CN114958693A - 一株枯草芽孢杆菌、一种重组枯草芽孢杆菌及其应用 - Google Patents
一株枯草芽孢杆菌、一种重组枯草芽孢杆菌及其应用 Download PDFInfo
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- CN114958693A CN114958693A CN202210770685.2A CN202210770685A CN114958693A CN 114958693 A CN114958693 A CN 114958693A CN 202210770685 A CN202210770685 A CN 202210770685A CN 114958693 A CN114958693 A CN 114958693A
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- bacillus subtilis
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- riboflavin
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Abstract
本发明提供了一株枯草芽孢杆菌、一种重组枯草芽孢杆菌及其应用，属于微生物发酵技术领域。本发明的枯草芽孢杆菌(Bacillussubtilis)RF1‑6是以核黄素高产菌株RF1作为出发菌株，经过基因改造和诱变，筛选得到的核黄素产量最高的突变株，保藏编号为CCTCCNO：M2022565。与核黄素高产菌株RF1相比，核黄素产量能够提高22.8％。
Description
技术领域
本发明属于微生物发酵技术领域，具体涉及一株枯草芽孢杆菌、一种重组枯草芽孢杆菌及其应用。
背景技术
核黄素，又称维生素B2，是一种独特的水溶性维生素，具有见光易分解的特性。它最初是在1879年从乳清中分离出来的，并被命名为牛奶色素。核黄素可以结晶成橘黄色的晶体，但其纯形态很难溶于水，可溶于氯化钠溶液，易溶于稀的氢氧化钠溶液，在碱性溶液中容易溶解，在强酸溶液中稳定。它主要由植物和微生物合成，是一种重要的动物营养物质。它对动物来说是一种重要的营养物质，需要从外界获取。核黄素在动物体内转化为两种活性物质:黄素腺嘌呤二核苷酸(FAD)和黄素单核核苷酸(FMN)，作为氧化还原酶氧化途径酶的辅因子，参与一系列氧化还原反应，其中一些对有氧细胞的功能至关重要。当缺乏时，就影响机体的生物氧化，使代谢发生障碍。其病变多表现为口、眼和外生殖器部位的炎症。
核黄素主要用于医药、食品添加剂、饲料加工等行业，也被用于临床上癌症治疗和预防。目前国内外广泛采用微生物发酵法工业生产核黄素，能够合成核黄素的微生物有细菌、真菌和霉菌，工业生产中主要以枯草芽孢杆菌和阿舒假囊酵母作为生产菌株。目前文献中报道的枯草芽孢杆菌合成核黄素的最高产量为15.7g/L(参见【Wang，Z.，Chen，T.，Ma，X.，Shen，Z.and Zhao，X.Enhancement of riboflavin production with Bacillus subtilisby expression and site-directed mutagenesis of zwf and gnd gene fromCorynebacterium glutamicum[J].Bioresource Technology,2011,102(4):3934-3940】)，但是这一产量仍无法满足工业生产需求。
发明内容
有鉴于此，本发明的目的在于提供一株枯草芽孢杆菌、一种重组枯草芽孢杆菌及其应用，本发明的枯草芽孢杆菌和重组枯草芽孢杆菌的核黄素产量高。
本发明提供了一株枯草芽孢杆菌(Bacillussubtilis)RF1-6，保藏编号为CCTCCNO：M 2022565。
本发明还提供了一种重组枯草芽孢杆菌，以上述方案所述枯草芽孢杆菌(Bacillussubtilis)RF1-6作为原始菌株，包含重组质粒；所述重组质粒上插入有zwf基因、ywlf基因和ribBA基因；所述重组质粒的原始质粒优选为pMA5-sat；所述zwf基因、ywlf基因和ribBA基因优选的以同源重组的方式连接到所述原始质粒上。
优选的，所述zwf基因、ywlf基因和ribBA基因顺次插入在所述重组质粒的EcoRI和KpnI酶切位点之间。
优选的，所述重组质粒上还插入有强启动子P43。
优选的，所述强启动子P43插入在zwf基因上游的EcoRI酶切位点处。
优选的，所述重组枯草芽孢杆菌中pgi基因敲低。
优选的，所述重组枯草芽孢杆菌中purR基因敲除。
优选的，所述重组枯草芽孢杆菌包括枯草芽孢杆菌RF1-6ZYRS，保藏编号为CCTCCNO：M 2022566。
本发明还提供了一种菌剂，包括上述方案所述枯草芽孢杆菌或者包括上述方案所述的重组枯草芽孢杆菌。
本发明还提供了上述方案所述的枯草芽孢杆菌RF1-6或者所述的重组枯草芽孢杆菌或者所述的菌剂在合成核黄素中的应用。
本发明提供了一株枯草芽孢杆菌(Bacillus subtilis)RF1-6，本发明的枯草芽孢杆菌(Bacillus subtilis)RF1-6是以核黄素高产菌株RF1作为出发菌株，经过基因改造和诱变，筛选得到的核黄素产量最高的突变株，保藏编号为CCTCCNO：M 2022565。与核黄素高产菌株RF1相比，核黄素产量能够提高22.8％。
本发明还提供了一株基于枯草芽孢杆菌RF1-6构建的工程菌枯草芽孢杆菌RF1-6ZYRS，枯草芽孢杆菌RF1-6ZYRS的核黄素产量最高产量达到25.2g/L，相对于所述枯草芽孢杆菌RF1-6最终产量提高69.58％。
生物保藏说明
枯草芽孢杆菌(Bacillus subtilis)RF1-6，于2022年5月9日保藏在中国典型培养物保藏中心，地址为湖北省武汉市武昌区八一路299号，保藏编号为：CCTCC NO：M 2022565。
枯草芽孢杆菌(Bacillus subtilis)RF1-6ZYRS，于2022年5月9日保藏在中国典型培养物保藏中心，地址为湖北省武汉市武昌区八一路299号，保藏编号为：CCTCC NO：M2022566。
附图说明
图1为本发明的诱变质粒图谱；
图2为报告系统工作原理；
图3为摇瓶复筛结果；
图4为突变株RF1-6发酵罐发酵结果；
图5为菌剂RF1-6ZYPS在5L发酵罐发酵结果。
具体实施方式
本发明提供了一株枯草芽孢杆菌(Bacillus subtilis)RF1-6，保藏编号为CCTCCNO：M 2022565。
在本发明中，所述枯草芽孢杆菌RF1-6以核黄素高产菌株RF1作为出发菌株，经过基因改造和诱变，筛选得到的核黄素产量最高的突变株。与核黄素高产菌株RF1相比，核黄素产量能够提高22.8％。
本发明还提供了一种重组枯草芽孢杆菌，以上述方案所述枯草芽孢杆菌(Bacillus subtilis)RF1-6作为原始菌株，包含重组质粒；所述重组质粒上插入有zwf基因、ywlf基因和ribBA基因。
本发明通过对核黄素高产菌株RF1-6进行代谢改造，增加核黄素代谢流，从而增加核黄素产量。
在本发明中，所述zwf基因、ywlf基因和ribBA基因优选的顺次插入在所述重组质粒的EcoRI和KpnI酶切位点之间；所述重组质粒上优选的还插入有强启动子P43；所述强启动子P43优选的插入在zwf基因上游的EcoRI酶切位点处。所述重组质粒的原始质粒优选为pMA5-sat；所述zwf基因、ywlf基因和ribBA基因优选的以同源重组的方式连接到所述原始质粒上。
在本发明中，zwf编码葡萄糖-6-磷酸酶脱氢酶，ribBA编码一种双功能GTP环水解酶II/3,4-二羟基-2-丁酮4-磷酸合酶，ywlf编码核糖5-磷酸异构酶B。这三个基因共同促进核黄素合成代谢流，提高核黄素产量。
本发明利用强启动子P43控制zwf基因、ywlf基因和ribBA基因表达。
在本发明中，所述重组枯草芽孢杆菌中pgi基因优选的敲低，通过sRNA敲低技术，降低基因pgi的表达水平，使代谢流流向磷酸戊糖途径。在本发明中，所述重组枯草芽孢杆菌中purR基因优选的敲除。通过将purR基因敲除，解除细胞内反馈抑制，使细胞内合成更多的前体物质GTP。
在本发明中，所述重组枯草芽孢杆菌优选的包括枯草芽孢杆菌RF1-6ZYRS，保藏编号为CCTCC NO：M 2022566。
在本发明中，所述枯草芽孢杆菌RF1-6ZYRS的核黄素产量最高产量达到25.2g/L，相对于所述枯草芽孢杆菌RF1-6最终产量提高69.58％。
本发明还提供了一种菌剂，包括上述方案所述枯草芽孢杆菌或者包括上述方案所述的重组枯草芽孢杆菌。
本发明还提供了上述方案所述的枯草芽孢杆菌RF1-6或者所述的重组枯草芽孢杆菌或者所述的菌剂在合成核黄素中的应用。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。
下述实施例中所涉及的培养基如下：
LB固体培养基：10g/L蛋白胨、5g/L的酵母膏、10g/L的NaCl、0.2g/L的琼脂粉。
LB液体培养基：10g/L蛋白胨、5g/L的酵母膏、10g/L的NaCl。
摇瓶发酵培养基：20g/L葡萄糖，20g/L酵母粉，4g/L柠檬酸铵，1g/L K2HPO4，1g/LKH2PO4，2g/LMgSO4·7H2O，0.04g/LMnCl2，0.06g/L CaCl2，2g/L CuSO4，pH 6.8。
种子培养基：40g/L葡萄糖、5g/L酵母膏、10g/L蛋白胨、10g/LNaCl和10μg/m L氯霉素。
分批补料发酵培养基：20g/L葡萄糖，20g/L酵母粉，6g/L(NH4)2HPO4，5g/LK2HPO4，1.5g/LMgSO4·7H2O，0.03g/LZnSO4·7H2O，0.05g/LMnCl2，0.02g/LFeSO4·7H2O。
补料培养基：600g/L葡萄糖，10g/L酵母粉，6g/L(NH4)2HPO4，5g/L K2HPO4，0.5g/LMgSO4·7H2O。
上述培养基均以水为溶剂。
下述实施例中所涉及的检测方法如下：
用分光光度计监测OD600 nm下细胞的生长。
将制备得到的发酵液用0.01M NaOH稀释，然后在12000rpm下离心2min，取上清液测定核黄素浓度。将上清液转移到新的EP管中，并稀释至合适的浓度范围(0.3～0.8)，使用分光光度计在OD444 nm处测量吸光度值。核黄素浓度根据核黄素浓度标准曲线计算。按照核黄素标准曲线计算公式为：OD444*稀释倍数*30/1000。
使用Glucose analysis(Model-SBA40，Shandong，China)测量葡萄糖浓度。
实施例1诱变质粒的构建
1.构建诱变质粒：从质粒MP6上扩增dam、seq基因，ugi基因来源于大肠杆菌基因组，将PCR产物通过琼脂糖凝胶电泳分离PCR产物，并切胶回收目的PCR产物。通过重叠PCR的方式将三个片段融合。首先将上下游片段按体积比1:1混合，加入等体积的PCR酶进行融合PCR反应，条件为98℃、3min，98℃、8s，61℃、5s，72℃、2min，扩增13个循环，以该步反应后的产物为模板，使用引物扩增融合片段，反应条件为：98℃、3min，98℃、10s，58℃、15s，72℃、1min，扩增34个循环。将PCR产物回收，并使用Gibson组装试剂盒，将融合片段连接于pBT2质粒的HindIII和BamHI位点上，由强启动子P43控制基因表达，构建pBT2-M3质粒。pBT2质粒为温度敏感型质粒，在42℃时该质粒会发生丢失，不会对突变株遗传稳定性产生影响。基因mutL来源于大肠杆菌，按照上述方法扩增并连于质粒pET28a上，构建质粒pET28a-mutL，利用反向扩增引物扩增质粒pET28a-mutL，在引物上含有突变位点，在基因mutL上引入突变，构建质粒突变的质粒pET28a-mutL(E32K)。以构建好的突变基因mutL为模板，扩增基因mutL的PCR片段，并按照上述的Gibson组装方式，将突变的mutl基因连于pBT2-M3质粒上，构建诱变质粒pBT2-M4。
2.报告质粒构建：从枯草芽孢杆菌168基因组上克隆rib操纵子上游5’端的FMNswitch序列，并扩增报告基因gfp，按照上述重叠PCR方法将FMN核糖开关与gfp融合，形成融合的FMNswitch-gfp片段。扩增基因amyE上游同源臂(1000bp)，下游同源臂(1000bp)和Marker片段(含有博来霉素抗性基因和lox66-lox71重组位点)，并通过琼脂糖凝胶电泳分离PCR产物，并切胶回收目的PCR产物。按照上述重叠PCR方法，将融合的PCR片段整合到核黄素高产菌株RF1基因组上。
3.突变文库构建：诱变质粒pBT2-M4导入含有报告质粒的核黄素高产菌株RF1中，将细胞培养至对数期(OD600＝0.6)，然后取对数期的细胞进行室温等离子诱变(ARTP)诱变处理，处理后的突变体文库在无机盐培养基中培养12h，取样收集菌体，用PBS缓冲液洗涤3次，然后将洗涤的细胞用PBS重选，然后稀释到合适的菌浓并用于流式细胞分选仪分选，选取荧光强度低于对照菌株(ARTP未处理)的菌群，并进行后培养。
4.将分离的突变体涂布与抗性平板，在37℃下培养24h后，用高通量菌落挑选仪将平板上荧光强度较弱的菌落挑至含有无机盐培养基的96孔板中，震荡培养24h测定OD444处的吸光值，核黄素在该波长下具有最大吸收峰，可以间接反映核黄素浓度。每轮挑取1000个细胞，进行复筛鉴定。计算核黄素合成能力(OD444/OD600)，挑选产量最高的突变株。
5.摇瓶复筛，将筛选到的突变株接种到含有50ml发酵培养基的250ml摇瓶中，200r/min，41℃培养48h，测定核黄素浓度，再次筛选产量最高的突变株。
6.将筛选到的突变株在平板上画线纯化，纯化后的菌落接种到LBG培养基中在42℃条件下培养，丢掉诱变质粒，并进行摇瓶发酵确定其最终产量。经过复筛，选择10株产量最高的突变株进行摇瓶发酵，结果显示，产量最高的突变株核黄素产量提高22.8％，命名为RF1-6，保藏编号为CCTCC NO：M 2022565。
5L发酵罐鉴定突变株核黄素产量，摇瓶实验筛选得到的高产突变株RF1-6在5L发酵罐测定核黄素产量，具体步骤如下：
(1)分别将在10mL LB培养基中培养24h的枯草芽孢杆菌菌株RF1-6按照体积比3％的接种量接种到100mL种子培养基中，温度为41℃，转速为180rpm，培养16h后，制备得到种子液(OD600＝21.2)；
(2)将制备得到的100mL种子液全部接种到含有1900mL的发酵培养基的5L发酵罐中，进行分批补料发酵。
通过控制补料培养基流量，使发酵液中的剩余葡萄糖浓度保持在不低于5g/L。发酵过程中，发酵液pH为6.8，加1M H2SO4和50％氨水。在开始分批进料前，转速保持在400rpm，然后提高到800rpm直到发酵结束，温度始终保持在41℃。补料发酵培养60h，测定核黄素产量，核黄素浓度达到14.86g/L。与出发菌株核黄素高产菌株RF1的核黄素浓度为9.8g/l相比，产量提高了48.9％。因此诱变系统与高通量筛选系统可有效改进工业菌株的性能，提高代谢物合成。
7.为了进一步提高核黄素产量，通过传统的基因操作对高产菌株进行代谢改造，增加核黄素代谢流，从而增加核黄素产量。首先我们构建过表达质粒，将基因zwf、ywlf和ribBA用同源重组的方式连接到pMA5-sat质粒上，利用强启动子P43控制基因表达，构建菌剂RF1-6ZYP，然后通过sRNA敲低技术，降低基因pgi的表达水平，使代谢流流向磷酸戊糖途径。为了增加前体物质GTP的合成，将purR基因敲除，解除细胞内反馈抑制，使细胞内合成更多的GTP。获得的菌剂命名为RF1-6ZYRS，保藏编号为CCTCC NO：M 2022566。经过一系列代谢改造获得的菌剂在5L发酵罐水平核黄素产量大幅度提高，其最高产量达到25.2g/L，最终产量提高69.58％。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (10)
1.一株枯草芽孢杆菌(Bacillus subtilis)RF1-6，保藏编号为CCTCC NO：M 2022565。
2.一种重组枯草芽孢杆菌，以权利要求1所述枯草芽孢杆菌(Bacillus subtilis)RF1-6作为原始菌株，包含重组质粒；所述重组质粒上插入有zwf基因、ywlf基因和ribBA基因；
所述重组质粒的原始质粒优选为pMA5-sat；
所述zwf基因、ywlf基因和ribBA基因优选的以同源重组的方式连接到所述原始质粒上。
3.根据权利要求2所述的重组枯草芽孢杆菌，其特征在于，所述zwf基因、ywlf基因和ribBA基因顺次插入在所述重组质粒的EcoRI和KpnI酶切位点之间。
4.根据权利要求3所述的重组枯草芽孢杆菌，其特征在于，所述重组质粒上还插入有强启动子P43。
5.根据权利要求4所述的重组枯草芽孢杆菌，其特征在于，所述强启动子P43插入在zwf基因上游的EcoRI酶切位点处。
6.根据权利要求2～5任意一项所述的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌中pgi基因敲低。
7.根据权利要求2～5任意一项所述的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌中purR基因敲除。
8.根据权利要求5所述的重组枯草芽孢杆菌，其特征在于，所述重组枯草芽孢杆菌包括枯草芽孢杆菌RF1-6ZYRS，保藏编号为CCTCC NO：M2022566。
9.一种菌剂，包括权利要求1所述枯草芽孢杆菌或者包括权利要求2～8任意一项所述的重组枯草芽孢杆菌。
10.权利要求1所述的枯草芽孢杆菌RF1-6或者权利要求2～8任意一项所述的重组枯草芽孢杆菌或者权利要求9所述的菌剂在合成核黄素中的应用。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568433A (zh) * | 2024-01-15 | 2024-02-20 | 黑龙江新和成生物科技有限公司 | 一种提高枯草芽孢杆菌发酵生产核黄素的方法 |
| CN118360309A (zh) * | 2024-04-18 | 2024-07-19 | 苏州华赛生物工程技术有限公司 | 下调pgi在生产腺苷中的应用 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1891813A (zh) * | 2006-05-17 | 2007-01-10 | 天津大学 | 产核黄素的工程菌株及其构建方法 |
| CN104531745A (zh) * | 2014-12-09 | 2015-04-22 | 江南大学 | 一种新型抗性质粒的构建及其在核黄素生产菌中的运用 |
| CN110591990A (zh) * | 2019-07-05 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | 一株高产核黄素工程菌株及其应用 |
| CN112538453A (zh) * | 2020-12-15 | 2021-03-23 | 通辽梅花生物科技有限公司 | 一种产核黄素的枯草芽孢杆菌及其构建方法与应用 |
| CN113073074A (zh) * | 2021-04-12 | 2021-07-06 | 江南大学 | 一种高效合成核黄素的基因工程菌及其应用 |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1891813A (zh) * | 2006-05-17 | 2007-01-10 | 天津大学 | 产核黄素的工程菌株及其构建方法 |
| CN104531745A (zh) * | 2014-12-09 | 2015-04-22 | 江南大学 | 一种新型抗性质粒的构建及其在核黄素生产菌中的运用 |
| CN110591990A (zh) * | 2019-07-05 | 2019-12-20 | 中国科学院天津工业生物技术研究所 | 一株高产核黄素工程菌株及其应用 |
| CN112538453A (zh) * | 2020-12-15 | 2021-03-23 | 通辽梅花生物科技有限公司 | 一种产核黄素的枯草芽孢杆菌及其构建方法与应用 |
| CN113073074A (zh) * | 2021-04-12 | 2021-07-06 | 江南大学 | 一种高效合成核黄素的基因工程菌及其应用 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568433A (zh) * | 2024-01-15 | 2024-02-20 | 黑龙江新和成生物科技有限公司 | 一种提高枯草芽孢杆菌发酵生产核黄素的方法 |
| CN117568433B (zh) * | 2024-01-15 | 2024-04-16 | 黑龙江新和成生物科技有限公司 | 一种提高枯草芽孢杆菌发酵生产核黄素的方法 |
| CN118360309A (zh) * | 2024-04-18 | 2024-07-19 | 苏州华赛生物工程技术有限公司 | 下调pgi在生产腺苷中的应用 |
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