f/1r和gltA-2f/2r，以B.subtilis A5基因组为模板，使用pfu高保真DNA聚合酶扩增分别得到gltA的上、下游同源臂；用引物gltA-1f/2r和glnA-1f/2r融合上、下游片段分别得到gltA*同源片段(含A1181V突变，原始gltA基因的核苷酸序列如SEQID NO:1所示，其编码蛋白的氨基酸序列如SEQ ID NO:2所示)，将片段与pKSU质粒经SalI/PstI双酶切、连接、转化等操作后得到质粒pKSU-gltA。通过电化学转化至B.subtilis A5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5ml LB液体培养基中，42℃200rpm培养12h并传一代，稀释涂布含5μg/mL的氯霉素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，得到在B.subtilis A5引入gltAA1181V的B.subtilis A9菌株。
实施例2工程菌株B.subtilis A10(P43-gltA)、B.subtilis A11(P43-glnA)的构建
用引物P43-gltA-1f/1r和P43-gltA-2f/2r，P43-glnA-1f/1r和P43-glnA-2f/2r，以B.subtilis A5基因组为模板，使用pfu高保真DNA聚合酶扩增分别得到gltA和glnA的上、下游同源臂；用引物P43-f/r以质粒为模板扩增得到P43启动子；用引物gltA-1f/2r和glnA-1f/2r融合P43启动子及上、下游片段分别得到gltA同源片段(完整gltA基因的核苷酸序列如SEQ ID NO:1所示，其编码蛋白的氨基酸序列如SEQ ID NO:2所示)和glnA同源片段(完整glnA基因的核苷酸序列如SEQ ID NO:3所示，其编码蛋白的氨基酸序列如SEQ ID NO:4所示)，将两个片段与pKSU质粒经SalI/PstI双酶切、连接、转化等操作后得到质粒pKSU-P43-gltA和pKSU-P43-glnA。通过电化学转化至B.subtilis A5中，筛选方法同实施例1，分别得到在B.subtilis A5引入P43-gltA的B.subtilis A10菌株，在B.subtilis A5引入P43-glnA的B.subtilis A11菌株。
实施例3工程菌株B.subtilis A12、B.subtilis A13的构建(引入P43-glnA)
将质粒pKSU-P43-glnA分别转化至B.subtilis A9和B.subtilis A10菌株中，获得工程菌B.subtilis A12(gltAA1181V&P43-glnA)、B.subtilis A13(P43-gltA&P43-glnA)，筛选方法同实施例1。
实施例4工程菌株B.subtilis A9、A10、A11、A12、A13和B.subtilis A5核苷合成能力对比
1、将菌株三区划线LB平板，37℃过夜培养；
2、挑单菌落接种至30mL种子培养基中，110rpm 36℃培养7～8h；
3、按10％v/v接种量转接至30ml发酵培养基中，摇床转速130rpm，35.5℃培养46h。
发酵结果见表2和图1，从结果可以看出，与出发菌株B.subtilis A5相比，工程菌的腺苷积累量都有提高，A9(gltAA1181V)、A10(P43-gltA)和A11(P43-glnA)单点工程菌分别积累腺苷0.4g/L、0.3g/L和0.6g/L，A11积累较多，gltAA1181V的引入增强了谷氨酸合成，为谷氨酰胺的合成提供前体；glnA增强了谷氨酰胺的合成，为核苷合成途径提供直接前体。双强化工程菌A12和A13积累腺苷1.3g/L和1.0g/L，同时鸟苷积累0.8g/L和0.7g/L，说明这两个位点的强化在核苷积累中起到了主要作用。
本发明提供的包含枯草芽孢杆菌谷氨酸合酶基因gltA强化和谷氨酰胺合成酶基因glnA强化的工程菌的用途，包括但不限于核苷。
表2各菌株产苷能力对比
菌株 腺苷g/L 鸟苷g/L 肌苷g/L
B.s A5 8.9 0.2 0.3
B.s A9 9.3 0.3 0.4
B.s A10 9.2 0.5 0.5
B.s A11 9.5 0.7 0.3
B.s A12 10.2 1 0.6
B.s A13 9.9 0.9 0.4
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> 高产核苷的枯草芽孢杆菌工程菌及其构建方法与应用
<130> KHP201115679.2
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4563
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 1
atgacgtaca atcaaatgcc aaaagctcaa ggtctctacc gtcctgaatt tgaacatgat 60
gcatgcggaa tcggcctata tgcacactta aagggcaagc agactcacga cattgtcaaa 120
caaggactta agatgctttg ccagctagac catagaggag ggcaaggcag tgatccggat 180
acaggagacg gagccggctt actggttcaa atccctgatg ctttctttag aaaagagtgc 240
aaaaacatca atctgccaga aaaagaacgt tacggggtag gaatggtctt tttctcacag 300
aaggaagatg aaagaaaaaa aattgaaaag caaatcaatg cactaattga acaagaaggc 360
caagtcgttc ttggatggag aactgtacct gtaaatgtcg gaaaaatcgg aacagtggcg 420
caaaaaagct gtccgtttgt ccgtcaggta tttatcggcg caagttctga tctaaaggac 480
aatttatctt ttgaacgaaa attgtatgtc attcgtaaac aggctgaaaa ttggggagta 540
acggaaggtc ttgattttta ttttgccagt ctttcaagcc agaccattgt ttacaaaggc 600
cttttaacac ctgaacaggt tgatgcattt tattctgatc tgcaagacga agcattcgtt 660
tctgcttttg cgcttgttca ttcacgcttc agtaccaata cattccctac atgggaaaga 720
gcgcatccta accgctattt ggttcacaac ggtgaaatta acacccttcg cggaaatatc 780
aactggatga gagcacgtga gcagcaattt gtttctgaaa gcttcggtga ggacttaaac 840
aagattttgc cgattctcaa tgctgatggc agtgactctt ccattttgga taacgcattt 900
gagttttttg tgatggctgg acgcaaaccg gcacatacgg caatgatgct cattccagag 960
ccttggacgg aaaatacgca tatgtccaag gaaaaaaggg cgttctacga gtaccatagc 1020
tccctgatgg agccttggga cggaccgaca gctatttcat ttactgacgg taaacaaatc 1080
ggggcgatcc ttgaccggaa cggtctccgt ccggcccgtt attatgtcac aaaagatgat 1140
tatattattt tctcatctga agtaggcgtt attgaagttg aacaggagaa cgttttatat 1200
aaaaaccgcc ttgagcctgg gaaaatgctt ttaatcgacc tggaagaagg ccgtattatc 1260
tccgatgagg aagtcaaaac acaaattgca actgagtatc cgtaccaaaa atggcttgaa 1320
gaagagcttg tacaagtaaa tcctgatccg gagtcaagag aagaagaaca atttagtgat 1380
cttcttactc gccagaaggc attcggatac acatatgagg atatccaaaa gtatctaatt 1440
cctgttatta aagaaggcaa agatcccctt ggttcaatgg ggaatgacgc tccgcttgct 1500
gtactttctg accgagcgca atcgctgttt aactacttta aacagctgtt tgcacaagta 1560
acgaacccgc caatcgacgc gattcgtgag cagcttgtca cttcaacgat gacttggctc 1620
ggtgcggaag gcgatcttct tcatccaagt gaacgaaatg ttcgccggat taaactgtat 1680
acacctgttt tatccaatga acagttttac gcattgaaaa cgattgttca tcctgattta 1740
aaaagccaaa aaattgatgt gctgttttca gaggatcttg aacgcggttt aaaggacatg 1800
ttcacacagg cagagaaagc catcagccaa ggcgtaagtt tgttaatttt atcagacaaa 1860
aagatgaacg aacgactgac accaattccg ccgcttctgg cagtaagcgc actgcaccag 1920
cacttaatcc gcaaagggct gcgtacgaag gtcagcatta ttgtggaatc gggagaagca 1980
cgtgaagtgc atcactttgc agcgttaatc ggttatggcg cagatgcgat taacccttat 2040
ctcgcttatg cgacctacaa gcaggaaatt gatgaaggcc gtttggatat cagctatgaa 2100
gaagcggtta gcaaatatgg aaaaagcatt actgagggcg tcgtaaaagt gatgtccaag 2160
atgggaattt caactgtgca aagctacaga ggcgcacaaa tctttgaggc ggtaggaatc 2220
agccgcgatg tgatcgaccg ctatttctca ggtactgcat cacagctagg cggcatcgat 2280
ttacaaacaa ttgcagaaga ggcacagcgc cgccaccggg aagcgtatca ggatgattac 2340
agcaaaacgc ttgaaccggg aagtgacttc caatggagaa atggcggaga gcatcatgcg 2400
tttaatccga aaacgattca tactttgcaa tgggcgtgcc gcagaaatga ttacaatctg 2460
tttaaacagt atacaaaagc ggctgatgaa gaacgcatcg gatttttgcg gaacttgttc 2520
gcatttgacg gaaaccgcaa gcctttgaag ttggaggagg ttgaatccgc tgaatcgatt 2580
gtcaaacgtt ttaaaacggg tgcgatgtca ttcgggtcct tgagtaagga agcgcacgaa 2640
gctttagcaa tcgcaatgaa ccgtcttgga ggaaaaagca acagcggtga aggcggagaa 2700
gatcccaaac gctttgttcc agatgagaac ggcgatgata gaagaagtgc gatcaaacaa 2760
attgcatccg gacggtttgg tgtcaaaagc cattacctcg tcaatgctga tgagctgcag 2820
attaaaatgg ctcaaggtgc taagccgggt gaaggcgggc agcttcctgg caacaaggta 2880
tatccatggg ttgccgatgt ccgtgggtca acgccaggtg tcggattaat ctcacctccg 2940
ccacaccatg acatttattc gattgaggat ttagcccagc tgatccacga tttgaaaaat 3000
gccaaccgtg acgcaagaat cagcgtaaag ctggtgtcaa aagcaggtgt aggaacaatc 3060
gctgcaggtg ttgccaaagc gactgcagat gtcattgtga tcagcggcta tgacggaggt 3120
acaggcgctt ctccgaaaac cagtattaaa catacagggc ttccgtggga gcttggcctt 3180
gcagaagcac atcaaacact aatgctgaac ggacttcgtg accgtgttgt attagaaaca 3240
gatggaaagc tcatgacggg ccgcgacgtt gtgatggctg ccttgctcgg cgccgaagaa 3300
tttggtttcg caacggctcc gttagttgta ctcggctgtg tcatgatgcg tgcctgccat 3360
ttggatacat gccctgtcgg tgtagcgaca caaaatccag agcttcgcaa aaagttcatg 3420
ggagatcctg accatattgt gaactatatg ctgttcattg ccgaagaagt tcgtgagtac 3480
atggctgcgt taggcttcaa gacatttgat gaaatgatcg gccgcactga tgtacttcat 3540
gcaagtgaac gggcaaagga gcactggaaa gcaagccagc ttgatttgtc tacccttctt 3600
tatcagcctg aaggggtgcg gacgttccaa tcgccgcaaa atcataaaat tgatcaatca 3660
cttgatatta caacaattct cccggccgta caagaagcca tcgaatctgg aaaagaagct 3720
gatatttcga ttgaaatcaa taatacaaat cgtgtagccg gaacgataac cggcagtgaa 3780
atctcaaagc gttacggaga agaaggtctt cctgaagata caatcaagct gcactttacc 3840
ggatcagccg gccaaagctt tggagctttc gttcctaaag ggatgacgct ttatttggac 3900
ggagactcaa atgattacgt cggaaaaggg ctttctggcg gaaaaatcat cgtcaagtca 3960
tcagaaggat tcaactctgc ttcggatgac aatgtcatta tcggcaacgt ggctttttac 4020
ggtgcgacaa gcggagaagc atatattaac gggcgtgctg gtgaacgctt tgccgttcga 4080
aacagcggtg tgaacgtagt tgtagaaggc atcggcgacc acggctgtga atacatgact 4140
ggcggcagcg ttgtcgttct cggtgatgta ggcaaaaact ttgcggcagg tatgtccggc 4200
ggaatcgctt atgtactgac cgaagatgtc aaagcgttta aacgcaaatg caaccttgag 4260
atgattttat ttgaatcatt agaggatgag aaagaaatcc agcaaatcaa agcaatgctt 4320
gaaagacata ctgcgtatac aaacagccaa aaagcagaag atctgctaga tcaatgggaa 4380
gacagtgtga aaaaattcgt caaagtcatt ccgaaaaact ataaacaaat gctcgcaagc 4440
atcgaagagc aaaaagctgc aggtttatca gatgaagaag ccatcatgtt cgcttttgag 4500
gccaacacga agccaaagca gaatacagca gcatcgggac aaaaacaagc ggtagtacag 4560
taa 4563
<210> 2
<211> 1520
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 2
Met Thr Tyr Asn Gln Met Pro Lys Ala Gln Gly Leu Tyr Arg Pro Glu
1 5 10 15
Phe Glu His Asp Ala Cys Gly Ile Gly Leu Tyr Ala His Leu Lys Gly
20 25 30
Lys Gln Thr His Asp Ile Val Lys Gln Gly Leu Lys Met Leu Cys Gln
35 40 45
Leu Asp His Arg Gly Gly Gln Gly Ser Asp Pro Asp Thr Gly Asp Gly
50 55 60
Ala Gly Leu Leu Val Gln Ile Pro Asp Ala Phe Phe Arg Lys Glu Cys
65 70 75 80
Lys Asn Ile Asn Leu Pro Glu Lys Glu Arg Tyr Gly Val Gly Met Val
85 90 95
Phe Phe Ser Gln Lys Glu Asp Glu Arg Lys Lys Ile Glu Lys Gln Ile
100 105 110
Asn Ala Leu Ile Glu Gln Glu Gly Gln Val Val Leu Gly Trp Arg Thr
115 120 125
Val Pro Val Asn Val Gly Lys Ile Gly Thr Val Ala Gln Lys Ser Cys
130 135 140
Pro Phe Val Arg Gln Val Phe Ile Gly Ala Ser Ser Asp Leu Lys Asp
145 150 155 160
Asn Leu Ser Phe Glu Arg Lys Leu Tyr Val Ile Arg Lys Gln Ala Glu
165 170 175
Asn Trp Gly Val Thr Glu Gly Leu Asp Phe Tyr Phe Ala Ser Leu Ser
180 185 190
Ser Gln Thr Ile Val Tyr Lys Gly Leu Leu Thr Pro Glu Gln Val Asp
195 200 205
Ala Phe Tyr Ser Asp Leu Gln Asp Glu Ala Phe Val Ser Ala Phe Ala
210 215 220
Leu Val His Ser Arg Phe Ser Thr Asn Thr Phe Pro Thr Trp Glu Arg
225 230 235 240
Ala His Pro Asn Arg Tyr Leu Val His Asn Gly Glu Ile Asn Thr Leu
245 250 255
Arg Gly Asn Ile Asn Trp Met Arg Ala Arg Glu Gln Gln Phe Val Ser
260 265 270
Glu Ser Phe Gly Glu Asp Leu Asn Lys Ile Leu Pro Ile Leu Asn Ala
275 280 285
Asp Gly Ser Asp Ser Ser Ile Leu Asp Asn Ala Phe Glu Phe Phe Val
290 295 300
Met Ala Gly Arg Lys Pro Ala His Thr Ala Met Met Leu Ile Pro Glu
305 310 315 320
Pro Trp Thr Glu Asn Thr His Met Ser Lys Glu Lys Arg Ala Phe Tyr
325 330 335
Glu Tyr His Ser Ser Leu Met Glu Pro Trp Asp Gly Pro Thr Ala Ile
340 345 350
Ser Phe Thr Asp Gly Lys Gln Ile Gly Ala Ile Leu Asp Arg Asn Gly
355 360 365
Leu Arg Pro Ala Arg Tyr Tyr Val Thr Lys Asp Asp Tyr Ile Ile Phe
370 375 380
Ser Ser Glu Val Gly Val Ile Glu Val Glu Gln Glu Asn Val Leu Tyr
385 390 395 400
Lys Asn Arg Leu Glu Pro Gly Lys Met Leu Leu Ile Asp Leu Glu Glu
405 410 415
Gly Arg Ile Ile Ser Asp Glu Glu Val Lys Thr Gln Ile Ala Thr Glu
420 425 430
Tyr Pro Tyr Gln Lys Trp Leu Glu Glu Glu Leu Val Gln Val Asn Pro
435 440 445
Asp Pro Glu Ser Arg Glu Glu Glu Gln Phe Ser Asp Leu Leu Thr Arg
450 455 460
Gln Lys Ala Phe Gly Tyr Thr Tyr Glu Asp Ile Gln Lys Tyr Leu Ile
465 470 475 480
Pro Val Ile Lys Glu Gly Lys Asp Pro Leu Gly Ser Met Gly Asn Asp
485 490 495
Ala Pro Leu Ala Val Leu Ser Asp Arg Ala Gln Ser Leu Phe Asn Tyr
500 505 510
Phe Lys Gln Leu Phe Ala Gln Val Thr Asn Pro Pro Ile Asp Ala Ile
515 520 525
Arg Glu Gln Leu Val Thr Ser Thr Met Thr Trp Leu Gly Ala Glu Gly
530 535 540
Asp Leu Leu His Pro Ser Glu Arg Asn Val Arg Arg Ile Lys Leu Tyr
545 550 555 560
Thr Pro Val Leu Ser Asn Glu Gln Phe Tyr Ala Leu Lys Thr Ile Val
565 570 575
His Pro Asp Leu Lys Ser Gln Lys Ile Asp Val Leu Phe Ser Glu Asp
580 585 590
Leu Glu Arg Gly Leu Lys Asp Met Phe Thr Gln Ala Glu Lys Ala Ile
595 600 605
Ser Gln Gly Val Ser Leu Leu Ile Leu Ser Asp Lys Lys Met Asn Glu
610 615 620
Arg Leu Thr Pro Ile Pro Pro Leu Leu Ala Val Ser Ala Leu His Gln
625 630 635 640
His Leu Ile Arg Lys Gly Leu Arg Thr Lys Val Ser Ile Ile Val Glu
645 650 655
Ser Gly Glu Ala Arg Glu Val His His Phe Ala Ala Leu Ile Gly Tyr
660 665 670
Gly Ala Asp Ala Ile Asn Pro Tyr Leu Ala Tyr Ala Thr Tyr Lys Gln
675 680 685
Glu Ile Asp Glu Gly Arg Leu Asp Ile Ser Tyr Glu Glu Ala Val Ser
690 695 700
Lys Tyr Gly Lys Ser Ile Thr Glu Gly Val Val Lys Val Met Ser Lys
705 710 715 720
Met Gly Ile Ser Thr Val Gln Ser Tyr Arg Gly Ala Gln Ile Phe Glu
725 730 735
Ala Val Gly Ile Ser Arg Asp Val Ile Asp Arg Tyr Phe Ser Gly Thr
740 745 750
Ala Ser Gln Leu Gly Gly Ile Asp Leu Gln Thr Ile Ala Glu Glu Ala
755 760 765
Gln Arg Arg His Arg Glu Ala Tyr Gln Asp Asp Tyr Ser Lys Thr Leu
770 775 780
Glu Pro Gly Ser Asp Phe Gln Trp Arg Asn Gly Gly Glu His His Ala
785 790 795 800
Phe Asn Pro Lys Thr Ile His Thr Leu Gln Trp Ala Cys Arg Arg Asn
805 810 815
Asp Tyr Asn Leu Phe Lys Gln Tyr Thr Lys Ala Ala Asp Glu Glu Arg
820 825 830
Ile Gly Phe Leu Arg Asn Leu Phe Ala Phe Asp Gly Asn Arg Lys Pro
835 840 845
Leu Lys Leu Glu Glu Val Glu Ser Ala Glu Ser Ile Val Lys Arg Phe
850 855 860
Lys Thr Gly Ala Met Ser Phe Gly Ser Leu Ser Lys Glu Ala His Glu
865 870 875 880
Ala Leu Ala Ile Ala Met Asn Arg Leu Gly Gly Lys Ser Asn Ser Gly
885 890 895
Glu Gly Gly Glu Asp Pro Lys Arg Phe Val Pro Asp Glu Asn Gly Asp
900 905 910
Asp Arg Arg Ser Ala Ile Lys Gln Ile Ala Ser Gly Arg Phe Gly Val
915 920 925
Lys Ser His Tyr Leu Val Asn Ala Asp Glu Leu Gln Ile Lys Met Ala
930 935 940
Gln Gly Ala Lys Pro Gly Glu Gly Gly Gln Leu Pro Gly Asn Lys Val
945 950 955 960
Tyr Pro Trp Val Ala Asp Val Arg Gly Ser Thr Pro Gly Val Gly Leu
965 970 975
Ile Ser Pro Pro Pro His His Asp Ile Tyr Ser Ile Glu Asp Leu Ala
980 985 990
Gln Leu Ile His Asp Leu Lys Asn Ala Asn Arg Asp Ala Arg Ile Ser
995 1000 1005
Val Lys Leu Val Ser Lys Ala Gly Val Gly Thr Ile Ala Ala Gly Val
1010 1015 1020
Ala Lys Ala Thr Ala Asp Val Ile Val Ile Ser Gly Tyr Asp Gly Gly
1025 1030 1035 1040
Thr Gly Ala Ser Pro Lys Thr Ser Ile Lys His Thr Gly Leu Pro Trp
1045 1050 1055
Glu Leu Gly Leu Ala Glu Ala His Gln Thr Leu Met Leu Asn Gly Leu
1060 1065 1070
Arg Asp Arg Val Val Leu Glu Thr Asp Gly Lys Leu Met Thr Gly Arg
1075 1080 1085
Asp Val Val Met Ala Ala Leu Leu Gly Ala Glu Glu Phe Gly Phe Ala
1090 1095 1100
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1105 1110 1115 1120
Leu Asp Thr Cys Pro Val Gly Val Ala Thr Gln Asn Pro Glu Leu Arg
1125 1130 1135
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1140 1145 1150
Ile Ala Glu Glu Val Arg Glu Tyr Met Ala Ala Leu Gly Phe Lys Thr
1155 1160 1165
Phe Asp Glu Met Ile Gly Arg Thr Asp Val Leu His Ala Ser Glu Arg
1170 1175 1180
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1185 1190 1195 1200
Tyr Gln Pro Glu Gly Val Arg Thr Phe Gln Ser Pro Gln Asn His Lys
1205 1210 1215
Ile Asp Gln Ser Leu Asp Ile Thr Thr Ile Leu Pro Ala Val Gln Glu
1220 1225 1230
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1235 1240 1245
Thr Asn Arg Val Ala Gly Thr Ile Thr Gly Ser Glu Ile Ser Lys Arg
1250 1255 1260
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Gly Ser Ala Gly Gln Ser Phe Gly Ala Phe Val Pro Lys Gly Met Thr
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Leu Tyr Leu Asp Gly Asp Ser Asn Asp Tyr Val Gly Lys Gly Leu Ser
1300 1305 1310
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1330 1335 1340
Gly Glu Ala Tyr Ile Asn Gly Arg Ala Gly Glu Arg Phe Ala Val Arg
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Asn Ser Gly Val Asn Val Val Val Glu Gly Ile Gly Asp His Gly Cys
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1380 1385 1390
Asn Phe Ala Ala Gly Met Ser Gly Gly Ile Ala Tyr Val Leu Thr Glu
1395 1400 1405
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1410 1415 1420
Glu Ser Leu Glu Asp Glu Lys Glu Ile Gln Gln Ile Lys Ala Met Leu
1425 1430 1435 1440
Glu Arg His Thr Ala Tyr Thr Asn Ser Gln Lys Ala Glu Asp Leu Leu
1445 1450 1455
Asp Gln Trp Glu Asp Ser Val Lys Lys Phe Val Lys Val Ile Pro Lys
1460 1465 1470
Asn Tyr Lys Gln Met Leu Ala Ser Ile Glu Glu Gln Lys Ala Ala Gly
1475 1480 1485
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1505 1510 1515 1520
<210> 3
<211> 1334
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 3
atggcaaagt acactagaga agatatcgaa aaattagtaa aagaagaaaa cgtgaagtat 60
atccgccttc aatttactga cattcttgga acaatcaaga atgttgagat tcctgtaagc 120
agcttggaaa agcgcttgat aataaagtca tgtttgacgg ttcttctatt gagggattcg 180
ttcgtatcga agagtcagac atgtacctgt atccagatct aaatacattt gttatcttcc 240
catggacagc tgaaaaaggt aaagtagcac gtttcatctg tgatatttac aatccggatg 300
gcacaccttt tgaaggtgac ccgcgaaaca acttaaaacg gattctgaaa gaaatggaag 360
acctcggctt cagtgatttt aaccttgggc ctgagcctga attcttctta ttcaaattgg 420
acgaaaaagg cgagccgacg cttgaactaa acgacaaagg cggatatttc gacttagctc 480
caactgattt aggagaaaac tgccgccgcg atatcgtact tgagcttgaa gagatgggct 540
ttgaaatcga agcgtctcac cacgaagtag cacctggtca gcacgaaatc gactttaaat 600
atgctggagc agtccgctct tgtgatgaca tccaaacatt taaactagtt gtcaaaacaa 660
ttgcccgtaa acacggcctg catgcgacat ttatgccaaa accattgttc ggtgtaaacg 720
gttcaggtat gcactgcaat ctatcactct tcaaaaatgg tgttaacgca ttctttgacg 780
aaaacgcaga tcttcagtta agtgaaacag cgaagcactt cattgcaggt atcgtgaagc 840
acgcaacaag ctttacagca gtaacaaacc cgacagtaaa ctcttacaaa cgtcttgttc 900
ctggctatga agcaccttgt tatgtagcat ggagcgcgca aaacagaagc ccgcttatcc 960
gtatcccggc ttctcgcggc atcagcacac gtgttgaagt acgcagtgta gacccagctg 1020
caaacccata ccttgcactt agcgtattgc ttgctgcagg attagacgga atcaaaaaca 1080
aactggaagc gccggctcca atcgaccgca acatctatgt gatgagcaaa gaagagcgca 1140
tggaaaacgg aatcgttgac cttccagcaa cacttgcgga agcactagaa gaattcaaat 1200
caaacgaagt catggtcaaa gcgctgggcg agcacctatt cgaacacttc atcgaagcaa 1260
aagaaatcga atgggatatg ttccgcacgc aagtacatcc ttgggaacgc gaacagtata 1320
tgtctcagta ttaa 1334
<210> 4
<211> 444
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 4
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Asn Val Lys Tyr Ile Arg Leu Gln Phe Thr Asp Ile Leu Gly Thr Ile
20 25 30
Lys Asn Val Glu Ile Pro Val Ser Gln Leu Gly Lys Ala Leu Asp Asn
35 40 45
Lys Val Met Phe Asp Gly Ser Ser Ile Glu Gly Phe Val Arg Ile Glu
50 55 60
Glu Ser Asp Met Tyr Leu Tyr Pro Asp Leu Asn Thr Phe Val Ile Phe
65 70 75 80
Pro Trp Thr Ala Glu Lys Gly Lys Val Ala Arg Phe Ile Cys Asp Ile
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Tyr Asn Pro Asp Gly Thr Pro Phe Glu Gly Asp Pro Arg Asn Asn Leu
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Lys Arg Ile Leu Lys Glu Met Glu Asp Leu Gly Phe Ser Asp Phe Asn
115 120 125
Leu Gly Pro Glu Pro Glu Phe Phe Leu Phe Lys Leu Asp Glu Lys Gly
130 135 140
Glu Pro Thr Leu Glu Leu Asn Asp Lys Gly Gly Tyr Phe Asp Leu Ala
145 150 155 160
Pro Thr Asp Leu Gly Glu Asn Cys Arg Arg Asp Ile Val Leu Glu Leu
165 170 175
Glu Glu Met Gly Phe Glu Ile Glu Ala Ser His His Glu Val Ala Pro
180 185 190
Gly Gln His Glu Ile Asp Phe Lys Tyr Ala Gly Ala Val Arg Ser Cys
195 200 205
Asp Asp Ile Gln Thr Phe Lys Leu Val Val Lys Thr Ile Ala Arg Lys
210 215 220
His Gly Leu His Ala Thr Phe Met Pro Lys Pro Leu Phe Gly Val Asn
225 230 235 240
Gly Ser Gly Met His Cys Asn Leu Ser Leu Phe Lys Asn Gly Val Asn
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Ala Phe Phe Asp Glu Asn Ala Asp Leu Gln Leu Ser Glu Thr Ala Lys
260 265 270
His Phe Ile Ala Gly Ile Val Lys His Ala Thr Ser Phe Thr Ala Val
275 280 285
Thr Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Tyr Glu
290 295 300
Ala Pro Cys Tyr Val Ala Trp Ser Ala Gln Asn Arg Ser Pro Leu Ile
305 310 315 320
Arg Ile Pro Ala Ser Arg Gly Ile Ser Thr Arg Val Glu Val Arg Ser
325 330 335
Val Asp Pro Ala Ala Asn Pro Tyr Leu Ala Leu Ser Val Leu Leu Ala
340 345 350
Ala Gly Leu Asp Gly Ile Lys Asn Lys Leu Glu Ala Pro Ala Pro Ile
355 360 365
Asp Arg Asn Ile Tyr Val Met Ser Lys Glu Glu Arg Met Glu Asn Gly
370 375 380
Ile Val Asp Leu Pro Ala Thr Leu Ala Glu Ala Leu Glu Glu Phe Lys
385 390 395 400
Ser Asn Glu Val Met Val Lys Ala Leu Gly Glu His Leu Phe Glu His
405 410 415
Phe Ile Glu Ala Lys Glu Ile Glu Trp Asp Met Phe Arg Thr Gln Val
420 425 430
His Pro Trp Glu Arg Glu Gln Tyr Met Ser Gln Tyr
435 440
<210> 5
<211> 1520
<212> PRT
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 5
Met Thr Tyr Asn Gln Met Pro Lys Ala Gln Gly Leu Tyr Arg Pro Glu
1 5 10 15
Phe Glu His Asp Ala Cys Gly Ile Gly Leu Tyr Ala His Leu Lys Gly
20 25 30
Lys Gln Thr His Asp Ile Val Lys Gln Gly Leu Lys Met Leu Cys Gln
35 40 45
Leu Asp His Arg Gly Gly Gln Gly Ser Asp Pro Asp Thr Gly Asp Gly
50 55 60
Ala Gly Leu Leu Val Gln Ile Pro Asp Ala Phe Phe Arg Lys Glu Cys
65 70 75 80
Lys Asn Ile Asn Leu Pro Glu Lys Glu Arg Tyr Gly Val Gly Met Val
85 90 95
Phe Phe Ser Gln Lys Glu Asp Glu Arg Lys Lys Ile Glu Lys Gln Ile
100 105 110
Asn Ala Leu Ile Glu Gln Glu Gly Gln Val Val Leu Gly Trp Arg Thr
115 120 125
Val Pro Val Asn Val Gly Lys Ile Gly Thr Val Ala Gln Lys Ser Cys
130 135 140
Pro Phe Val Arg Gln Val Phe Ile Gly Ala Ser Ser Asp Leu Lys Asp
145 150 155 160
Asn Leu Ser Phe Glu Arg Lys Leu Tyr Val Ile Arg Lys Gln Ala Glu
165 170 175
Asn Trp Gly Val Thr Glu Gly Leu Asp Phe Tyr Phe Ala Ser Leu Ser
180 185 190
Ser Gln Thr Ile Val Tyr Lys Gly Leu Leu Thr Pro Glu Gln Val Asp
195 200 205
Ala Phe Tyr Ser Asp Leu Gln Asp Glu Ala Phe Val Ser Ala Phe Ala
210 215 220
Leu Val His Ser Arg Phe Ser Thr Asn Thr Phe Pro Thr Trp Glu Arg
225 230 235 240
Ala His Pro Asn Arg Tyr Leu Val His Asn Gly Glu Ile Asn Thr Leu
245 250 255
Arg Gly Asn Ile Asn Trp Met Arg Ala Arg Glu Gln Gln Phe Val Ser
260 265 270
Glu Ser Phe Gly Glu Asp Leu Asn Lys Ile Leu Pro Ile Leu Asn Ala
275 280 285
Asp Gly Ser Asp Ser Ser Ile Leu Asp Asn Ala Phe Glu Phe Phe Val
290 295 300
Met Ala Gly Arg Lys Pro Ala His Thr Ala Met Met Leu Ile Pro Glu
305 310 315 320
Pro Trp Thr Glu Asn Thr His Met Ser Lys Glu Lys Arg Ala Phe Tyr
325 330 335
Glu Tyr His Ser Ser Leu Met Glu Pro Trp Asp Gly Pro Thr Ala Ile
340 345 350
Ser Phe Thr Asp Gly Lys Gln Ile Gly Ala Ile Leu Asp Arg Asn Gly
355 360 365
Leu Arg Pro Ala Arg Tyr Tyr Val Thr Lys Asp Asp Tyr Ile Ile Phe
370 375 380
Ser Ser Glu Val Gly Val Ile Glu Val Glu Gln Glu Asn Val Leu Tyr
385 390 395 400
Lys Asn Arg Leu Glu Pro Gly Lys Met Leu Leu Ile Asp Leu Glu Glu
405 410 415
Gly Arg Ile Ile Ser Asp Glu Glu Val Lys Thr Gln Ile Ala Thr Glu
420 425 430
Tyr Pro Tyr Gln Lys Trp Leu Glu Glu Glu Leu Val Gln Val Asn Pro
435 440 445
Asp Pro Glu Ser Arg Glu Glu Glu Gln Phe Ser Asp Leu Leu Thr Arg
450 455 460
Gln Lys Ala Phe Gly Tyr Thr Tyr Glu Asp Ile Gln Lys Tyr Leu Ile
465 470 475 480
Pro Val Ile Lys Glu Gly Lys Asp Pro Leu Gly Ser Met Gly Asn Asp
485 490 495
Ala Pro Leu Ala Val Leu Ser Asp Arg Ala Gln Ser Leu Phe Asn Tyr
500 505 510
Phe Lys Gln Leu Phe Ala Gln Val Thr Asn Pro Pro Ile Asp Ala Ile
515 520 525
Arg Glu Gln Leu Val Thr Ser Thr Met Thr Trp Leu Gly Ala Glu Gly
530 535 540
Asp Leu Leu His Pro Ser Glu Arg Asn Val Arg Arg Ile Lys Leu Tyr
545 550 555 560
Thr Pro Val Leu Ser Asn Glu Gln Phe Tyr Ala Leu Lys Thr Ile Val
565 570 575
His Pro Asp Leu Lys Ser Gln Lys Ile Asp Val Leu Phe Ser Glu Asp
580 585 590
Leu Glu Arg Gly Leu Lys Asp Met Phe Thr Gln Ala Glu Lys Ala Ile
595 600 605
Ser Gln Gly Val Ser Leu Leu Ile Leu Ser Asp Lys Lys Met Asn Glu
610 615 620
Arg Leu Thr Pro Ile Pro Pro Leu Leu Ala Val Ser Ala Leu His Gln
625 630 635 640
His Leu Ile Arg Lys Gly Leu Arg Thr Lys Val Ser Ile Ile Val Glu
645 650 655
Ser Gly Glu Ala Arg Glu Val His His Phe Ala Ala Leu Ile Gly Tyr
660 665 670
Gly Ala Asp Ala Ile Asn Pro Tyr Leu Ala Tyr Ala Thr Tyr Lys Gln
675 680 685
Glu Ile Asp Glu Gly Arg Leu Asp Ile Ser Tyr Glu Glu Ala Val Ser
690 695 700
Lys Tyr Gly Lys Ser Ile Thr Glu Gly Val Val Lys Val Met Ser Lys
705 710 715 720
Met Gly Ile Ser Thr Val Gln Ser Tyr Arg Gly Ala Gln Ile Phe Glu
725 730 735
Ala Val Gly Ile Ser Arg Asp Val Ile Asp Arg Tyr Phe Ser Gly Thr
740 745 750
Ala Ser Gln Leu Gly Gly Ile Asp Leu Gln Thr Ile Ala Glu Glu Ala
755 760 765
Gln Arg Arg His Arg Glu Ala Tyr Gln Asp Asp Tyr Ser Lys Thr Leu
770 775 780
Glu Pro Gly Ser Asp Phe Gln Trp Arg Asn Gly Gly Glu His His Ala
785 790 795 800
Phe Asn Pro Lys Thr Ile His Thr Leu Gln Trp Ala Cys Arg Arg Asn
805 810 815
Asp Tyr Asn Leu Phe Lys Gln Tyr Thr Lys Ala Ala Asp Glu Glu Arg
820 825 830
Ile Gly Phe Leu Arg Asn Leu Phe Ala Phe Asp Gly Asn Arg Lys Pro
835 840 845
Leu Lys Leu Glu Glu Val Glu Ser Ala Glu Ser Ile Val Lys Arg Phe
850 855 860
Lys Thr Gly Ala Met Ser Phe Gly Ser Leu Ser Lys Glu Ala His Glu
865 870 875 880
Ala Leu Ala Ile Ala Met Asn Arg Leu Gly Gly Lys Ser Asn Ser Gly
885 890 895
Glu Gly Gly Glu Asp Pro Lys Arg Phe Val Pro Asp Glu Asn Gly Asp
900 905 910
Asp Arg Arg Ser Ala Ile Lys Gln Ile Ala Ser Gly Arg Phe Gly Val
915 920 925
Lys Ser His Tyr Leu Val Asn Ala Asp Glu Leu Gln Ile Lys Met Ala
930 935 940
Gln Gly Ala Lys Pro Gly Glu Gly Gly Gln Leu Pro Gly Asn Lys Val
945 950 955 960
Tyr Pro Trp Val Ala Asp Val Arg Gly Ser Thr Pro Gly Val Gly Leu
965 970 975
Ile Ser Pro Pro Pro His His Asp Ile Tyr Ser Ile Glu Asp Leu Ala
980 985 990
Gln Leu Ile His Asp Leu Lys Asn Ala Asn Arg Asp Ala Arg Ile Ser
995 1000 1005
Val Lys Leu Val Ser Lys Ala Gly Val Gly Thr Ile Ala Ala Gly Val
1010 1015 1020
Ala Lys Ala Thr Ala Asp Val Ile Val Ile Ser Gly Tyr Asp Gly Gly
1025 1030 1035 1040
Thr Gly Ala Ser Pro Lys Thr Ser Ile Lys His Thr Gly Leu Pro Trp
1045 1050 1055
Glu Leu Gly Leu Ala Glu Ala His Gln Thr Leu Met Leu Asn Gly Leu
1060 1065 1070
Arg Asp Arg Val Val Leu Glu Thr Asp Gly Lys Leu Met Thr Gly Arg
1075 1080 1085
Asp Val Val Met Ala Ala Leu Leu Gly Ala Glu Glu Phe Gly Phe Ala
1090 1095 1100
Thr Ala Pro Leu Val Val Leu Gly Cys Val Met Met Arg Ala Cys His
1105 1110 1115 1120
Leu Asp Thr Cys Pro Val Gly Val Ala Thr Gln Asn Pro Glu Leu Arg
1125 1130 1135
Lys Lys Phe Met Gly Asp Pro Asp His Ile Val Asn Tyr Met Leu Phe
1140 1145 1150
Ile Ala Glu Glu Val Arg Glu Tyr Met Ala Ala Leu Gly Phe Lys Thr
1155 1160 1165
Phe Asp Glu Met Ile Gly Arg Thr Asp Val Leu His Val Ser Glu Arg
1170 1175 1180
Ala Lys Glu His Trp Lys Ala Ser Gln Leu Asp Leu Ser Thr Leu Leu
1185 1190 1195 1200
Tyr Gln Pro Glu Gly Val Arg Thr Phe Gln Ser Pro Gln Asn His Lys
1205 1210 1215
Ile Asp Gln Ser Leu Asp Ile Thr Thr Ile Leu Pro Ala Val Gln Glu
1220 1225 1230
Ala Ile Glu Ser Gly Lys Glu Ala Asp Ile Ser Ile Glu Ile Asn Asn
1235 1240 1245
Thr Asn Arg Val Ala Gly Thr Ile Thr Gly Ser Glu Ile Ser Lys Arg
1250 1255 1260
Tyr Gly Glu Glu Gly Leu Pro Glu Asp Thr Ile Lys Leu His Phe Thr
1265 1270 1275 1280
Gly Ser Ala Gly Gln Ser Phe Gly Ala Phe Val Pro Lys Gly Met Thr
1285 1290 1295
Leu Tyr Leu Asp Gly Asp Ser Asn Asp Tyr Val Gly Lys Gly Leu Ser
1300 1305 1310
Gly Gly Lys Ile Ile Val Lys Ser Ser Glu Gly Phe Asn Ser Ala Ser
1315 1320 1325
Asp Asp Asn Val Ile Ile Gly Asn Val Ala Phe Tyr Gly Ala Thr Ser
1330 1335 1340
Gly Glu Ala Tyr Ile Asn Gly Arg Ala Gly Glu Arg Phe Ala Val Arg
1345 1350 1355 1360
Asn Ser Gly Val Asn Val Val Val Glu Gly Ile Gly Asp His Gly Cys
1365 1370 1375
Glu Tyr Met Thr Gly Gly Ser Val Val Val Leu Gly Asp Val Gly Lys
1380 1385 1390
Asn Phe Ala Ala Gly Met Ser Gly Gly Ile Ala Tyr Val Leu Thr Glu
1395 1400 1405
Asp Val Lys Ala Phe Lys Arg Lys Cys Asn Leu Glu Met Ile Leu Phe
1410 1415 1420
Glu Ser Leu Glu Asp Glu Lys Glu Ile Gln Gln Ile Lys Ala Met Leu
1425 1430 1435 1440
Glu Arg His Thr Ala Tyr Thr Asn Ser Gln Lys Ala Glu Asp Leu Leu
1445 1450 1455
Asp Gln Trp Glu Asp Ser Val Lys Lys Phe Val Lys Val Ile Pro Lys
1460 1465 1470
Asn Tyr Lys Gln Met Leu Ala Ser Ile Glu Glu Gln Lys Ala Ala Gly
1475 1480 1485
Leu Ser Asp Glu Glu Ala Ile Met Phe Ala Phe Glu Ala Asn Thr Lys
1490 1495 1500
Pro Lys Gln Asn Thr Ala Ala Ser Gly Gln Lys Gln Ala Val Val Gln
1505 1510 1515 1520

Claims (10)

1.发酵生产核苷的方法或提高核苷发酵产量的方法，其特征在于，包括以下步骤：
(1)增强枯草芽孢杆菌(Bacillus subtilis)染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA；和/或
增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA；
(2)将步骤(1)获得的基因增强菌株用于核苷的发酵生产。
2.根据权利要求1所述的方法，其特征在于，增强的途径选自以下1)～3)，或任选的组合：
1)通过点突变而增强；
2)通过改变染色体上所述基因的启动子序列而增强；
3)通过将强启动子与所述基因可操作地连接而增强。
3.根据权利要求2所述的方法，其特征在于，通过点突变增强谷氨酸合酶基因gltA，使其编码的谷氨酸合酶的第1181位丙氨酸突变为缬氨酸；和/或
通过将强启动子与谷氨酸合酶基因gltA可操作地连接而增强；和/或
通过将强启动子与谷氨酰胺合成酶基因glnA可操作地连接而增强；
优选地，所述强启动子为来自枯草芽孢杆菌cdd基因的P43。
4.根据权利要求1所述的方法，其特征在于，所述枯草芽孢杆菌为B.subtilis A5。
5.根据权利要求1-4任一项所述的方法，其特征在于，所述核苷选自腺苷、肌苷、鸟苷和黄苷中的至少一种。
6.高产核苷的枯草芽孢杆菌工程菌，其特征在于，所述工程菌是通过增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA和/或编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA，获得的基因增强菌株；
优选地，所述枯草芽孢杆菌为B.subtilis A5。
7.根据权利要求6所述的工程菌，其特征在于，增强的途径选自以下a)～c)，或任选的组合：
a)通过点突变增强谷氨酸合酶基因gltA，使其编码的谷氨酸合酶的第1181位丙氨酸突变为缬氨酸；
b)通过将强启动子与谷氨酸合酶基因gltA可操作地连接；
c)通过将强启动子与谷氨酰胺合成酶基因glnA可操作地连接；
优选地，所述强启动子为来自枯草芽孢杆菌cdd基因的P43。
8.根据权利要求7所述的工程菌，其特征在于，增强的途径为a)和c)组合。
9.高产核苷的枯草芽孢杆菌工程菌的构建方法，其特征在于，包括：利用基因工程手段增强枯草芽孢杆菌染色体上编码NCBI参考序列WP_009967365.1的谷氨酸合酶基因gltA和/或编码NCBI参考序列WP_003231737.1的谷氨酰胺合成酶基因glnA，获得基因增强菌株；
增强的途径选自以下a)～c)，或任选的组合：
a)通过点突变增强谷氨酸合酶基因gltA，使其编码的谷氨酸合酶的第1181位丙氨酸突变为缬氨酸；
b)通过将强启动子与谷氨酸合酶基因gltA可操作地连接；
c)通过将强启动子与谷氨酰胺合成酶基因glnA可操作地连接；
优选地，所述枯草芽孢杆菌为B.subtilis A5；所述强启动子优选来自枯草芽孢杆菌cdd基因的P43启动子。
10.权利要求6-8任一项所述的工程菌或按照权利要求9所述方法构建的工程菌在发酵生产核苷中的应用；
所述核苷选自腺苷、肌苷、鸟苷和黄苷中的至少一种。
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CN111471635A (zh) * 2020-04-13 2020-07-31 江南大学 一种提高枯草芽孢杆菌核酸含量的方法
CN113025550A (zh) * 2021-05-24 2021-06-25 中国科学院天津工业生物技术研究所 高产维生素b2枯草芽孢杆菌工程菌株、其构建及应用
CN113151238A (zh) * 2021-05-24 2021-07-23 廊坊梅花生物技术开发有限公司 磷酸戊糖变位酶突变体及其在构建高产核苷的枯草芽孢杆菌中的应用
CN113278596A (zh) * 2021-05-24 2021-08-20 廊坊梅花生物技术开发有限公司 可提高芽孢杆菌核苷产量的突变体及其应用
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