CN112574934B - 高产鸟苷的工程菌及其构建方法与应用 - Google Patents
高产鸟苷的工程菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明提供高产鸟苷的工程菌及其构建方法与应用。本发明还提供发酵生产嘌呤核苷的方法，包括：1)改造细菌染色体上编码NCBI参考序列WP_038462632.1或WP_014469845.1的GMP合成酶基因，使其编码的GMP合成酶的第234位缬氨酸被其他氨基酸取代，导致该酶活性增强；和/或2)改造细菌染色体上编码NCBI参考序列WP_013353436.1的GMP还原酶基因，使其编码的GMP还原酶的第8位谷氨酸突变为终止密码子，导致该酶活性减弱或失活；以及3)用步骤1)和/或步骤2)改造得到的细菌发酵生产嘌呤核苷；所述细菌是具有嘌呤核苷生产能力的微生物。包含上述两种突变的微生物可用于发酵生产鸟苷并提高鸟苷发酵产量。
Description
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，涉及高产鸟苷的工程 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)。
鸟嘌呤核苷，又名9-β-D呋喃核糖鸟嘌呤，以下也称鸟苷，具有多种用途，在食 品和医药行业有着广泛的作用。在食品领域，鸟苷是鸟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。
目前，微生物发酵是生产鸟苷的主要方法，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，定向选育鸟苷高产菌株；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、能够高产鸟苷的生产菌株。
发明内容
本发明的目的是提供一种发酵生产嘌呤核苷的方法或提高嘌呤核苷发酵产量的方法。
本发明的另一目的是提供高产鸟苷的工程菌及其构建方法与应用。
为了实现本发明目的，第一方面，本发明提供一种发酵生产嘌呤核苷的方法或 提高嘌呤核苷发酵产量的方法，包括以下步骤：
(1)改造细菌染色体上编码NCBI参考序列WP_038462632.1或WP_014469845.1 的GMP合成酶基因，使其编码的GMP合成酶的第234位缬氨酸被其他氨基酸(如异亮 氨酸)取代，导致该酶活性增强；和/或
(2)改造细菌染色体上编码NCBI参考序列WP_013353436.1的GMP还原酶基因， 使其编码的GMP还原酶的第8位谷氨酸突变为终止密码子，导致该酶活性减弱或失 活；以及
(3)用步骤(1)和/或步骤(2)改造得到的细菌发酵生产嘌呤核苷；
其中，所述细菌是具有嘌呤核苷生产能力的微生物。
优选地，所述细菌为枯草芽孢杆菌(Bacillus subtilis)或解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
本发明中，所述嘌呤核苷优选为鸟苷。
第二方面，本发明提供一种高产鸟苷的工程菌，所述工程菌是通过增强原始菌 株中编码NCBI参考序列WP_038462632.1或WP_014469845.1的基因，获得的基因增强 菌株；和/或
所述工程菌是通过弱化原始菌株中编码NCBI参考序列WP_013353436.1的基因，获得的基因弱化菌株；所述弱化包括敲除或降低基因的表达。
其中，所述原始菌株是具有嘌呤核苷生产能力的微生物。
进一步地，改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列 WP_038462632.1或WP_014469845.1的GMP合成酶基因，使其编码的GMP合成酶的第 234位缬氨酸被异亮氨酸取代，导致该酶活性增强；和/或
改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列 WP_013353436.1的GMP还原酶基因，使其编码的GMP还原酶的第8位谷氨酸突变为 终止密码子，导致该酶活性减弱或失活。
第三方面，本发明提供高产鸟苷的工程菌的构建方法，包括：利用基因工程手 段增强原始菌株中编码NCBI参考序列WP_038462632.1或WP_014469845.1的基因，获 得基因增强菌株；和/或
利用基因工程手段弱化原始菌株中编码NCBI参考序列WP_013353436.1的基因，获得的基因弱化菌株；所述弱化包括敲除或降低基因的表达。
其中，所述原始菌株是具有嘌呤核苷生产能力的微生物。
进一步地，改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列 WP_038462632.1或WP_014469845.1的GMP合成酶基因，使其编码的GMP合成酶的第 234位缬氨酸被异亮氨酸取代，导致该酶活性增强；和/或
改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列 WP_013353436.1的GMP还原酶基因，使其编码的GMP还原酶的第8位谷氨酸突变为 终止密码子，导致该酶活性减弱或失活。
上述改造的方法包括但不限于诱变、定点突变、同源重组等中的至少一种。
第四方面，本发明提供所述工程菌或按照上述方法构建的工程菌在发酵生产鸟苷中的应用。
第五方面，本发明提供GMP合成酶突变体，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GMP合成酶突变体的基因或含有该基因的生物 材料在发酵生产嘌呤核苷或提高嘌呤核苷(如鸟苷)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：
本发明提供的发酵生产鸟苷的方法，包括选育并培养以下微生物：其中GMP合 成酶第234位缬氨酸被其他氨基酸取代，导致该酶活性增强；且GMP还原酶第8位谷 氨酸突变为终止密码子，导致该酶活性减弱或失活。包含上述两种突变的微生物可 用于发酵生产鸟苷并提高鸟苷发酵产量。
具体实施方式
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。
菌株简写如下：Bacillus.subtilis JZB15083：B.s 83；Bacillus.subtilis 831：B.s 831； Bacillus.subtilis 832：B.s 832；Bacillus.subtilis 833：B.s 833；Bacillus.subtilis ATCC13952：B.s ATCC13952；Bacillus amyloliquefaciens 834：B.a834；Bacillus amyloliquefaciens 835：B.a 835；Bacillus amyloliquefaciens 836：B.a836；Bacillus amyloliquefaciens DSM7(ATCC23350)：B.a DSM7。
以下实施例中使用的培养基及培养条件如下：
种子培养基(g/L)：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，pH7.0～7.2。
发酵培养基(g/L)：葡萄糖120，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，pH7.0～7.2；
发酵条件：35.5℃发酵70h。
LB液体培养基(g/L)的配制：蛋白胨10，酵母提取物5，NaCl 10，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') |
| upp-1f | acgcgtcgaccaatccattccatgaagttctgg |
| upp-1r | caaaaaggagctgaacacagtatctgtacggaacaaaataaatcaga |
| upp-2f | tctgatttattttgttccgtacagatactgtgttcagctcctttttg |
| upp-2r | aactgcaggcataagccgaaactgatcgtt |
| guaA-1f | caaaataaggatcctctagagtcgacatgacgaagttagtgaatgaa |
| guaA-1r | atcaaaacggctatgacagaag |
| guaA-2f | cttctgtcatagccgttttgat |
| guaA-2r | ccagtgccaagcttgcatgcctgcagttattcccactcaatcgtcgcagg |
| guaC-1f | caaaataaggatcctctagagtcgacgacagccgtgaacaacgcgaagg |
| guaC-1r | ctgaatatcttagtaatcgaatac |
| guaC-2f | gtattcgattactaagatattcag |
| guaC-2r | cggccagtgccaagcttgcatgcctgcagtgccatccgcctgttccgaag |
实施例1诱变筛选获得鸟苷高产菌株
前期B.subtilis ATCC13952菌株经过多轮诱变筛选后，获得了B.subtilisJZB15083 菌株，该菌在摇瓶产鸟苷水平为18g/L，转化率15％，为一株鸟苷高产菌。
通过比较基因组学分析，B.subtilis JZB15083具有多个基因发生突变，guaAV234I、guaCG8*这两个位点的突变可能是促进鸟苷高产的因素。因此将这两个位点在野生菌 中进行点突变，进行验证。
实施例2 B.subtilis ATCC13952(Δupp)菌株的获得
因本发明采用的基因无痕编辑方法是基于温敏质粒介导的两步整合及upp反筛原理，编辑过程可参考文献A markerless gene replacement methodforB.amyloliquefaciensLL3and its usein genome reduction and improvement ofpoly-γ-glutamic acid production[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。因此 需先将目标菌株基因组上的upp基因删除，防止其对筛选的干扰。以枯草芽孢杆菌 B.subtilis ATCC13952为出发菌株，用引物upp-1f/1r、upp-2f/2r，以B.subtilisATCC13952 基因组为模板，使用pfu DNA聚合酶扩增分别得到1019bp和925bp的上、下游同源臂， 用引物upp-1f/2r扩增得到上、下游融合片段，将片段与pKSV7质粒经SalI/PstI双酶切、 连接、转化等操作后得到质粒pKSV7-Δupp，转化至B.subtilis ATCC13952，用含2.5 μg/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液体中，42℃、200rpm培养12h并传一代，菌液稀释后 涂布于含0.8μM 5-FU的LB平板筛选二次重组子，获得B.subtilis ATCC13952(Δupp) 菌株。用于后续工程菌株的构建。
实施例3 B.amyloliquefaciens DSM7(Δupp)菌株的获得
将上述实施例2中获得的pKSV7-Δupp质粒转化至B.amyloliquefaciens DSM7菌株 中，经过相同的筛选方法获得B.amyloliquefaciens DSM7(Δupp)菌株。用于后续工程菌株的构建。
实施例4枯草芽孢杆菌中guaAV234I点突变菌株的获得
以B.s ATCC13952(Δupp)菌株为出发菌。用引物guaA-1f/1r和guaA-2f/2r，以B.sATCC13952(野生型菌株)基因组为模板，使用pfu高保真DNA聚合酶扩增分别得到 guaA的上、下游同源臂；用引物guaA-1f/2r融合上、下游片段得到含有V234I点突变 的guaA片段(guaAV234I片段的核苷酸序列如SEQ ID NO:1所示，其编码蛋白的氨基酸 序列如SEQ ID NO:2所示)；将guaAV234I片段与pKSU质粒(pKSU质粒由南开大学王 淑芳教授惠赠，参见Amarkerless gene replacement method forB.amyloliquefaciensLL3and its useingenome reduction and improvement of poly-γ-glutamic acid production[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)经XbaI/PstI双酶切、连接、转化等操作后得到重组 质粒pKSU-guaAV234I。转化至B.s ATCC13952(Δupp)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5ml LB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guaAV234I点突变菌株，命名为B.s 831。
实施例5枯草芽孢杆菌中guaC E8*点突变菌株的获得
以B.s ATCC13952(Δupp)菌株为出发菌。用引物guaC-1f/1r和guaC-2f/2r，以B.sATCC13952基因组为模板，使用pfu高保真DNA聚合酶扩增分别得到guaC的上、下游 同源臂；用引物guaC-1f/2r融合上下游片段得到含有E8*点突变的guaC片段(核苷酸 序列如SEQ IDNo:3所示，其编码蛋白的氨基酸序列如SEQ ID No:4所示)；将guaC与 pKSU质粒经SalI/PstI双酶切、连接、转化等操作后得到出质粒pKSU-guaCE8*。转化 至B.s ATCC13952(Δupp)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 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guaCE8*点突变菌株，命名为B.s 832。
实施例6枯草芽孢杆菌中guaAV234I和guaCE8*双点突变菌株的获得
以B.s 831菌株为出发菌，将上述获得的pKSU-guaCE8*质粒转化至B.s 831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，筛选获得同时含有guaAV234I和 guaCE8*点突变的菌株，命名为B.s 833。
实施例7解淀粉芽孢杆菌中guaAV234I点突变菌株的获得
以B.a DSM7(ATCC23350)(Δupp)菌株为出发菌。用引物guaA-1f/1r和guaA-2f/2r， 以B.a DSM7基因组为模板，使用pfu高保真DNA聚合酶扩增分别得到guaA的上、下游 同源臂；用引物guaA-1f/2r融合上、下游片段得到含有guaAV234I点突变的片段 (guaAV234I片段的核苷酸序列如SEQ ID NO:5所示，其编码蛋白的氨基酸序列如SEQ ID NO:6所示)；将guaAV234I片段与pKSU质粒经XbaI/PstI双酶切、连接、转化等操作 后得到重组质粒pKSU-guaAV234I。转化至B.a DSM7(Δupp)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5ml LB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guaAV234I点突变菌株， 命名为B.a 834。
实施例8解淀粉芽孢杆菌中guaCE8*点突变菌株的获得
以B.a DSM7(ATCC23350)(Δupp)菌株为出发菌。用引物guaC-1f/1r和 guaC-2f/2r，以B.a DSM7基因组为模板，使用pfu高保真DNA聚合酶扩增分别得 到guaC的上、下游同源臂；用引物guaC-1f/2r融合上下游片段得到含有E8*点突变 的guaC片段(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guaC与pKSU质粒经SalI/PstI双酶切、连接、转化等操作后得到出 质粒pKSU-guaC E8*。转化至B.a DSM7(Δupp)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5ml LB液体培养基中，42℃200 rpm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guaC E8*点突变菌株命名为B.a 835。
实施例9解淀粉芽孢杆菌中guaAV234I和guaCE8*双点突变菌株的获得
以B.a 834菌株为出发菌，将pKSU-guaCE8*质粒转化至B.a834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，筛选获得同时含有 guaAV234I和guaCE8*点突变的菌株，命名为B.a 836。
实施例10工程菌株核苷合成能力对比
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种子培养基中，110rpm 37℃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℃培养70h。
4、使用液相色谱仪对发酵液中的鸟苷进行检测，经检测，工程菌株所含的突变 对腺苷生产能力也有所改善(表2)。
表2三株工程菌摇瓶发酵产鸟苷及腺苷评估结果(三次重复均值)
| 菌株 | 鸟苷产量(g/L) | 腺苷产量(g/L) |
| B.s ATCC13952(Δupp) | 0.08 | 0.05 |
| B.s 831 | 0.8 | 0.1 |
| B.s 832 | 0.2 | 0 |
| B.s 833 | 1.2 | 0.8 |
| B.a DSM7(Δupp) | 0.06 | 0.05 |
| B.a 834 | 0.6 | 0.1 |
| B.a 835 | 0.3 | 0.2 |
| B.a 836 | 1.1 | 0.3 |
上述实验结果表明，guaAV234I和/或guaCE8*点突变不仅对提高鸟苷产量有效，对 提高腺苷产量也有一定的效果。
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> 高产鸟苷的工程菌及其构建方法与应用
<130> KHP201115677.0
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atgacgaagt tagtgaatga aatgattctt gttcttgatt tcggcagtca gtataaccag 60
ctgattaccc gccggatccg tgaatttggc gtatatagcg agctgcatcc ccatacgttg 120
acagctgagg aaatcaaaga aatgaatcca aaaggaatta tcctttcagg cggaccgaac 180
agtgtatatg atgaaggatc tttccgctgt gacgaaaaaa tctttgaact cgatattcct 240
gtattgggca tctgctacgg tatgcagctg atgactcatt acctcggagg gaaagtcgag 300
gcggcaagcc agcgcgaata cggaaaagcg aacattcaga ttcaaggaac tcctgacctg 360
ttcaaagatc ttccggaaga acaagtggta tggatgagcc acggcgactt agtcgtacaa 420
gtgccggaag ggtttacggt agatgcgaca agccatcact gcccgaactc agcgatgagt 480
aaaaaagaca aaaaatggta cggcgttcag ttccacccgg aagtccgcca ttcagaatac 540
ggaaatgacc ttctgaaaaa ctttgtcttc ggtccttgtg aatgtaaagg cgaatggtca 600
atggaaaact ttatcgaaat cgaaatgcaa aaaatccgcg aaacagtcgg agacaaacaa 660
gtgctttgcg ctttgagcgg cggagttgat tcttctgtca tagccgtttt gattcataaa 720
gcgatcggcg accagctgac ttgtattttc gtcgaccacg gcctgctccg taaaggcgaa 780
gcggaaggcg tcatgaaaac gttcagcgaa ggctttaata tgaatgtcat taaagttgat 840
gcgaaagaca gattcttaaa taagctgaaa ggtgtttctg atcctgagca aaaacgcaaa 900
atcatcggca acgaattcat ttacgtattt gatgatgaag cggtcaagct gaaaggaatc 960
gattaccttg cgcaaggaac gctttacaca gacattattg aaagcggaac ggcaacagcg 1020
caaacgatca aatcgcacca caatgtcggc ggtcttcctg aagatatgca gtttgaactg 1080
atcgaaccgc ttaacactct tttcaaagat gaagtgcgcg cgctcggcac agagctcggc 1140
attccggatg atatcgtatg gcgtcagccg ttcccgggac ctggtcttgg catccgcgta 1200
ctcggcgaag taacggaaga aaaacttgaa atcgttcgtg aatcagacgc gattctgcgc 1260
gaagaagtgg caaaccacgg ccttgagcgc gacatctggc agtacttcac ggttcttcct 1320
gacatccgca gcgtcggcgt catgggagat gcgagaacgt atgattacac aatcggtatc 1380
cgtgccgtaa cttcaatcga cggcatgaca tctgactggg cgcgtatccc ttgggatgtg 1440
cttgaagtga tttcgacacg tatcgtcaat gaagtgaaac acatcaaccg cgtcgtgtat 1500
gatattacaa gtaagccgcc tgcgacgatt gagtgggaat aa 1542
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Met Thr Lys Leu Val Asn Glu Met Ile Leu Val Leu Asp Phe Gly Ser
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Ser Glu Leu His Pro His Thr Leu Thr Ala Glu Glu Ile Lys Glu Met
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Asn Pro Lys Gly Ile Ile Leu Ser Gly Gly Pro Asn Ser Val Tyr Asp
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Glu Gly Ser Phe Arg Cys Asp Glu Lys Ile Phe Glu Leu Asp Ile Pro
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Val Leu Gly Ile Cys Tyr Gly Met Gln Leu Met Thr His Tyr Leu Gly
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Gly Lys Val Glu Ala Ala Ser Gln Arg Glu Tyr Gly Lys Ala Asn Ile
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Gln Ile Gln Gly Thr Pro Asp Leu Phe Lys Asp Leu Pro Glu Glu Gln
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Val Val Trp Met Ser His Gly Asp Leu Val Val Gln Val Pro Glu Gly
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Phe Thr Val Asp Ala Thr Ser His His Cys Pro Asn Ser Ala Met Ser
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Lys Lys Asp Lys Lys Trp Tyr Gly Val Gln Phe His Pro Glu Val Arg
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His Ser Glu Tyr Gly Asn Asp Leu Leu Lys Asn Phe Val Phe Gly Pro
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Cys Glu Cys Lys Gly Glu Trp Ser Met Glu Asn Phe Ile Glu Ile Glu
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Leu Ser Gly Gly Val Asp Ser Ser Val Ile Ala Val Leu Ile His Lys
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Ala Ile Gly Asp Gln Leu Thr Cys Ile Phe Val Asp His Gly Leu Leu
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Arg Lys Gly Glu Ala Glu Gly Val Met Lys Thr Phe Ser Glu Gly Phe
260 265 270
Asn Met Asn Val Ile Lys Val Asp Ala Lys Asp Arg Phe Leu Asn Lys
275 280 285
Leu Lys Gly Val Ser Asp Pro Glu Gln Lys Arg Lys Ile Ile Gly Asn
290 295 300
Glu Phe Ile Tyr Val Phe Asp Asp Glu Ala Val Lys Leu Lys Gly Ile
305 310 315 320
Asp Tyr Leu Ala Gln Gly Thr Leu Tyr Thr Asp Ile Ile Glu Ser Gly
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Thr Ala Thr Ala Gln Thr Ile Lys Ser His His Asn Val Gly Gly Leu
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Pro Glu Asp Met Gln Phe Glu Leu Ile Glu Pro Leu Asn Thr Leu Phe
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Lys Asp Glu Val Arg Ala Leu Gly Thr Glu Leu Gly Ile Pro Asp Asp
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Glu
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atggaaaatg tattcgatta ctaagatatt cagctgattc ccgcaaaatg cattgtgaac 60
agccgttcag aatgtgatac atcagttacg ttgggcggac acacatttaa acttccggtc 120
gtgccagcca acatgcagac ggttatagat gaaaacatcg ctgcctggct cgcggaaaac 180
gggtattttt atatcatgca ccgcttcgag ccggaaaaac gcctggcttt tgttcaggat 240
atgaaagcgc gcggactgat ttcttccatc agtgtcggcg tgaaggaaaa tgattatgaa 300
ttcattcggg agctgaaagc acagcagctt gtgccggatt atatcaccat cgatatcgcg 360
cacggtcatt ccaatgccgt gatcagcatg atccaattta ttaaagaaca tgttccggaa 420
agctttgtca ttgcagggaa cgtcgggacg cctgaagccg taagggagct ggaacgcgcc 480
ggcgctgacg cgacaaaggt gggaatcggc cccggtaaag tctgcatcac aaagattaaa 540
acaggcttcg gaacaggcgg atggcaatta gccgcgcttc gctggtgcgc aaaagcggca 600
agcaaaccga tcattgccga cggcggcatc cgcacgcacg gggatattgc gaagtccatc 660
cgtttcggcg cgtctatggt catgatcgga tcattattcg ccggtcacga ggaatcaccg 720
ggacaaacgg tcgaaatcga cggaaagctt tataaagaat atttcggctc cgcttccgaa 780
tttcaaaaag gcgaaaagaa aaatgtagag ggcaaaaaaa tgcatgtcga gcataaaggg 840
tctctgcagg acacgctgat tgaaatggag caggatctgc aatcttccat ctcctacgcc 900
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ttcaacggcg acagatttta a 981
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Asp Tyr Glu Phe Ile Arg Glu Leu Lys Ala Gln Gln Leu Val Pro Asp
100 105 110
Tyr Ile Thr Ile Asp Ile Ala His Gly His Ser Asn Ala Val Ile Ser
115 120 125
Met Ile Gln Phe Ile Lys Glu His Val Pro Glu Ser Phe Val Ile Ala
130 135 140
Gly Asn Val Gly Thr Pro Glu Ala Val Arg Glu Leu Glu Arg Ala Gly
145 150 155 160
Ala Asp Ala Thr Lys Val Gly Ile Gly Pro Gly Lys Val Cys Ile Thr
165 170 175
Lys Ile Lys Thr Gly Phe Gly Thr Gly Gly Trp Gln Leu Ala Ala Leu
180 185 190
Arg Trp Cys Ala Lys Ala Ala Ser Lys Pro Ile Ile Ala Asp Gly Gly
195 200 205
Ile Arg Thr His Gly Asp Ile Ala Lys Ser Ile Arg Phe Gly Ala Ser
210 215 220
Met Val Met Ile Gly Ser Leu Phe Ala Gly His Glu Glu Ser Pro Gly
225 230 235 240
Gln Thr Val Glu Ile Asp Gly Lys Leu Tyr Lys Glu Tyr Phe Gly Ser
245 250 255
Ala Ser Glu Phe Gln Lys Gly Glu Lys Lys Asn Val Glu Gly Lys Lys
260 265 270
Met His Val Glu His Lys Gly Ser Leu Gln Asp Thr Leu Ile Glu Met
275 280 285
Glu Gln Asp Leu Gln Ser Ser Ile Ser Tyr Ala Gly Gly Asn Lys Leu
290 295 300
Glu Ala Ile Arg Asn Val Asp Tyr Val Ile Val Lys Asn Ser Ile Phe
305 310 315 320
Asn Gly Asp Arg Phe
325
<210> 5
<211> 1542
<212> DNA
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 5
atgacgaagt tagtgaatga aatgattctt gttcttgatt tcggcagtca gtataaccag 60
ctgattaccc gccggatccg tgaatttggc gtatatagcg agctgcatcc ccatacgttg 120
acagctgagg aaatcaaaga aatgaatcca aaaggaatta tcctttcagg cggaccgaac 180
agtgtatatg atgaaggatc tttccgctgt gacgaaaaaa tctttgaact cgatattcct 240
gtattgggca tctgctacgg tatgcagctg atgactcatt acctcggagg gaaagtcgag 300
gcggcaagcc agcgcgaata cggaaaagcg aacattcaga ttcaaggaac tcctgacctg 360
ttcaaagatc ttccggaaga acaagtggta tggatgagcc acggcgactt agtcgtacaa 420
gtgccggaag ggtttacggt agatgcgaca agccatcact gcccgaactc agcgatgagt 480
aaaaaagaca aaaaatggta cggcgttcag ttccacccgg aagtccgcca ttcagaatac 540
ggaaatgacc ttctgaaaaa ctttgtcttc ggtccttgtg aatgtaaagg cgaatggtca 600
atggaaaact ttatcgaaat cgaaatgcaa aaaatccgcg aaacagtcgg agacaaacaa 660
gtgctttgcg ctttgagcgg cggagttgat tcttctgtca tagccgtttt gattcataaa 720
gcgatcggcg accagctgac ttgtattttc gtcgaccacg gcctgctccg taaaggcgaa 780
gcggaaggcg tcatgaaaac gttcagcgaa ggctttaata tgaatgtcat taaagttgat 840
gcgaaagaca gattcttaaa taagctgaaa ggtgtttctg atcctgagca aaaacgcaaa 900
atcatcggca acgaattcat ttacgtattt gatgatgaag cggtcaagct gaaaggaatc 960
gattaccttg cgcaaggaac gctttacaca gacattattg aaagcggaac ggcaacagcg 1020
caaacgatca aatcgcacca caatgtcggc ggtcttcctg aagatatgca gtttgaactg 1080
atcgaaccgc ttaacactct tttcaaagat gaagtgcgcg cgctcggcac agagctcggc 1140
attccggatg atatcgtatg gcgtcagccg ttcccgggac ctggtcttgg catccgcgta 1200
ctcggcgaag taacggaaga aaaacttgaa atcgttcgtg aatcagacgc gattctgcgc 1260
gaagaagtgg caaaccacgg ccttgagcgc gacatctggc agtacttcac ggttcttcct 1320
gacatccgca gcgtcggcgt catgggagat gcgagatcgt atgattacac aatcggtatc 1380
cgtgccgtaa cttcaatcga cggcatgaca tctgactggg cgcgtatccc ttgggatgtg 1440
cttgaagtga tttcgacacg tatcgtcaat gaagtgaaac acatcaaccg cgtcgtgtat 1500
gatattacaa gtaagccgcc tgcgacgatt gagtgggaat aa 1542
<210> 6
<211> 513
<212> PRT
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 6
Met Thr Lys Leu Val Asn Glu Met Ile Leu Val Leu Asp Phe Gly Ser
1 5 10 15
Gln Tyr Asn Gln Leu Ile Thr Arg Arg Ile Arg Glu Phe Gly Val Tyr
20 25 30
Ser Glu Leu His Pro His Thr Leu Thr Ala Glu Glu Ile Lys Glu Met
35 40 45
Asn Pro Lys Gly Ile Ile Leu Ser Gly Gly Pro Asn Ser Val Tyr Asp
50 55 60
Glu Gly Ser Phe Arg Cys Asp Glu Lys Ile Phe Glu Leu Asp Ile Pro
65 70 75 80
Val Leu Gly Ile Cys Tyr Gly Met Gln Leu Met Thr His Tyr Leu Gly
85 90 95
Gly Lys Val Glu Ala Ala Ser Gln Arg Glu Tyr Gly Lys Ala Asn Ile
100 105 110
Gln Ile Gln Gly Thr Pro Asp Leu Phe Lys Asp Leu Pro Glu Glu Gln
115 120 125
Val Val Trp Met Ser His Gly Asp Leu Val Val Gln Val Pro Glu Gly
130 135 140
Phe Thr Val Asp Ala Thr Ser His His Cys Pro Asn Ser Ala Met Ser
145 150 155 160
Lys Lys Asp Lys Lys Trp Tyr Gly Val Gln Phe His Pro Glu Val Arg
165 170 175
His Ser Glu Tyr Gly Asn Asp Leu Leu Lys Asn Phe Val Phe Gly Pro
180 185 190
Cys Glu Cys Lys Gly Glu Trp Ser Met Glu Asn Phe Ile Glu Ile Glu
195 200 205
Met Gln Lys Ile Arg Glu Thr Val Gly Asp Lys Gln Val Leu Cys Ala
210 215 220
Leu Ser Gly Gly Val Asp Ser Ser Val Ile Ala Val Leu Ile His Lys
225 230 235 240
Ala Ile Gly Asp Gln Leu Thr Cys Ile Phe Val Asp His Gly Leu Leu
245 250 255
Arg Lys Gly Glu Ala Glu Gly Val Met Lys Thr Phe Ser Glu Gly Phe
260 265 270
Asn Met Asn Val Ile Lys Val Asp Ala Lys Asp Arg Phe Leu Asn Lys
275 280 285
Leu Lys Gly Val Ser Asp Pro Glu Gln Lys Arg Lys Ile Ile Gly Asn
290 295 300
Glu Phe Ile Tyr Val Phe Asp Asp Glu Ala Val Lys Leu Lys Gly Ile
305 310 315 320
Asp Tyr Leu Ala Gln Gly Thr Leu Tyr Thr Asp Ile Ile Glu Ser Gly
325 330 335
Thr Ala Thr Ala Gln Thr Ile Lys Ser His His Asn Val Gly Gly Leu
340 345 350
Pro Glu Asp Met Gln Phe Glu Leu Ile Glu Pro Leu Asn Thr Leu Phe
355 360 365
Lys Asp Glu Val Arg Ala Leu Gly Thr Glu Leu Gly Ile Pro Asp Asp
370 375 380
Ile Val Trp Arg Gln Pro Phe Pro Gly Pro Gly Leu Gly Ile Arg Val
385 390 395 400
Leu Gly Glu Val Thr Glu Glu Lys Leu Glu Ile Val Arg Glu Ser Asp
405 410 415
Ala Ile Leu Arg Glu Glu Val Ala Asn His Gly Leu Glu Arg Asp Ile
420 425 430
Trp Gln Tyr Phe Thr Val Leu Pro Asp Ile Arg Ser Val Gly Val Met
435 440 445
Gly Asp Ala Arg Ser Tyr Asp Tyr Thr Ile Gly Ile Arg Ala Val Thr
450 455 460
Ser Ile Asp Gly Met Thr Ser Asp Trp Ala Arg Ile Pro Trp Asp Val
465 470 475 480
Leu Glu Val Ile Ser Thr Arg Ile Val Asn Glu Val Lys His Ile Asn
485 490 495
Arg Val Val Tyr Asp Ile Thr Ser Lys Pro Pro Ala Thr Ile Glu Trp
500 505 510
Glu
Claims (6)
1.发酵生产嘌呤核苷的方法或提高嘌呤核苷发酵产量的方法，其特征在于，包括以下步骤：
(1)改造细菌染色体上编码NCBI参考序列WP_038462632.1或WP_014469845.1的GMP合成酶基因，使其编码的GMP合成酶的第234位氨基酸被异亮氨酸取代，导致该酶活性增强；和/或
(2)改造细菌染色体上编码NCBI参考序列WP_013353436.1的GMP还原酶基因，使其编码的GMP还原酶的第8位谷氨酸突变为终止密码子，导致该酶活性减弱或失活；以及
(3)用步骤(1)和/或步骤(2)改造得到的细菌发酵生产嘌呤核苷；
其中，所述细菌为枯草芽孢杆菌(Bacillus subtilis)或解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。
2.根据权利要求1所述的方法，其特征在于，所述嘌呤核苷为鸟苷。
3.高产鸟苷的工程菌，其特征在于，所述工程菌是通过改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列WP_038462632.1或WP_014469845.1的GMP合成酶基因，使其编码的GMP合成酶的第234位氨基酸被异亮氨酸取代，导致该酶活性增强的菌株；和/或
所述工程菌是通过改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列WP_013353436.1的GMP还原酶基因，使其编码的GMP还原酶的第8位谷氨酸突变为终止密码子，导致该酶活性减弱或失活的菌株。
4.高产鸟苷的工程菌的构建方法，其特征在于，包括：
改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列WP_038462632.1或WP_014469845.1的GMP合成酶基因，使其编码的GMP合成酶的第234位氨基酸被异亮氨酸取代，导致该酶活性增强；和/或
改造枯草芽孢杆菌或解淀粉芽孢杆菌染色体上编码NCBI参考序列WP_013353436.1的GMP还原酶基因，使其编码的GMP还原酶的第8位谷氨酸突变为终止密码子，导致该酶活性减弱或失活。
5.根据权利要求4所述的方法，其特征在于，改造的方法选自诱变、定点突变、同源重组中的至少一种。
6.权利要求3所述的工程菌或按照权利要求4或5所述方法构建的工程菌在发酵生产鸟苷中的应用。
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| CN113151238B (zh) * | 2021-05-24 | 2022-09-30 | 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 |
| CN113278596B (zh) * | 2021-05-24 | 2022-07-29 | 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 |
| CN116157524B (zh) * | 2021-09-23 | 2023-10-20 | Cj第一制糖株式会社 | 新型谷氨酰胺-水解gmp合酶变体和用于使用其生产嘌呤核苷酸的方法 |
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