CN114231477A - 高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用 - Google Patents
高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用 Download PDFInfo
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Abstract
本发明公开了一种高产β‑烟酰胺单核苷酸的基因工程菌株,构建步骤如下:过表达NAD+合成酶基因NadE、烟酸磷酸核糖转移酶基因PncB和转运蛋白基因PnuC三种基因中的任意一个或两个或三个。本发明基因工程菌是以枯草芽孢杆菌或大肠杆菌或酵母为底盘菌株,在底盘菌株中至少过表达NAD+合成酶NadE、烟酸磷酸核糖基转移酶PncB与转运蛋白PnuC其中的一个基因获得的,从而实现微生物发酵高产β‑烟酰胺单核苷酸。本发明无需使用ATP、烟酰胺等昂贵原料,经济效益显著。
Description
技术领域
本发明属生物技术领域,尤其是一种高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用。
背景技术
β-烟酰胺单核酸(Nicotinamide mononucleotide,NMN)是合成辅酶I的前体物质,研究表明其具有抗衰老活性,此外,NMN对胰岛素的分泌也能起到调节作用,对mRNA的表达水平也有影响,NMN在医疗健康领域有着广泛的应用前景。
在早期,限于研究技术水平,NMN的获取只能通过实验室合成,限制了NMN在多个方面的推广应用。近些年来,逐渐出现了工业化量产的方法。
2002年王波等人的专利尼克酰胺腺嘌呤二核苷酸的制备方法(CN.102876759)中提出以烟酰胺与四乙酰核糖为起始原料,经三氟甲磺酸三甲基硅酯(TMSOTf)缩合、脱乙酰基经活性炭色谱分离并重结晶得12,收率为58%,再经三氯氧磷/磷酸三甲酯磷酸化制得β-NMN。该方法避免了溴化氢和二氧化硫的使用,脱乙酰基无需碱的加入,采用一锅法制备中间体,但同时产物产生消旋,α异构体约为13%,且两种异构体的分离困难,为工业化生产带来一定困难。2018年魏霞蔚等人的专利制备β-烟酰胺单核苷酸或β-烟酰胺核糖的方法(CN109053838)改进了上述路线,以烟酸乙酯与四乙酰核糖为起始原料,经TMSOTf缩合、脱乙酰基、三氯氧磷/磷酸三甲酯磷酸化、氨解共4步反应制得β-NMN,此方法总收率约为64%。2018年,索韦等人的专利烟酰胺单核苷酸的有效合成(CN 107613990)将缩酮化保护的烟酰胺核糖磷酸化、脱保护制备β-NMN,总收率约为69%。化学合成方法在一定程度上解决了量产的问题,但也存在其它不利因素,一方面部分化学原料毒性较大,工人工作环境较差,另一方面部分原料价格昂贵成本居高不下。
2018年,竺伟等人的专利固定化全细胞一步酶法催化制备β-烟酰胺单核苷酸(CN2018109407295)中以D-5-磷酸核糖和烟酰胺为原料,在ATP存在下,用固定化含有磷酸核糖焦磷酸合成酶(PRPPs)和烟酰胺磷酸核糖转移酶(NAMPT)的基因工程菌全细胞催化,一步实现高效生物合成β-NMN。2018年,傅荣昭等人的专利一种烟酰胺磷酸核糖转移酶突变体及其应用(CN 2018023206)中通过基因定点突变的方法人工构建了多种具有高催化活性的NAMPT突变体,以烟酰胺(NAM)和磷酸核糖焦磷酸(PRPP)为底物催化合成了β-NMN。与现有天然的NAMPT突变体相比,人工获得的突变体相比,人工获得的突变体催化活性更强,工业应用价值更高。同年,GeorgeMarinescu等人的文章(β-nicotinamide mononucleotide(NMN)production in Escherichia coli.Scientific Reports,2018,8(1):12278.)中也提出通过构建烟酰胺磷酸核糖转移酶(NAMPT)和磷酸核糖焦磷酸合酶(PRPPs)双基因共表达载体,以大肠杆菌为底盘细胞,以烟酰胺为原料发酵生成β-NMN。2019年,祝俊等人的专利一种烟酰胺核糖激酶(NRK)突变体及其应用(CN110373398A)中以烟酰胺核糖(NR)为原料,使用该突变酶催化烟酰胺核糖10~50g/L,pH 6~7的条件下,配合ATP作用,催化合成β-NMN,最高转化率大于90%。2020年,陶荣盛等人的专利一种制备烟酰胺单核苷酸的方法(CN112159831 A)提供了一种制备烟酰胺单核苷酸的方法,其以次黄苷酸和/或鸟苷酸、ATP和烟酰胺为原料,用核苷酶、核糖磷酸焦磷酸激酶和烟酰胺磷酸核糖转移酶进行联合催化反应,一锅法合成得到烟酰胺单核苷酸。现有生物法虽然能够绿色生产NMN,但需使用ATP、烟酰胺、D-5-磷酸核糖、次黄苷酸等为原料,生产成本依然十分昂贵。
因此,本发明旨在提出一种通过构建NAD+合成酶NadE、烟酸磷酸核糖基转移酶PncB、转运蛋白PnuC的基因工程菌株,以葡萄糖或甘油等廉价原料直接发酵生产NMN的新方法。
发明内容
本发明目的在于克服现有技术中的生产原料昂贵、毒性较大、步骤繁琐、产生α异构体副产物和无机盐杂质等不足之处,提供一种高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用。
本发明解决技术问题所采用的技术方案是:
一种高产β-烟酰胺单核苷酸的基因工程菌株,构建步骤如下:
过表达NAD+合成酶基因NadE、烟酸磷酸核糖转移酶基因PncB和转运蛋白基因PnuC三种基因中的任意一个或两个或三个。
进一步地,所述基因工程菌株使用的底盘菌株包括枯草芽孢杆菌、大肠杆菌、酿酒酵母、毕赤酵母菌。
进一步地,所述枯草芽孢杆菌包括B.subtilis 168、B.subtilis WB600、B.subtilis WB800;
或者,所述大肠杆菌包括E.coli BL21(DE3)、E.coli BL21(DE3)pLysS、E.coliRosetta(DE3)、E.coli JM109(DE3);
或者,构建时使用的过表达载体质粒包括pMA5、pWB980、pHT43、pHT01和pET22b、pET28a、pET30a、pUC57。
进一步地,过表达的NAD+合成酶基因NadE的氨基酸序列与序列SEQ ID No.1具有至少95%的一致性;
或者,过表达的烟酸磷酸糖基转移酶基因PncB的氨基酸序列与序列SEQ ID No.2具有至少95%的一致性;
或者,过表达的转运蛋白基因PnuC的核苷酸序列与序列SEQ ID No.3具有至少95%的一致性;
或者,过表达NAD+合成酶基因NadE的氨基酸序列与序列SEQ ID No.4具有95%的一致性;
或者,过表达的烟酸磷酸糖基转移酶基因PncB的氨基酸序列与序列SEQ ID No.5具有至少95%的一致性。
进一步地,所述基因工程菌株包括Bacillus subtilis NadE(B.S.N)或Bacillussubtilis 168NadE1(B.S168.N1)、Bacillus subtilis PncB1(B.S.P1)或Bacillussubtilis PncB2(B.S.P2)或Bacillus subtilis WB800PncB(B.S800.P3)或Bacillussubtilis WB600PncB(B.S800.P4)、Bacillus subtilis NadE-PncB1(B.S.PN1)或Bacillussubtilis NadE-PncB2(B.S.PN2)或Bacillus subtilis PncB2-PnuC(B.S.PP)或E.coliBL21(DE3)NadE(E.N)或E.coli BL21(DE3)pLysS NadE2(E.N2)或E.coli Rosetta(DE3)NadE3(E.N3)或E.coli BL21(DE3)PncB(E.P)或E.coli JM109(DE3)PncB(E.P2)或E.coliBL21(DE3)NadE-PncB(E.NP)。
如上所述的基因工程的构建方法,其特征在于:步骤如下:
一、重组质粒的构建:
⑴目的片段基因的获得:根据目的基因设计引物扩增,或者根据基因序列直接合成;根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入酶切位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF1/PncB-RR1,在基因两端分别引入酶切位点;PCR分别扩增Bacillus subtilis 168中NadE、PncB,Bacillusmycoides中PnuC基因根据基因序列直接合成;
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF2/NadE-RR2,在基因两端分别引入酶切位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF2/PncB-RR2,在基因两端分别引入酶切位点;PCR分别扩增大肠杆菌BL21中的NadE、PncB目的基因;
⑵重组质粒的构建:将扩增的B.subtilis168中的NadE基因片段纯化回收后与单酶切后的质粒pMA5质粒使用非连接酶依赖型单片段快速克隆试剂盒进行同源重组,得到重组产物质粒pMA5-NadE;将扩增的B.subtilis168中的PncB基因片段纯化回收后与单酶切后的质粒pMA5质粒进行同源重组,得到重组产物质粒pMA5-PncB;设计引物pP-FF1/pP-RR1,以重组质粒pMA5-PncB为模板,扩增HpaⅡpromoter和PncB序列,将扩增的HpaⅡpromoter和PncB基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒进行同源重组,得到重组产物质粒pMA5-NadE-PncB;用单酶切质粒pMA5-PncB,将合成的Bacillus mycoides中的PnuC片段与单酶切后的质粒pMA5-PncB同源重组,得到重组质粒pMA5-PncB-PnuC;
用内切酶单酶切载体质粒pET22b,将扩增的E.coli BL21的NadE基因片段纯化回收后与单酶切后的质粒pET22b质粒进行同源重组,得到重组质粒pET22b-NadE,用内切酶单酶切质粒pET22b-NadE,将扩增的E.coli BL21中的PncB基因片段纯化回收后与单酶切后的质粒pET22b-NadE质粒进行同源重组,得到重组质粒pET22b-NadE-PncB;
二、过表达基因工程菌株的构建:
将构建的重组质粒分别转化到大肠杆菌DH5α感受态细胞中,通过菌落PCR验证,筛选大肠杆菌DH5α阳性转化子,于含有抗生素的LB液体培养基中,30~37℃恒温震荡过夜培养,-80℃保存菌种备用;提取大肠杆菌DH5α阳性转化子中的重组质粒,将提取得到的重组质粒转化至B.subtilis或E.coli底盘菌株中,涂布于含有抗生素的抗性平板中,进行菌落PCR验证,挑选B.subtilis或E.coli菌株中的阳性转化子于含有抗生素的液体培养基中,37℃恒温震荡过夜培养,-80℃保存菌种备用,得到高产β-烟酰胺单核苷酸基因工程菌株。
一种重组载体,所述载体含有如权利要求1至6任一项所述的基因工程菌株的氨基酸序列。
如上所述的基因工程菌株在β-烟酰胺单核苷酸生产方面中的应用。
利用如上所述的基因工程菌株生产β-烟酰胺单核苷酸的方法,步骤如下:
通过发酵培养所述高产β-烟酰胺单核苷酸基因工程菌株,从发酵液中提取β-烟酰胺单核苷酸。
进一步地,发酵培养时的发酵培养基碳源为甘油或葡萄糖,氮源为蛋白胨或酵母提取物,培养温度为30-37℃;
或者,提取β-烟酰胺单核苷酸使用反相高效液相色谱制备柱纯化,固定相为十八烷基硅烷键合硅胶。
本发明取得的有益效果是:
1、本发明基因工程菌是以枯草芽孢杆菌或大肠杆菌为底盘菌株,在底盘菌株中至少过表达NAD+合成酶NadE、烟酸磷酸核糖基转移酶PncB与转运蛋白PnuC其中的一个基因获得的,从而实现微生物发酵高产β-烟酰胺单核苷酸。
2、本发明方法是通过构建过表达NAD+合成酶NadE、烟酸磷酸核糖基转移酶PncB、转运蛋白PnuC的基因工程菌株,以葡萄糖或甘油等廉价原料发酵生产β-烟酰胺单核苷酸的新方法。
3、本发明方法中无需额外添加ATP、烟酰胺等昂贵原料,显著降低了生产成本。
4、本发明方法中NAD+合成酶NadE、烟酸磷酸核糖基转移酶PncB均来源于同源序列,无需考虑不同宿主之间其密码子的偏好性。
5、本发明不涉及有毒试剂,减少了环境污染,为后续纯化操作降低难度。
附图说明
图1为本发明中使用的pMA5质粒图谱;
图2为本发明中枯草芽孢杆菌中使用的重组质粒的构建图;其中:a为本发明中以pMA5为基础构建pMA5-NadE重组质粒的构建图;b为本发明中以pMA5为基础构建pMA5-PncB1重组质粒的构建图;c为本发明中以pMA5为基础构建pMA5-PncB2重组质粒的构建图;d为本发明中以pMA5-NadE为基础构建pMA5-NadE-PncB1重组质粒的构建图;e为本发明中以pMA5-NadE为基础构建pMA5-NadE-PncB2重组质粒的构建图;f为本发明中以pMA5-PncB为基础构建pMA5-PncB-PnuC重组质粒的构建图;
图3为本发明中使用的pET22b质粒图谱;
图4为本发明中大肠杆菌中使用的重组质粒的构建图;其中:a为本发明中以pET22b为基础构建pET22b-NadE重组质粒的构建图;b为本发明中以pET22b为基础构建pET22b-PncB重组质粒的构建图;c为本发明中以pET22b-NadE为基础构建pET22b-NadE-PncB重组质粒的构建图;
图5为本发明中不同基因工程菌株发酵结果图;其中a、b为枯草芽孢杆菌基因工程菌株发酵结果图;c为本发明中大肠杆菌基因工程菌株摇瓶发酵结果图;d为本发明中不同碳源对基因工程菌株发酵结果的影响图;其中,B.S:枯草芽孢杆菌出发原始菌株;B.S.N:枯草芽孢杆菌WB600-pMA5-NadE;B.S168.N1:枯草芽孢杆菌168-pWB980-NadE;B.S.P1:枯草芽孢杆菌WB600-pMA5-PncB(酶切位点为HinDIII),B.S.P2:枯草芽孢杆菌WB600-pMA5-PncB(酶切位点为NdeI);B.S800.P3:枯草芽孢杆菌WB800-pHT3-PncB;B.S800.P4:枯草芽孢杆菌WB800-pH01-PncB;B.S.PN1:枯草芽孢杆菌WB600-pMA5-NadE-PncB(酶切位点为HinDIII);B.S.PN2:枯草芽孢杆菌WB600-pMA5-NadE-PncB(酶切位点为NheI);B.S.PP:枯草芽孢杆菌WB600-pMA5-PncB(酶切位点为NdeI)-PnuC(酶切位点为HinDIII);E.coli:大肠杆菌BL21(DE3);E.N:大肠杆菌BL21(DE3)-pET22b-NadE;E.N2:大肠杆菌BL21(DE3)pLysS-pET28a-NadE;E.N3:大肠杆菌Rosetta(DE3)-pET30a-NadE;E.P:大肠杆菌BL21(DE3)-pET22b-PncB;E.P2:大肠杆菌JM109(DE3)-pUC57-PncB;E.NP:大肠杆菌BL21(DE3)-pET22b-NadE-PncB。
具体实施方式
为更好理解本发明,下面结合实施例对本发明做进一步地详细说明,但是本发明要求保护的范围并不局限于实施例所表示的范围。
本发明中所使用的的原料,如无特殊说明,均为常规市售产品,本发明中所使用的方法,如无特殊说明,均为本领域常规方法,本发明所使用的各物质质量均为常规使用质量。
一种高产β-烟酰胺单核苷酸的基因工程菌株,构建步骤如下:
过表达NAD+合成酶基因NadE、烟酸磷酸核糖转移酶基因PncB和转运蛋白基因PnuC三种基因中的任意一个或两个或三个。
较优地,所述基因工程菌株使用的底盘菌株包括但不限于枯草芽孢杆菌、大肠杆菌、酿酒酵母、毕赤酵母菌等适合构建表达系统的微生物,均在本发明权利保护中;
较优地,所述枯草芽孢杆菌包括但不限于B.subtilis 168、B.subtilis WB600、B.subtilisWB 800等,均在本发明权利保护中;
所述大肠杆菌包括但不限于E.coli BL21(DE3)、E.coli BL21(DE3)pLysS、E.coliRosetta(DE3)、E.coli JM109(DE3)等,均在本发明权利保护中;
构建时使用的过表达载体质粒包括但不限于pMA5、pWB980、pHT43、pHT01和pET22b、pET28a、pET30a、pUC57等,还包括其他适用于构建过表达基因工程菌株的其他载体质粒。
构建时使用的质粒为pMA5,含有两个多克隆位点区域,两个启动子,更符合多基因表达载体构建;
或者,构建时使用的质粒为pET22b,含有信号肽PelB,能够将蛋白定位在细胞外周质腔。
较优地,过表达的NAD+合成酶基因NadE的氨基酸序列与序列SEQ ID No.1具有至少95%的一致性;
或者,过表达的烟酸磷酸糖基转移酶基因PncB的氨基酸序列与序列SEQ ID No.2具有至少95%的一致性;
或者,过表达的转运蛋白基因PnuC的氨基酸序列与序列SEQ ID No.3具有至少95%的一致性;
或者,过表达NAD+合成酶基因NadE的氨基酸序列与序列SEQ ID No.4具有至少95%的一致性;
或者,过表达的烟酸磷酸糖基转移酶基因PncB的氨基酸序列与序列SEQ ID No.5具有至少95%的一致性。
较优地,所述基因工程菌株包括Bacillus subtilis NadE(B.S.N)或Bacillussubtilis 168NadE1(B.S168.N1)、Bacillus subtilis PncB1(B.S.P1)或Bacillussubtilis PncB2(B.S.P2)或Bacillus subtilis WB800PncB(B.S800.P3)或Bacillussubtilis WB600PncB(B.S800.P4)、Bacillus subtilis NadE-PncB1(B.S.PN1)或Bacillussubtilis NadE-PncB2(B.S.PN2)或Bacillus subtilis PncB2-PnuC(B.S.PP)或E.coliBL21(DE3)NadE(E.N)或E.coli BL21(DE3)pLysS NadE2(E.N2)或E.coli Rosetta(DE3)NadE3(E.N3)或E.coli BL21(DE3)PncB(E.P)或E.coli JM109(DE3)PncB(E.P2)或E.coliBL21(DE3)NadE-PncB(E.NP)。
如上所述的基因工程的构建方法,步骤如下:
一、重组质粒的构建:
⑴目的片段基因的获得:目的片段基因的获得根据目的基因设计引物扩增,或者根据基因序列直接合成;根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入酶切位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF1/PncB-RR1,在基因两端分别引入酶切位点;PCR分别扩增Bacillus subtilis 168中NadE、PncB,Bacillus mycoides中PnuC基因根据基因序列直接合成;
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF2/NadE-RR2,在基因两端分别引入酶切位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF2/PncB-RR2,在基因两端分别引入酶切位点;PCR分别扩增大肠杆菌BL21中的NadE、PncB目的基因;
⑵重组质粒的构建:将扩增的B.subtilis168中的NadE基因片段纯化回收后与单酶切后的质粒pMA5质粒使用非连接酶依赖型单片段快速克隆试剂盒进行同源重组,得到重组产物质粒pMA5-NadE;将扩增的B.subtilis168中的PncB基因片段纯化回收后与单酶切后的质粒pMA5质粒进行同源重组,得到重组产物质粒pMA5-PncB;设计引物pP-FF1/pP-RR1,以重组质粒pMA5-PncB为模板,扩增HpaⅡpromoter和PncB序列,将扩增的HpaⅡpromoter和PncB基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒进行同源重组,得到重组产物质粒pMA5-NadE-PncB;用单酶切质粒pMA5-PncB,将合成的Bacillus mycoides中的PnuC片段与单酶切后的质粒pMA5-PncB同源重组,得到重组质粒pMA5-PncB-PnuC;
用合适的内切酶单酶切载体质粒pET22b,将扩增的E.coli BL21的NadE基因片段纯化回收后与单酶切后的pET22b质粒进行同源重组,得到重组质粒pET22b-NadE,用内切酶单酶切质粒pET22b-NadE,将扩增的E.coli BL21中的PncB基因片段纯化回收后与单酶切后的pET22b-NadE质粒进行同源重组,得到连接产物重组质粒pET22b-NadE-PncB;
二、过表达基因工程菌株的构建:
将构建的重组质粒分别转化到大肠杆菌DH5α感受态细胞中,通过菌落PCR验证,筛选大肠杆菌DH5α阳性转化子,于含有抗生素的LB液体培养基中,30~37℃恒温震荡过夜培养,-80℃保存菌种备用;提取大肠杆菌DH5α阳性转化子中的重组质粒,将提取得到的重组质粒转化至B.subtilis 168、B.subtilis WB600、B.subtilis WB800、E.coli BL21(DE3)、E.coli BL21(DE3)pLysS等底盘菌株中,涂布于含有抗生素的抗性平板中,进行菌落PCR验证,挑选B.subtilis WB600、E.coli BL21(DE3)等阳性转化子于含有抗生素的液体培养基中,37℃恒温震荡过夜培养,-80℃保存菌种备用,得到高产β-烟酰胺单核苷酸基因工程菌株。
一种重组载体,所述载体含有如上所述的基因工程菌株的基因编码序列。
如上所述的基因工程菌株在β-烟酰胺单核苷酸生产方面中的应用。
利用如上所述的基因工程菌株生产β-烟酰胺单核苷酸的方法,步骤如下:
通过发酵培养所述高产β-烟酰胺单核苷酸基因工程菌株,从发酵液中提取β-烟酰胺单核苷酸。
较优地,发酵培养时的发酵培养基碳源为甘油或葡萄糖,氮源为蛋白胨或酵母提取物,培养温度为30-37℃;
或者,提取β-烟酰胺单核苷酸使用反相高效液相色谱制备柱纯化,固定相为十八烷基硅烷键合硅胶。
较优地,编码NAD+合成酶NadE基因来源于枯草芽孢杆菌,其氨基酸序列如SEQ IDNo.1所示。
较优地,编码烟酸磷酸核糖基转移酶PncB基因来源于枯草芽孢杆菌,其氨基酸序列如SEQ ID No.2所示。
较优地,编码转运蛋白PnuC基因来源于蕈状芽孢杆菌,其氨基酸序列如SEQ IDNo.3所示。
较优地,编码NAD+合成酶NadE基因来源于大肠杆菌,其氨基酸序列如SEQ ID No.4所示。
较优地,编码烟酸磷酸核糖基转移酶PncB基因来源于大肠杆菌,其氨基酸序列如SEQ ID No.5所示。
较优地,底盘菌株可以是适合的枯草芽孢杆菌,优选的是Bacillus subtilisWB600。
较优地,底盘菌株可以是适合的大肠杆菌,优选的是E.coli BL21(DE3)。
较优地,利用上述基因工程菌株发酵法生产β-烟酰胺单核苷酸的方法,包括以下步骤:
将高产β-烟酰胺单核苷酸基因工程菌株接种到含有以甘油或葡萄糖为碳源、以蛋白胨、酵母提取物为氮源的培养基中,30~37℃培养12-24h,发酵结束后将发酵液进行超声处理,低温离心收集上清,将上清用0.22μm的滤膜过滤,即含有β-烟酰胺单核苷酸粗样品的溶液。
将上述含有β-烟酰胺单核苷酸粗样品的溶液用反向色谱制备柱纯化β-烟酰胺单核苷酸,色谱柱固定相为十八烷基硅烷键合硅胶,流动相A为盐酸溶液配成的pH3-7的溶液,流动相B为乙醇,进行梯度洗脱纯化,收集含有β-烟酰胺单核苷酸的洗脱峰溶液,并用膜浓缩设备进行纳滤浓缩后,用真空冷冻干燥机冻干,获得纯化的β-NMN。
具体地,相关制备及检测如下:
一种高产β-烟酰胺单核苷酸工程菌株的构建方法,步骤如下:
首先构建过表达NAD+合成酶NadE、烟酸磷酸核糖基转移酶PncB、转运蛋白PnuC的基因的重组质粒,将构建得到的重组质粒转化至底盘菌株中,涂布于含有抗生素的抗性平板中,挑选阳性转化子于液体种子培养基中,30~37℃过夜培养,之后转接至发酵培养基中,30~37℃培养12-24h,收集菌体,破碎菌体后离心,收集上清液,上清液过滤后用反向制备色谱纯化得到β-烟酰胺单核苷酸产品。
较优地,所述携带目的基因NadE、PncB、PnuC的重组质粒的构建步骤如下:
⑴目的基因的获得
1)枯草芽孢杆菌NadE基因的获得
根据NCBI查询得到的枯草芽孢杆菌NadE基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入BamHI酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶BamHI的位点,PCR扩增枯草芽孢杆菌168中的NadE基因,所述引物NadE-FF1/NadE-RR1的序列为:
NadE-FF1:即5’-aaagtgaaatcagggggatccATGAGCATGCAGGAAAAGATTATG-3’,下划线序列为BamHI酶切位点;
NadE-RR1:即5’-gagctcgactctagaggatccTTATTTCCACCAGTCATCAAACATAGA-3’,下划线序列为BamHI酶切位点;
所述枯草芽孢杆菌NAD+合成酶基因NadE的氨基酸序列为SEQ ID No.1,也可根据SEQ ID No.1序列直接合成枯草芽孢杆菌NadE基因;
本申请中的NAD+合成酶基因NadE的氨基酸序列,包括由如SEQ ID No.1所示氨基酸序列及进行1个或多个氨基酸取代而得到的同功能氨基酸序列。本领域技术人员可以根据本申请公开的NadE基因氨基酸序列,根据现有分子生物学技术,采用克隆或合成的方法或其他适合的方法获得本申请的NadE基因。另外不同菌株、菌种或其他物种来源的NadE基因具有相似功能,因此,编码上述NadE基因的氨基酸序列并不仅限于如SEQ ID No.1所示的氨基酸序列。如果编码得到的蛋白与SEQ ID No.1所述蛋白没有明显的功能差异,也包括在本发明的范围内。
2)枯草芽孢杆菌PncB基因的获得
根据NCBI查询得到的枯草芽孢杆菌PncB酶基因设计引物序列PncB-FF1/PncB-RR1,在基因两端分别引入Nde I或HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶位点,PCR扩增枯草芽孢杆菌168中的PncB基因,所述引物PncB-FF1/PncB-RR1的序列为:
PncB-FF1:即5’-aaaaggagcgatttacatatgGTGTTAGAGTACGGATTTAAAGATGACA-3’,下划线序列为Nde I酶切位点;
PncB-RR1:即5’-acaaactgcataactcatatgTTATTCTTCCTCAAGCTCTTCTTCAA-3’,下划线序列为Nde I酶切位点;
PncB-FF2:即5’-cggtacctctagaagaagcttGTGTTAGAGTACGGATTTAAAGATGACA-3’,下划线序列为HinD III酶切位点;
PncB-RR2:即5’-ctttaccttgtctccaagcttTTATTCTTCCTCAAGCTCTTCTTCAA-3’,下划线序列为HinD III酶切位点;
所述枯草芽孢杆菌烟酸磷酸糖基转移酶基因PncB的序列为SEQ ID No.2,也可根据SEQ ID No.2序列直接合成枯草芽孢杆菌PncB基因;
本申请中的烟酸磷酸糖基转移酶基因PncB氨基酸序列,包括由如SEQ ID No.2所示氨基酸序列及进行1个或多个氨基酸取代而得到的同功能氨基酸序列。本领域技术人员可以根据本申请公开的PncB氨基酸序列,根据现有分子生物学技术,采用克隆或合成的方法或其他适合的方法获得本申请的PncB基因。另外不同菌株、菌种或其他物种来源的PncB基因具有相似功能,因此,编码上述PncB基因的氨基酸序列并不仅限于如SEQ ID No.2所示的氨基酸序列。如果编码得到的蛋白与SEQ ID No.2所述蛋白没有明显的功能差异,也包括在本发明的范围内。
3)大肠杆菌NadE基因的获得
根据NCBI查询得到的大肠杆菌NadE酶基因设计引物序列NadE-FF2/NadE-RR2,在基因两端分别引入HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶HinD III的位点;PCR扩增大肠杆菌BL21中的NadE基因。所述引物NadE-FF2/NadE-RR2的序列为:
NadE-FF2:即5’-ctcgagtgcggccgcaagcttATGACATTGCAACAACAAATAATAAAGG-3’,下划线序列为HinD III酶切位点;
NadE-RR2:即5’-tcgagctccgtcgacaagcttTTACTTTTTCCAGAAATCATCGAAAA-3’,下划线序列为HinD III酶切位点;
所述大肠杆菌NAD+合成酶NadE的氨基酸序列为SEQ ID No.4;也可根据SEQ IDNo.4序列直接合成大肠杆菌NadE基因。
4)大肠杆菌PncB基因的获得
根据NCBI查询得到的大肠杆菌PncB酶基因设计引物序列PncB-FF2/PncB-RR2,在基因两端分别引入EcoR I酶切位点,在氨基酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶EcoR I的位点,PCR扩增大肠杆菌BL21中的PncB基因。所述引物PncB-FF2/PncB-RR2的序列为:
PncB-FF3:即5’-ttgtcgacggagctcgaattcATGACACAATTCGCTTCTCCTGT-3’,下划线序列为EcoR I酶切位点;
PncB-RR3:即5’-aattaattcggatccgaattcTTAACTGGCTTTTTTAATATGCGG-3’,下划线序列为EcoR I酶切位点;
所述大肠杆菌烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.5;也可根据SEQ ID No.5序列直接合成大肠杆菌PncB基因。
5)蕈状芽孢杆菌PnuC基因的获得
根据NCBI查询得到的蕈状芽孢杆菌中转运蛋白PnuC氨基酸序列SEQ ID No.3直接合成PnuC基因。
本申请中的转运蛋白基因PunC氨基酸序列,包括由如SEQ ID No.3所示氨基酸序列及进行1个或多个氨基酸取代而得到的同功能氨基酸序列。本领域技术人员可以根据本申请公开的PunC氨基酸序列,根据现有分子生物学技术,采用克隆或合成的方法或其他适合的方法获得本申请的PunC基因。另外不同菌株、菌种或其他物种来源的PunC基因具有相似功能,因此,编码上述PunC基因的氨基酸序列并不仅限于如SEQ ID No.3所示的氨基酸序列。如果编码得到的蛋白与SEQ ID No.3所述蛋白没有明显的功能差异,也包括在本发明的范围内。
(2)重组质粒的构建
在一个实施例中,用BamHI单酶切质粒pMA5(如图1),将扩增的枯草芽孢杆菌168中的NadE基因片段纯化回收后与单酶切后的质粒pMA5质粒进行同源重组,得到连接产物重组质粒pMA5-NadE,如图2a。
在一个实施例中,用HinD III单酶切质粒pMA5并纯化回收,将扩增的B.subtilis168中的PncB1基因片段纯化回收后与单酶切后的质粒pMA5质粒用ClonExpress II OneStep Cloning Kit进行重组,得到重组产物质粒pMA5-PncB1,如图2b。
在一个实施例中,用Nde I单酶切质粒pMA5并纯化回收,将扩增的B.subtilis 168中的PncB2基因片段纯化回收后与单酶切后的质粒pMA5质粒用ClonExpress II One StepCloning Kit进行同源重组,得到重组产物质粒pMA5-PncB2,如图2c。
在一个实施例中,用HinD III单酶切质粒pMA5-NadE并纯化回收,将扩增的B.subtilis 168中的PncB1基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒用ClonExpress II One Step Cloning Kit进行重组,得到重组产物质粒pMA5-NadE-PncB1,如图2d。
在一个实施例中,设计引物pP-FF1/pP-RR1,以重组质粒pMA5-PncB2为模板,扩增HpaⅡpromoter和PncB2序列,用Nhe I单酶切质粒pMA5-NadE并纯化回收,将扩增的HpaⅡpromoter和PncB2基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒进行同源重组,得到重组产物质粒pMA5-NadE-PncB2,如图2e。
在一个实施例中,用HinD III单酶切质粒pMA5-PncB2并纯化回收,将扩增的Bacillus mycoides中PnuC片段纯化回收后与单酶切后的质粒pMA5-PncB2同源重组,得到重组质粒pMA5-PncB2-PnuC,如图2f。
在一个实施例中,用HinD III单酶切质粒pET22b(如图3),将扩增的E.coli BL21的NadE基因片段纯化回收后与单酶切后的质粒pET22b质粒进行同源重组,得到连接产物重组质粒pET22b-NadE,如图4a。
在一个实施例中,用EcoR I单酶切质粒pET22b,将扩增的E.coli BL21的PncB基因片段纯化回收后与单酶切后的质粒pET22b质粒进行同源重组,得到连接产物重组质粒pET22b-PncB,如图4b。
在一个实施例中,用EcoR I单酶切质粒pET22b-NadE,将扩增的E.coli BL21中的PncB基因片段纯化回收后与单酶切后的质粒pET22b-NadE质粒进行同源重组,得到连接产物重组质粒pET22b-NadE-PncB,如图4c。
将构建的重组质粒pMA5-NadE、pMA5-PncB1、pMA5-PncB2、pMA5-NadE-PncB1、pMA5-NadE-PncB2、pMA5-PncB2-PunC、pET22b-NadE、pET22b-PncB、pET22b-NadE-PncB分别转化到大肠杆菌DH5α感受态细胞中,挑取大肠杆菌DH5α阳性转化子备用。
(3)基因工程菌株的构建:分别提取大肠杆菌DH5α阳性转化子中的重组质粒pMA5-NadE、pMA5-PncB1、pMA5-PncB2、pMA5-NadE-PncB1、pMA5-NadE-PncB2、pMA5-PncB2-PunC,分别将构建得到的重组质粒转化至B.subtilis 168、B.subtilis WB600、B.subtilis WB800感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,挑选阳性转化子得到最终的过表达基因工程菌,将这些基因工程菌于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用;将重组质粒pET22b-NadE、pET22b-PncB、pET22b-NadE-PncB化学法转化至大肠杆菌E.coli BL21(DE3)、E.coli BL21(DE3)pLysS等感受态细胞中,涂布于含有氨苄青霉素的LB抗性平板中,进行菌落PCR验证,挑选阳性转化子得到最终的过表达基因工程菌,将这些基因工程菌于含有氨苄青霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
(4)基因工程菌株发酵生产β-NMN
利用如上所述的过表达基因工程菌发酵生产β-NMN的方法,步骤如下:
将基因工程菌接种在5mL含有卡那霉素或氨苄青霉素的LB或TB液体种子培养基中,在30~37℃、180~220r/min条件下过夜培养,将过夜培养的发酵液按1~2%的接种量接种到含氨苄青霉素或卡那霉素抗生素的LB或TB发酵培养基中培养12~24小时。如工程菌底盘菌为枯草芽孢杆菌,接种发酵培养基后可连续培养12-24小时;如工程菌底盘菌为大肠杆菌,接种发酵培养基后需要将菌株培养至OD600在0.6-0.8之间,添加终浓度0.4-1mM的IPTG诱导蛋白表达12-24小时,诱导温度20~25℃。
然后,离心或过滤收集发酵液中菌体,按照4mg/mL的终浓度添加溶菌酶,并超声破碎,功率400W,破碎3s,停顿2s,共30个周期,4000r/min,离心5min,将上清液用0.22μm的滤膜过滤,即为含β-NMN的粗提液,-20冷冻保存备用。若工程菌为大肠杆菌,则将上述过夜诱导的发酵液4000r/min,离心10min,收集菌体。菌体细胞用菌液同体积蒸馏水重新悬浮,利用超声波细胞破碎仪破碎大肠杆菌细胞,后与枯草芽孢杆菌同样方法处理收集β-NMN粗品。
本发明中使用的培养基组成可以如下:
LB培养基:酵母提取物5-10g/L,胰蛋白胨10-20g/L,NaCl 5-10g/L,pH自然,加水定容至1L。
TB培养基:蛋白胨12-20g/L,酵母提取物24-40g/L,K2HPO472-100 mmol/L,KH2PO417-30mmol/L,甘油5-10mL/L或葡萄糖10-15g/L,PH7.0-7.4。
本发明中使用的PCR反应体系和条件可以如下
PCR反应体系:2×phanta maxbuffer 10-25μL,dNTP mixture(10mM)0.4-1μL,模板(20ng/ul)0.4-1μL,上、下游引物(10μM)各0.8-2μL,DMSO0.4-2μL,phanta max×Super-Fidelity DNAPolymerase 0.4-1μL,补超纯水至20-50μL。
PCR反应条件:95℃预变性3-5min;95℃变性15-30s,50-65℃退火15-30s,72℃延伸15s-2min,共循环30-35次,72℃延伸5min,4-16℃结束反应。
更为具体地,通过相关实施例来具体说明。
实施例1
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入BamHI酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶BamHI的位点。PCR扩增枯草芽孢杆菌168中目的基因NadE。
PCR反应体系:2×phantamaxbuffer 10μL,dNTP mixture(10mM)0.4μL,模板(20ng/ul)0.4μL,上、下游引物(10μM)各0.8μL,DMSO0.4μL,phantamax×Super-FidelityDNAPolymerase 0.4μL,补超纯水至20μL。
PCR反应条件:95℃预变性3min;95℃变性15s,58-63℃退火15s,72℃延伸30smin,共循环30次,72℃延伸5min,4℃结束反应。
NadE-FF1:即5’-aaagtgaaatcagggggatccATGAGCATGCAGGAAAAGATTATG-3’,下划线序列为BamHI酶切位点;
NadE-RR1:即5’-gagctcgactctagaggatccTTATTTCCACCAGTCATCAAACATAGA-3’,下划线序列为BamHI酶切位点;
所述NAD+合成酶基因NadE的氨基酸序列为SEQ ID No.1;
重组质粒的构建:
用BamHI单酶切质粒pMA5,将扩增的枯草芽孢杆菌168中的NadE基因片段纯化回收后与单酶切后的质粒pMA5质粒进行同源重组,得到连接产物重组质粒pMA5-NadE(图2a)。
重组质粒的转化:
取连接产物重组质粒pMA5-NadE,加入到在冰浴中已融化的含有大肠杆菌DH5α感受态细胞的离心管中,轻弹管壁,混匀,冰浴30min。42℃热激90s,然后立即冰浴5min(此过程不要移动)。无菌条件下,向离心管中加入900μLLB培养基,吹打混匀后,37℃、180r/min振荡培养45min。将离心管12000r/min离心1min,移除900μL上清,用移液器将剩余液体吹打混匀,涂布到含有氨苄青霉素的LB固体平板上。将LB平板37℃倒置培养过夜,至单菌落清晰可辨,挑取阳性转化子进行菌落PCR验证,得到大肠杆菌DH5α转化子E.coli DH5αNadE。
基因工程菌株的获得:
提取E.coli DH5αNadE中的重组质粒pMA5-NadE,将构建得到的重组质粒转化至枯草芽孢杆菌WB600感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,将得到的基因工程菌株Bacillus subtilis NadE(B.S.N)于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
利用如上所述的基因工程菌株B.S.N过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pMA5-NadE的基因工程菌株B.S.N接种到含有卡那霉素的TB培养基的试管中培养,37℃和220r/min转速下培养12h。再以1%的接种量转入50mL含有卡那霉素的TB培养基中表达,37℃,220r/min发酵24h后收集发酵液,加入终浓度为4mg/mL的溶菌酶,并做超声处理,细胞破碎后,4000r/min,冷冻离心5min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱法检测β-烟酰胺单核苷酸,结果如图5a所示,β-NMN的浓度970±38mg/L,较对照菌株Bacillus subtilisWB600(B.S)发酵结果提高了37%左右。
β-NMN的提纯方法:
将含有β-烟酰胺单核苷酸的发酵液用膜浓缩设备先后进行微滤(微滤膜孔径为0.2-1μm)和纳滤(截留分子量200),得到澄清滤液,即浓缩的β-NMN粗品溶液;将获得的粗品溶液pH值调至3-7,进样上反相高效液相色谱制备柱,固定相为十八烷基硅烷键合硅胶,流动相A为盐酸溶液配成的pH3-7的溶液,流动相B为乙醇,进行梯度洗脱纯化,得到纯化的样品溶液;将纯化的样品溶液用膜浓缩设备进行纳滤后,用真空冷冻干燥机冻干,获得纯化的β-NMN。
β-NMN的分析方法:
利用HPLC分析β-NMN的浓度,其中液相的配备要求如,分析的柱子型号:HypersilBDS C18,5μm,4.6×200mm;流动相:10%乙腈和90%25mM磷酸盐缓冲液(pH 6.0)(3.4g磷酸二氢钾溶于850mL超纯水,用85%磷酸调pH至6.0,加100mL乙腈,再用超纯水加至1L);流速:1mL/min;柱温25℃;检测波长254nm,β-NMN的出峰时间在1.35min。
实施例2
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF1/PncB-RR1,在基因两端分别引入HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶位点。PCR扩增枯草芽孢杆菌168中PncB基因片段。
PCR反应体系:2×phantamaxbuffer 10μL,dNTP mixture(10mM)0.4μL,模板(20ng/ul)0.4μL,上、下游引物(10μM)各0.8μL,DMSO0.4μL,phantamax×Super-FidelityDNAPolymerase 0.4μL,补超纯水至20μL。
PCR反应条件:95℃预变性3min;95℃变性15s,58-63℃退火15s,72℃延伸40smin,共循环30次,72℃延伸5min,4℃结束反应。
所述引物序列PncB-FF1/PncB-RR1为:
PncB-FF1:即5’-cggtacctctagaagaagcttGTGTTAGAGTACGGATTTAAAGATGACA-3’,下划线序列为HinD III酶切位点;
PncB-RR1:即5’-ctttaccttgtctccaagcttTTATTCTTCCTCAAGCTCTTCTTCAA-3’,下划线序列为HinD III酶切位点;
所述烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.2;
重组质粒的构建
用HinD III单酶切质粒pMA5并纯化回收,将扩增的B.subtilis168中的PncB1基因片段纯化回收后与单酶切后的质粒pMA5质粒用ClonExpress II One Step Cloning Kit进行重组,得到重组产物质粒pMA5-PncB1(图2b)。
重组质粒的转化:
取连接产物pMA5-PncB1加入到在冰浴中已融化的含有大肠杆菌DH5α感受态细胞的离心管中,轻弹管壁,混匀,冰浴30min。42℃热激90s,然后立即冰浴5min(此过程不要移动)。无菌条件下,向离心管中加入900μLLB培养基,吹打混匀后,37℃、180r/min振荡培养45min。将离心管12000r/min离心1min,移除900μL上清,用移液器将剩余液体吹打混匀,涂布到含有氨苄青霉素的LB固体平板上。将LB平板37℃倒置培养过夜,至单菌落清晰可辨,挑取阳性转化子进行菌落PCR验证,得到菌株E.coli DH5αPncB1。
基因工程菌株的获得:
提取菌株E.coli DH5αPncB1中的重组质粒pMA5-PncB1,将构建得到的重组质粒转化至枯草芽孢杆菌WB600感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,得到最终的基因工程菌株Bacillus subtilis PncB1(B.S.P1),挑选基因工程菌株Bacillus subtilis PncB1(B.S.P1)于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
利用如上所述的基因工程菌株B.S.P1过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pMA5-PncB1的基因工程菌株B.S.P1接种到含有卡那霉素的TB培养基的试管中培养,37℃和220r/min转速下培养12h。再以1%的接种量转入50mL含有卡那霉素的TB培养基中表达,37℃,220r/min发酵24h后收集发酵液,加入终浓度为4mg/mL的溶菌酶,并做超声处理,细胞破碎后,4000r/min,冷冻离心5min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5a所示,β-NMN的浓度1097±11mg/L,较对照菌株Bacillus subtilisWB600(B.S)发酵结果提高了55%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例3
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF2/PncB-RR2,在基因两端分别引入Nde I酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶位点。PCR扩增枯草芽孢杆菌168中PncB片段。
PCR反应体系:2×phantamaxbuffer 10μL,dNTP mixture(10mM)0.4μL,模板(20ng/ul)0.4μL,上、下游引物(10μM)各0.8μL,DMSO0.4μL,phantamax×Super-FidelityDNAPolymerase 0.4μL,补超纯水至20μL。
PCR反应条件:95℃预变性3min;95℃变性15s,58-63℃退火15s,72℃延伸40smin,共循环30次,72℃延伸5min,4℃结束反应。
所述引物PncB-FF2/PncB-RR2的序列为:
PncB-FF2:即5’-aaaaggagcgatttacatatgGTGTTAGAGTACGGATTTAAAGATGACA-3’,下划线序列为Nde I酶切位点;
PncB-RR2:即5’-acaaactgcataactcatatgTTATTCTTCCTCAAGCTCTTCTTCAA-3’,下划线序列为Nde I酶切位点;
所述烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.2;
重组质粒的构建:
用Nde I单酶切质粒pMA5并纯化回收,将扩增的B.subtilis168中的PncB2基因片段纯化回收后与单酶切后的质粒pMA5质粒用ClonExpress II One Step Cloning Kit进行同源重组,得到重组产物质粒pMA5-PncB2(图2c)。
重组质粒的转化:
取连接产物pMA5-PncB2,加入到在冰浴中已融化的含有大肠杆菌DH5α感受态细胞的离心管中,轻弹管壁,混匀,冰浴30min。42℃热激90s,然后立即冰浴5min(此过程不要移动)。无菌条件下,向离心管中加入900μLLB培养基,吹打混匀后,37℃、180r/min振荡培养45min。将离心管12000r/min离心1min,移除900μL上清,用移液器将剩余液体吹打混匀,涂布到含有氨苄青霉素的LB固体平板上。将LB平板37℃倒置培养过夜,至单菌落清晰可辨,挑取阳性转化子进行菌落PCR验证,得到菌株E.coli DH5αPncB2。
基因工程菌株的获得:
提取E.coli DH5αPncB2菌株中的重组质粒pMA5-PncB2,将构建得到的重组质粒转化至枯草芽孢杆菌WB600感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,得到最终的基因工程菌株Bacillus subtilis PncB2(B.S.P2),挑选基因工程菌株Bacillus subtilis PncB2(B.S.P2)于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
利用如上所述的基因工程菌株B.S.P2过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pMA5-PncB2的基因工程菌株B.S.P2接种到含有卡那霉素的TB培养基的试管中培养,37℃和220r/min转速下培养12h。再以1%的接种量转入50mL含有卡那霉素的TB培养基中表达,37℃,220r/min发酵24h后收集发酵液,加入终浓度为4mg/mL的溶菌酶,并做超声处理,细胞破碎后,4000r/min,冷冻离心5min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5a所示,β-NMN的浓度1160±27mg/L,较对照菌株Bacillus subtilisWB600(B.S)发酵结果提高了64%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例4
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入BamHI酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶BamHI的位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF1/PncB-RR1,在基因两端分别引入HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶位点。PCR分别扩增枯草芽孢杆菌168中NadE、PncB的目的基因。
NadE-FF1:即5’-aaagtgaaatcagggggatccATGAGCATGCAGGAAAAGATTATG-3’,下划线序列为BamHI酶切位点;
NadE-RR1:即5’-gagctcgactctagaggatccTTATTTCCACCAGTCATCAAACATAGA-3’,下划线序列为BamHI酶切位点;
所述NAD+合成酶基因NadE的氨基酸序列为SEQ ID No.1;
PncB-FF1:即5’-cggtacctctagaagaagcttGTGTTAGAGTACGGATTTAAAGATGACA-3’,下划线序列为HinD III酶切位点;
PncB-RR1:即5’-ctttaccttgtctccaagcttTTATTCTTCCTCAAGCTCTTCTTCAA-3’,下划线序列为HinD III酶切位点;
所述烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.2;
重组质粒的构建:
用HinD III单酶切质粒pMA5-NadE并纯化回收,将扩增的B.subtilis168中的PncB1基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒用ClonExpress II One StepCloning Kit进行重组,得到重组产物质粒pMA5-NadE-PncB1(图2d)。
重组质粒的转化:
取连接产物pMA5-NadE-PncB1加入到在冰浴中已融化的含有大肠杆菌DH5α感受态细胞的离心管中,轻弹管壁,混匀,冰浴30min。42℃热激90s,然后立即冰浴5min(此过程不要移动)。无菌条件下,向离心管中加入900μLLB培养基,吹打混匀后,37℃、180r/min振荡培养45min。将离心管12000r/min离心1min,移除900μL上清,用移液器将剩余液体吹打混匀,涂布到含有氨苄青霉素的LB固体平板上。将LB平板37℃倒置培养过夜,至单菌落清晰可辨,挑取阳性转化子进行菌落PCR验证,得到菌株E.coli DH5αNadE-PncB1。
基因工程菌株的获得:
提取菌株E.coli DH5αNadE-PncB1中的重组质粒pMA5-NadE-PncB1,将构建得到的重组质粒转化至枯草芽孢杆菌WB600感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,得到最终的基因工程菌株Bacillus subtilis NadE-PncB1(B.S.PN1),挑选基因工程菌株Bacillus subtilis NadE-PncB1(B.S.PN1)于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
利用如上所述的基因工程菌株B.S.PN1过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pMA5-NadE-PncB1的基因工程菌株B.S.PN1接种到含有卡那霉素的TB培养基的试管中培养,37℃和220r/min转速下培养12h。再以1%的接种量转入50mL含有卡那霉素的TB培养基中表达,37℃,220r/min发酵24h后收集发酵液,加入终浓度为4mg/mL的溶菌酶,并做超声处理,细胞破碎后,4000r/min,冷冻离心5min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5a所示,β-NMN的浓度942±30mg/L,较对照菌株Bacillus subtilisWB600(B.S)发酵结果提高了33%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例5
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入BamH I酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶BamHI的位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF2/PncB-RR2,在基因两端分别引入Nde I酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶位点。PCR分别扩增枯草芽孢杆菌168中NadE、PncB的目的基因。
NadE-FF1:即5’-aaagtgaaatcagggggatccATGAGCATGCAGGAAAAGATTATG-3’,下划线序列为BamHI酶切位点;
NadE-RR1:即5’-gagctcgactctagaggatccTTATTTCCACCAGTCATCAAACATAGA-3’,下划线序列为BamHI酶切位点;
所述NAD+合成酶基因NadE的氨基酸序列为SEQ ID No.1;
所述引物PncB-FF2/PncB-RR2的序列为:
PncB-FF2:即5’-aaaaggagcgatttacatatgGTGTTAGAGTACGGATTTAAAGATGACA-3’,下划线序列为Nde I酶切位点;
PncB-RR2:即5’-acaaactgcataactcatatgTTATTCTTCCTCAAGCTCTTCTTCAA-3’,下划线序列为Nde I酶切位点;
所述烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.2;
重组质粒的构建:
设计引物pP-FF1/pP-RR1,以重组质粒pMA5-PncB2为模板,扩增HpaⅡpromoter和PncB2序列,用Nhe I单酶切质粒pMA5-NadE并纯化回收,将扩增的HpaⅡpromoter和PncB2基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒进行同源重组,得到重组产物质粒pMA5-NadE-PncB2(图2e)。
所述引物pP-FF1/pP-RR1的序列为:
pP-FF1:即5’-tagagtcgagctcaagctagcGTGGAGATTTTTTGAGTGATCTTCTC-3’,下划线序列为Nhe I酶切位点;
pP-RR1:即5’-tctggtacgtaccaagctagcCTTTTTGCATTCTACAAACTGCATAA-3’,下划线序列为Nhe I酶切位点;
重组质粒的转化:
取连接产物pMA5-NadE-PncB2加入到在冰浴中已融化的含有大肠杆菌DH5α感受态细胞的离心管中,轻弹管壁,混匀,冰浴30min。42℃热激90s,然后立即冰浴5min(此过程不要移动)。无菌条件下,向离心管中加入900μLLB培养基,吹打混匀后,37℃、180r/min振荡培养45min。将离心管12000r/min离心1min,移除900μL上清,用移液器将剩余液体吹打混匀,涂布到含有氨苄青霉素的LB固体平板上。将LB平板37℃倒置培养过夜,至单菌落清晰可辨,挑取阳性转化子进行菌落PCR验证,得到菌株E.coli DH5αNadE-PncB2。
基因工程菌株的获得:
提取菌株E.coli DH5αNadE-PncB2中的重组质粒pMA5-NadE-PncB2,将构建得到的重组质粒转化至枯草芽孢杆菌WB600感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,得到最终的基因工程菌株Bacillus subtilis NadE-PncB2(B.S.PN2),挑选基因工程菌株Bacillus subtilis NadE-PncB2(B.S.PN2)于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
利用如上所述的基因工程菌株B.S.PN2过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pMA5-NadE-PncB2的基因工程菌株B.S.PN2接种到含有卡那霉素的TB培养基的试管中培养,37℃和220r/min转速下培养12h。再以1%的接种量转入50mL含有卡那霉素的TB培养基中表达,37℃,220r/min发酵24h后收集发酵液,加入终浓度为4mg/mL的溶菌酶,并做超声处理,细胞破碎后,4000r/min,冷冻离心5min,将上清用0.22μm的滤膜过滤,得到含有NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5a所示,β-NMN的浓度820±20mg/L,较对照菌株Bacillus subtilisWB600(B.S)发酵结果提高了16%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例6
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获取:
根据NCBI查询得到的蕈状芽孢杆菌PunC酶基因序列直接合成该目的基因,在基因两端分别引入HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶位点。PCR扩增蕈状芽孢杆菌中PunC片段。
所述转运蛋白基因PunC的氨基酸序列为SEQ ID No.3。
重组质粒的构建:
用HinD III单酶切质粒pMA5-PncB2并纯化回收,将扩增的PnuC片段纯化回收后与单酶切后的质粒pMA5-PncB2同源重组,得到重组质粒pMA5-PncB2-PnuC(图2f)。
重组质粒的转化:
取连接产物pMA5-PncB2-PnuC加入到在冰浴中已融化的含有大肠杆菌DH5α感受态细胞的离心管中,轻弹管壁,混匀,冰浴30min。42℃热激90s,然后立即冰浴5min(此过程不要移动)。无菌条件下,向离心管中加入900μLLB培养基,吹打混匀后,37℃、180r/min振荡培养45min。将离心管12000r/min离心1min,移除900μL上清,用移液器将剩余液体吹打混匀,涂布到含有氨苄青霉素的LB固体平板上。将LB平板37℃倒置培养过夜,至单菌落清晰可辨,挑取大肠杆菌DH5α阳性转化子进行菌落PCR验证,得到菌株E.coli DH5αPncB2-PnuC。
基因工程菌株的获得:
提取菌株E.coli DH5αPncB2-PnuC中的重组质粒pMA5-PncB2-PnuC,将构建得到的重组质粒转化至枯草芽孢杆菌WB600感受态细胞中,涂布于含有卡那霉素的LB抗性平板中,进行菌落PCR验证,得到最终的基因工程菌株Bacillus subtilis PncB2-PnuC(B.S.PP),挑选基因工程菌株Bacillus subtilis PncB2-PnuC(B.S.PP)于含有卡那霉素的LB液体培养基中,37℃恒温震荡过夜培养,-80℃保存备用。
利用如上所述的基因工程菌株B.S.PP过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pMA5-PncB2-PnuC的基因工程菌株B.S.PP接种到含有卡那霉素的TB培养基的试管中培养,37℃和220r/min转速下培养12h。再以1%的接种量转入50mL TB培养基中表达,37℃,220r/min发酵24h后收集发酵液,加入终浓度为4mg/mL的溶菌酶,并做超声处理,细胞破碎后,4000r/min,冷冻离心5min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5b所示,β-NMN的浓度987±24mg/L,较对照菌株Bacillus subtilisWB600(B.S)发酵结果提高了40%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例7
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF2/NadE-RR2,在基因两端分别引入HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶HinD III的位点;PCR扩增大肠杆菌BL21中NadE的目的基因。
PCR反应体系:2×phantamaxbuffer 10μL,dNTP mixture(10mM)0.4μL,模板(20ng/ul)0.4μL,上、下游引物(10μM)各0.8μL,DMSO0.4μL,phantamax×Super-FidelityDNAPolymerase 0.4μL,补超纯水至20μL。
PCR反应条件:95℃预变性3min;95℃变性15s,58-63℃退火15s,72℃延伸30smin,共循环30次,72℃延伸5min,16℃结束反应。
所述引物NadE-FF2/NadE-RR2的序列为:
NadE-FF2:即5’-ctcgagtgcggccgcaagcttATGACATTGCAACAACAAATAATAAAGG-3’,下划线序列为HinD III酶切位点;
NadE-RR2:即5’-tcgagctccgtcgacaagcttTTACTTTTTCCAGAAATCATCGAAAA-3’,下划线序列为HinD III酶切位点;
所述NAD+合成酶NadE的氨基酸序列为SEQ ID No.4;
重组质粒的构建与转化:
用HinD III单酶切质粒pET22b,将扩增的大肠杆菌BL21的NadE基因片段纯化回收后与单酶切后的质粒pET22b质粒进行同源重组,得到连接产物重组质粒pET22b-NadE(图4a),化学法导入大肠杆菌BL21(DE3)感受态细胞,筛选转化子保存,即为大肠杆菌工程菌株E.coli BL21(DE3)NadE(E.N)。
利用如上所述的基因工程菌株E.N过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pET22b-NadE的基因工程菌株E.N接种到含有5mL含有氨苄青霉素抗性的LB培养基的试管中培养,37℃和180r/min转速下培养12h。再以1%的接种量转入50mL含有氨苄青霉素抗性的LB培养基中表达,37℃表达至OD达到0.8,用0.4mM的IPTG诱导,同时温度调至20℃,诱导表达12h后提取菌体,并做超声处理,后8000r/min冷冻离心10min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5c所示,β-NMN的浓度10±0.5mg/L,较对照菌株E.coli BL21(DE3)(E.coli)发酵结果提高了108%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例8
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF3/PncB-RR3,在基因两端分别引入EcoR I酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶EcoR I的位点;PCR扩增大肠杆菌BL21中PncB的目的基因。
PCR反应体系:2×phantamaxbuffer 10μL,dNTP mixture(10mM)0.4μL,模板(20ng/ul)0.4μL,上、下游引物(10μM)各0.8μL,DMSO0.4μL,phantamax×Super-FidelityDNAPolymerase 0.4μL,补超纯水至20μL。
PCR反应条件:95℃预变性3min;95℃变性15s,58-63℃退火15s,72℃延伸40smin,共循环30次,72℃延伸5min,4℃结束反应。
所述引物PncB-FF3/PncB-RR3的序列为:
PncB-FF3:即5’-ttgtcgacggagctcgaattcATGACACAATTCGCTTCTCCTGT-3’,下划线序列为EcoR I酶切位点;
PncB-RR3:即5’-aattaattcggatccgaattcTTAACTGGCTTTTTTAATATGCGG-3’,下划线序列为EcoR I酶切位点;
所述烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.5;
重组质粒的构建与转化:
用EcoR I单酶切质粒pET22b,将扩增的大肠杆菌BL21的PncB基因片段纯化回收后与单酶切后的质粒pET22b质粒进行同源重组,得到连接产物重组质粒pET22b-PncB(图4b),化学法导入大肠杆菌BL21(DE3)感受态细胞,筛选转化子保存,即为大肠杆菌工程菌株E.coli BL21(DE3)PncB(E.P)。
利用如上所述的基因工程菌株E.P过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pET22b-PncB的基因工程菌株E.P接种到含有5mL含有氨苄青霉素抗性的LB培养基的试管中培养,37℃和180r/min转速下培养12h。再以1%的接种量转入50mL含有氨苄青霉素抗性的LB培养基中表达,37℃表达至OD达到0.8,用0.4mM的IPTG诱导,同时温度调至20℃,诱导表达12h后提取菌体,并做超声处理,后8000r/min冷冻离心10min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5c所示,β-NMN的浓度11±1.0mg/L,较对照菌株E.coli BL21(DE3)(E.coli)发酵结果提高了129%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例9
一种过表达合成β-烟酰胺单核苷酸的方法,步骤如下:
目的片段基因的获得:
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF2/NadE-RR2,在基因两端分别引入HinD III酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶HinD III的位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF3/PncB-RR3,在基因两端分别引入EcoR I酶切位点,在核苷酸序列的上下游5’端各自添加6个核苷酸,形成限制性内切酶EcoR I的位点;PCR分别扩增大肠杆菌BL21中的目的基因。所述引物NadE-FF2/NadE-RR2的序列为:
NadE-FF2:即5’-ctcgagtgcggccgcaagcttATGACATTGCAACAACAAATAATAAAGG-3’,下划线序列为HinD III酶切位点;
NadE-RR2:即5’-tcgagctccgtcgacaagcttTTACTTTTTCCAGAAATCATCGAAAA-3’,下划线序列为HinD III酶切位点;
所述NAD+合成酶NadE的氨基酸序列为SEQ ID No.4;
所述引物PncB-FF3/PncB-RR3的序列为:
PncB-FF3:即5’-ttgtcgacggagctcgaattcATGACACAATTCGCTTCTCCTGT-3’,下划线序列为EcoR I酶切位点;
PncB-RR3:即5’-aattaattcggatccgaattcTTAACTGGCTTTTTTAATATGCGG-3’,下划线序列为EcoR I酶切位点;
所述烟酸磷酸糖基转移酶基因PncB的氨基酸序列为SEQ ID No.5;
重组质粒的构建与转化:
用EcoR I单酶切质粒pET22b-NadE,将扩增的E.coli BL21中的PncB基因片段纯化回收后与单酶切后的质粒pET22b-NadE质粒进行同源重组,得到连接产物重组质粒pET22b-NadE-PncB(图4c),化学法导入大肠杆菌BL21(DE3)感受态细胞,筛选转化子保存,即为大肠杆菌工程菌株E.coli BL21(DE3)NadE-PncB(E.NP)。
利用如上所述的基因工程菌株E.NP过表达合成β-烟酰胺单核苷酸的方法,具体步骤如下:
将含有重组质粒pET22b-NadE-PncB的基因工程菌株E.NP接种到含有5mL含有氨苄青霉素抗性的LB培养基的试管中培养,37℃和180r/min转速下培养12h。再以1%的接种量转入50mL含有氨苄青霉素抗性的LB培养基中表达,37℃表达至OD达到0.8,用0.4mM的IPTG诱导,同时温度调至20℃,诱导表达12h后提取菌体,并做超声处理,后8000r/min冷冻离心10min,将上清用0.22μm的滤膜过滤,得到含有β-NMN产物的发酵液上清,用高效液相色谱检测β-烟酰胺单核苷酸,结果如图5c所示,β-NMN的浓度11±0.8mg/L,较对照菌株E.coliBL21(DE3)(E.coli)发酵结果提高了131%左右。
β-NMN的提纯方法与分析方法参照实施例1。
实施例10
不同碳源对枯草芽孢杆菌基因工程菌株发酵生成NMN的影响,碳源为甘油或葡萄糖;
本实施例中,涉及两种不同碳源的培养基,一种培养基配方是:蛋白胨12g/L,酵母提取物24g/L,K2HPO472 mmol/L,KH2PO417mmol/L,甘油5mL/L,PH7.0-7.4;另一种培养基配方是:蛋白胨12g/L,酵母提取物24g/L,K2HPO472 mmol/L,KH2PO417mmol/L,葡萄糖12g/L,PH7.0-7.4;
本实施例中,优选地,选择产量较高的基因工程菌株B.S.P2,按实施例3的方法发酵、制备β-NMN粗样品。
β-NMN检测结果如图5d所示,基因工程菌株以葡萄糖或者甘油为碳源,对其最终的NMN浓度无显著影响。
NMN的提纯方法与分析方法参照实施例1。
实施例11
构建过表达NadE增加β-NMN累积水平的B.subtilis168基因工程菌株
以Bacillus subtilis 168为底盘细胞,萎缩芽孢杆菌(Bacillus atrophaeusUCMB-5137)中NadE为目的片段,利用质粒pWB980构建重组质粒pWB980-NadE,按实施例1所述方法完成重组质粒的转化,得到最终的基因工程菌株Bacillus subtilis 168NadE1(B.S168.N1)。其中,NadE氨基酸序列与SEQ ID No.1序列相似性99%,GenBank登录号为ID:AKL85421.1。
基因工程菌株B.S168.N1发酵过程具体步骤参照实施例1,培养温度30℃,发酵结果如图5a所示,β-NMN的浓度734±12mg/L,较对照菌株B.S168发酵结果提高了3%左右。
实施例12
构建过表达NadE增加β-NMN累积水平的E.coli BL21(DE3)pLysS基因工程菌株
以E.coli BL21(DE3)pLysS为底盘细胞,恶臭假单胞菌BIRD-1(Pseudomonasputida BIRD-1)中的NadE为目的基因,利用质粒pET28a构建重组质粒pET28a-NadE,按实施例7所述方法完成重组质粒的转化,得到最终的基因工程菌株E.coliBL21(DE3)pLysS NadE2(E.N2)。其中,NadE氨基酸序列与SEQ ID No.4序列相似性96%,GenBank登录号为ID:ADR62218.1。
基因工程菌株E.coli.N2发酵过程中除培养温度30℃外其余具体步骤参照实施例7,发酵结果如图5c所示,β-NMN的浓度10±1.2mg/L,较对照菌株E.coli BL21(DE3)pLysS发酵结果提高了110%左右。
实施例13
构建过表达NadE增加β-NMN累积水平的E.coli Rosetta(DE3)基因工程菌株
以E.coli Rosetta(DE3)为底盘细胞,链球菌NCTC 12261(Streptococcus mitisNCTC 12261)中的NadE为目的基因,利用质粒pET30a构建重组质粒pET30a-NadE,按实施例7所述方法完成重组质粒的转化,得到最终的基因工程菌株E.coli Rosetta(DE3)NadE3(E.N3)。其中,NadE氨基酸序列与SEQ ID No.4序列相似性99%,GenBank登录号为ID:QBZ12090.1。
基因工程菌株E.N3发酵过程具体步骤参照实施例7,发酵结果如图5c所示,β-NMN的浓度11±1mg/L,较对照菌株E.coli Rosetta(DE3)发酵结果提高了57%左右。
实施例14
构建过表达PncB增加β-NMN累积水平的B.subtilisWB800基因工程菌株
以Bacillus subtilisWB800为底盘细胞,犬式布鲁氏菌(Brucella canisstr.Oliveri)中的PncB为目的基因,利用质粒pHT43构建重组质粒pHT43-PncB,按实施例1所述方法完成重组质粒的转化,得到最终的基因工程菌株Bacillus subtilis WB800PncB(B.S800.P3)。其中,PncB氨基酸序列与SEQ ID No.2序列相似性95%,GenBank登录号为ID:CDL75536.1。
基因工程菌株B.S.P3发酵过程具体步骤参照实施例1,发酵结果如图5a所示,β-NMN的浓度750±10mg/L,较对照菌株B.SWB800发酵结果提高了5%左右。
实施例15
构建过表达PncB增加β-NMN累积水平的B.subtilisWB800基因工程菌株
以Bacillus subtilisWB800为底盘细胞,嗜麦芽窄食单胞菌(Stenotrophomonasmaltophilia)中的PncB为目的基因,利用质粒pHT01构建重组质粒pHT01-PncB,按实施例2所述方法完成重组质粒的转化,得到最终的基因工程菌株Bacillus subtilis WB600PncB(B.S800.P4)。其中,PncB氨基酸序列与SEQ ID No.2序列相似性97%,GenBank登录号为ID:CRX68438.1。
基因工程菌株B.S800.P4发酵过程具体步骤参照实施例2,发酵结果如图5a所示,β-NMN的浓度722±10mg/L,较对照菌株B.SWB800发酵结果提高了2%左右。
实施例16
构建过表达PncB增加β-NMN累积水平的E.coli JM109(DE3)基因工程菌株
以E.coli JM109(DE3)为底盘细胞,恶臭假单胞菌BIRD-1(PseudomonasputidaBIRD-1)中的PncB为目的基因,利用质粒pUC57构建重组质粒pUC57-PncB,按实施例8所述方法完成重组质粒的转化,得到最终的基因工程菌株E.coli JM109(DE3)PncB(E.P2)。其中,PncB氨基酸序列与SEQ ID No.5序列相似性98%,GenBank登录号为ID:ADR62217.1。
基因工程菌株E.P2发酵过程具体步骤参照实施例8,发酵结果如图5c所示,β-NMN的浓度12±1mg/L,较对照菌株E.coli JM109(DE3)发酵结果提高了71%左右。
本发明中使用到的相关基因序列可以如下:
1.SEQ ID No.1:枯草芽孢杆菌中NAD+合成酶NadE氨基酸序列
MSMQEKIMRELHVKPSIDPKQEIEDRVNFLKQYVKKTGAKGFVLGISGGQDSTLAGRLAQLAVESIREEGGDAQFIAVRLPHGTQQDEDDAQLALKFIKPDKSWKFDIKSTVSAFSDQYQQETGDQLTDFNKGNVKARTRMIAQYAIGGQEGLLVLGTDHAAEAVTGFFTKYGDGGADLLPLTGLTKRQGRTLLKELGAPERLYLKEPTADLLDEKPQQSDETELGISYDEIDDYLEGKEVSAKVSEALEKRYSMTEHKRQVPASMFDDWWK*
2.SEQ ID No.2:枯草芽孢杆菌中烟酸磷酸糖基转移酶PncB氨基酸序列
VLEYGFKDDSLSLHTDLYQINMAETYWRDGIHEKKAIFELFFRRLPFENGYAVFAGLEKAIEYLENFKFTDSDLSYLQDELGYHEDFIEYLRGLSFTGSLYSMKEGELVFNNEPIMRVEAPLVEAQLIETALLNIVNYQTLIATKAARIKGVIGDEVALEFGTRRAHEMDAAMWGARAALIGGFSATSNVRAGKRFNIPVSGTHAHALVQAYRDEYTAFKKYAETHKDCVFLVDTYDTLRSGMPNAIRVAKEFGDRINFIGIRLDSGDLAYLSKKARKMLDEAGFTDAKVIASSDLDEHTIMNLKAQGARIDVWGVGTKLITAYDQPALGAVYKLVAIEEDGKMVDTIKISSNPEKVTTPGRKKVYRIINQSNHHSEGDYIALYDEQVNDQKRLRMFHPVHTFISKFVTNFYAKDLHELIFEKGILCYQNPEISDIQQYVQDNLSLLWEEYKRISKPEEYPVDLSEDCWSNKMQRIHEVKSRIEEELEEE*
3.SEQ ID No.3:蕈状芽孢杆菌中烟酸磷酸糖基转移酶PnuC氨基酸序列
MVRSPLFLLISSIICILVGFYIRSSYIEIFASVMGIINVWLLAREKVSNFLFGMITVAVFLYIFTTQGLYAMAVLAAFQFIFNVYGWYYWIARSGEEKVKPTVRLDLKGWIIYILFILVAWIGWGYYQVRYLESTNPYLDALNAVLGLVAQFMLSRKILENWHLWILYNIVSIVIYISTGLYVMLVLAIINLFLCIDGLLEWKKNHKERERVNNYI
4.SEQ ID No.4:大肠杆菌中NAD+合成酶NadE氨基酸序列
MTLQQQIIKALGAKPQINAEEEIRRSVDFLKSYLRTYPFIKSLVLGISGGQDSTLAGKLCQMAINELRQETGNESLQFIAVRLPYGVQADEQDCQDAIAFIQPDRVLTVNIKGAVLASEQALREAGIELSDFVRGNEKARERMKAQYSIAGMTSGVVVGTDHAAEAITGFFTKYGDGGTDINPLYRLNKRQGKQLLAALGCPEHLYKKAPTADLEDDRPSLPDEVALGVTYDNIDDYLEGKNVPQQVARTIENWYLKTEHKRRPPITVFDDFWKK
5.SEQ ID No.5:大肠杆菌中烟酸磷酸糖基转移酶PncB氨基酸序列
MTQFASPVLHSLLDTDAYKLHMQQAVFHHYYDVHVAAEFRCRGDDLLGIYADAIREQIQAMQHLRLQDDEYQWLSALPFFKADYLNWLREFRFNPEQVTVSNDNGKLDIRLSGPWREVILWEVPLLAVISEMVHRYRSPQADVAQALDTLESKLVDFSALTAGLDMSRFHLMDFGTRRRFSREVQETIVKRLQQESWFVGTSNYDLARRLSLTPMGTQAHEWFQAHQQISPDLANSQRAALAAWLEEYPDQLGIALTDCITMDAFLRDFGVEFASRYQGLRHDSGDPVEWGEKAIAHYEKLGIDPQSKTLVFSDNLDLRKAVELYRHFSSRVQLSFGIGTRLTCDIPQVKPLNIVIKLVECNGKPVAKLSDSPGKTICHDKAFVRALRKAFDLPHIKKAS
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
序列表
<110> 天津科技大学
<120> 高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
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<212> PRT
<213> 枯草芽孢杆菌中NAD+合成酶NadE氨基酸序列(Unknown)
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<210> 2
<211> 490
<212> PRT
<213> 枯草芽孢杆菌中烟酸磷酸糖基转移酶PncB氨基酸序列(Unknown)
<400> 2
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260 265 270
Ser Lys Lys Ala Arg Lys Met Leu Asp Glu Ala Gly Phe Thr Asp Ala
275 280 285
Lys Val Ile Ala Ser Ser Asp Leu Asp Glu His Thr Ile Met Asn Leu
290 295 300
Lys Ala Gln Gly Ala Arg Ile Asp Val Trp Gly Val Gly Thr Lys Leu
305 310 315 320
Ile Thr Ala Tyr Asp Gln Pro Ala Leu Gly Ala Val Tyr Lys Leu Val
325 330 335
Ala Ile Glu Glu Asp Gly Lys Met Val Asp Thr Ile Lys Ile Ser Ser
340 345 350
Asn Pro Glu Lys Val Thr Thr Pro Gly Arg Lys Lys Val Tyr Arg Ile
355 360 365
Ile Asn Gln Ser Asn His His Ser Glu Gly Asp Tyr Ile Ala Leu Tyr
370 375 380
Asp Glu Gln Val Asn Asp Gln Lys Arg Leu Arg Met Phe His Pro Val
385 390 395 400
His Thr Phe Ile Ser Lys Phe Val Thr Asn Phe Tyr Ala Lys Asp Leu
405 410 415
His Glu Leu Ile Phe Glu Lys Gly Ile Leu Cys Tyr Gln Asn Pro Glu
420 425 430
Ile Ser Asp Ile Gln Gln Tyr Val Gln Asp Asn Leu Ser Leu Leu Trp
435 440 445
Glu Glu Tyr Lys Arg Ile Ser Lys Pro Glu Glu Tyr Pro Val Asp Leu
450 455 460
Ser Glu Asp Cys Trp Ser Asn Lys Met Gln Arg Ile His Glu Val Lys
465 470 475 480
Ser Arg Ile Glu Glu Glu Leu Glu Glu Glu
485 490
<210> 3
<211> 216
<212> PRT
<213> 蕈状芽孢杆菌中烟酸磷酸糖基转移酶PnuC氨基酸序列(Unknown)
<400> 3
Met Val Arg Ser Pro Leu Phe Leu Leu Ile Ser Ser Ile Ile Cys Ile
1 5 10 15
Leu Val Gly Phe Tyr Ile Arg Ser Ser Tyr Ile Glu Ile Phe Ala Ser
20 25 30
Val Met Gly Ile Ile Asn Val Trp Leu Leu Ala Arg Glu Lys Val Ser
35 40 45
Asn Phe Leu Phe Gly Met Ile Thr Val Ala Val Phe Leu Tyr Ile Phe
50 55 60
Thr Thr Gln Gly Leu Tyr Ala Met Ala Val Leu Ala Ala Phe Gln Phe
65 70 75 80
Ile Phe Asn Val Tyr Gly Trp Tyr Tyr Trp Ile Ala Arg Ser Gly Glu
85 90 95
Glu Lys Val Lys Pro Thr Val Arg Leu Asp Leu Lys Gly Trp Ile Ile
100 105 110
Tyr Ile Leu Phe Ile Leu Val Ala Trp Ile Gly Trp Gly Tyr Tyr Gln
115 120 125
Val Arg Tyr Leu Glu Ser Thr Asn Pro Tyr Leu Asp Ala Leu Asn Ala
130 135 140
Val Leu Gly Leu Val Ala Gln Phe Met Leu Ser Arg Lys Ile Leu Glu
145 150 155 160
Asn Trp His Leu Trp Ile Leu Tyr Asn Ile Val Ser Ile Val Ile Tyr
165 170 175
Ile Ser Thr Gly Leu Tyr Val Met Leu Val Leu Ala Ile Ile Asn Leu
180 185 190
Phe Leu Cys Ile Asp Gly Leu Leu Glu Trp Lys Lys Asn His Lys Glu
195 200 205
Arg Glu Arg Val Asn Asn Tyr Ile
210 215
<210> 4
<211> 275
<212> PRT
<213> 大肠杆菌中NAD+合成酶NadE氨基酸序列(Unknown)
<400> 4
Met Thr Leu Gln Gln Gln Ile Ile Lys Ala Leu Gly Ala Lys Pro Gln
1 5 10 15
Ile Asn Ala Glu Glu Glu Ile Arg Arg Ser Val Asp Phe Leu Lys Ser
20 25 30
Tyr Leu Arg Thr Tyr Pro Phe Ile Lys Ser Leu Val Leu Gly Ile Ser
35 40 45
Gly Gly Gln Asp Ser Thr Leu Ala Gly Lys Leu Cys Gln Met Ala Ile
50 55 60
Asn Glu Leu Arg Gln Glu Thr Gly Asn Glu Ser Leu Gln Phe Ile Ala
65 70 75 80
Val Arg Leu Pro Tyr Gly Val Gln Ala Asp Glu Gln Asp Cys Gln Asp
85 90 95
Ala Ile Ala Phe Ile Gln Pro Asp Arg Val Leu Thr Val Asn Ile Lys
100 105 110
Gly Ala Val Leu Ala Ser Glu Gln Ala Leu Arg Glu Ala Gly Ile Glu
115 120 125
Leu Ser Asp Phe Val Arg Gly Asn Glu Lys Ala Arg Glu Arg Met Lys
130 135 140
Ala Gln Tyr Ser Ile Ala Gly Met Thr Ser Gly Val Val Val Gly Thr
145 150 155 160
Asp His Ala Ala Glu Ala Ile Thr Gly Phe Phe Thr Lys Tyr Gly Asp
165 170 175
Gly Gly Thr Asp Ile Asn Pro Leu Tyr Arg Leu Asn Lys Arg Gln Gly
180 185 190
Lys Gln Leu Leu Ala Ala Leu Gly Cys Pro Glu His Leu Tyr Lys Lys
195 200 205
Ala Pro Thr Ala Asp Leu Glu Asp Asp Arg Pro Ser Leu Pro Asp Glu
210 215 220
Val Ala Leu Gly Val Thr Tyr Asp Asn Ile Asp Asp Tyr Leu Glu Gly
225 230 235 240
Lys Asn Val Pro Gln Gln Val Ala Arg Thr Ile Glu Asn Trp Tyr Leu
245 250 255
Lys Thr Glu His Lys Arg Arg Pro Pro Ile Thr Val Phe Asp Asp Phe
260 265 270
Trp Lys Lys
275
<210> 5
<211> 400
<212> PRT
<213> 大肠杆菌中烟酸磷酸糖基转移酶PncB氨基酸序列(Unknown)
<400> 5
Met Thr Gln Phe Ala Ser Pro Val Leu His Ser Leu Leu Asp Thr Asp
1 5 10 15
Ala Tyr Lys Leu His Met Gln Gln Ala Val Phe His His Tyr Tyr Asp
20 25 30
Val His Val Ala Ala Glu Phe Arg Cys Arg Gly Asp Asp Leu Leu Gly
35 40 45
Ile Tyr Ala Asp Ala Ile Arg Glu Gln Ile Gln Ala Met Gln His Leu
50 55 60
Arg Leu Gln Asp Asp Glu Tyr Gln Trp Leu Ser Ala Leu Pro Phe Phe
65 70 75 80
Lys Ala Asp Tyr Leu Asn Trp Leu Arg Glu Phe Arg Phe Asn Pro Glu
85 90 95
Gln Val Thr Val Ser Asn Asp Asn Gly Lys Leu Asp Ile Arg Leu Ser
100 105 110
Gly Pro Trp Arg Glu Val Ile Leu Trp Glu Val Pro Leu Leu Ala Val
115 120 125
Ile Ser Glu Met Val His Arg Tyr Arg Ser Pro Gln Ala Asp Val Ala
130 135 140
Gln Ala Leu Asp Thr Leu Glu Ser Lys Leu Val Asp Phe Ser Ala Leu
145 150 155 160
Thr Ala Gly Leu Asp Met Ser Arg Phe His Leu Met Asp Phe Gly Thr
165 170 175
Arg Arg Arg Phe Ser Arg Glu Val Gln Glu Thr Ile Val Lys Arg Leu
180 185 190
Gln Gln Glu Ser Trp Phe Val Gly Thr Ser Asn Tyr Asp Leu Ala Arg
195 200 205
Arg Leu Ser Leu Thr Pro Met Gly Thr Gln Ala His Glu Trp Phe Gln
210 215 220
Ala His Gln Gln Ile Ser Pro Asp Leu Ala Asn Ser Gln Arg Ala Ala
225 230 235 240
Leu Ala Ala Trp Leu Glu Glu Tyr Pro Asp Gln Leu Gly Ile Ala Leu
245 250 255
Thr Asp Cys Ile Thr Met Asp Ala Phe Leu Arg Asp Phe Gly Val Glu
260 265 270
Phe Ala Ser Arg Tyr Gln Gly Leu Arg His Asp Ser Gly Asp Pro Val
275 280 285
Glu Trp Gly Glu Lys Ala Ile Ala His Tyr Glu Lys Leu Gly Ile Asp
290 295 300
Pro Gln Ser Lys Thr Leu Val Phe Ser Asp Asn Leu Asp Leu Arg Lys
305 310 315 320
Ala Val Glu Leu Tyr Arg His Phe Ser Ser Arg Val Gln Leu Ser Phe
325 330 335
Gly Ile Gly Thr Arg Leu Thr Cys Asp Ile Pro Gln Val Lys Pro Leu
340 345 350
Asn Ile Val Ile Lys Leu Val Glu Cys Asn Gly Lys Pro Val Ala Lys
355 360 365
Leu Ser Asp Ser Pro Gly Lys Thr Ile Cys His Asp Lys Ala Phe Val
370 375 380
Arg Ala Leu Arg Lys Ala Phe Asp Leu Pro His Ile Lys Lys Ala Ser
385 390 395 400
<210> 6
<211> 45
<212> DNA
<213> NadE-FF1(Unknown)
<400> 6
aaagtgaaat cagggggatc catgagcatg caggaaaaga ttatg 45
<210> 7
<211> 48
<212> DNA
<213> NadE-RR1(Unknown)
<400> 7
gagctcgact ctagaggatc cttatttcca ccagtcatca aacataga 48
<210> 8
<211> 49
<212> DNA
<213> PncB-FF1(Unknown)
<400> 8
aaaaggagcg atttacatat ggtgttagag tacggattta aagatgaca 49
<210> 9
<211> 47
<212> DNA
<213> PncB-RR1(Unknown)
<400> 9
acaaactgca taactcatat gttattcttc ctcaagctct tcttcaa 47
<210> 10
<211> 49
<212> DNA
<213> PncB-FF2(Unknown)
<400> 10
cggtacctct agaagaagct tgtgttagag tacggattta aagatgaca 49
<210> 11
<211> 47
<212> DNA
<213> PncB-RR2(Unknown)
<400> 11
ctttaccttg tctccaagct tttattcttc ctcaagctct tcttcaa 47
<210> 12
<211> 49
<212> DNA
<213> NadE-FF2(Unknown)
<400> 12
ctcgagtgcg gccgcaagct tatgacattg caacaacaaa taataaagg 49
<210> 13
<211> 47
<212> DNA
<213> NadE-RR2(Unknown)
<400> 13
tcgagctccg tcgacaagct tttacttttt ccagaaatca tcgaaaa 47
<210> 14
<211> 44
<212> DNA
<213> PncB-FF3(Unknown)
<400> 14
ttgtcgacgg agctcgaatt catgacacaa ttcgcttctc ctgt 44
<210> 15
<211> 45
<212> DNA
<213> PncB-RR3(Unknown)
<400> 15
aattaattcg gatccgaatt cttaactggc ttttttaata tgcgg 45
Claims (10)
1.一种高产β-烟酰胺单核苷酸的基因工程菌株,其特征在于:构建步骤如下:
过表达NAD+合成酶基因NadE、烟酸磷酸核糖转移酶基因PncB和转运蛋白基因PnuC三种基因中的任意一个或两个或三个。
2.根据权利要求1所述的高产β-烟酰胺单核苷酸的基因工程菌株,其特征在于:所述基因工程菌株使用的底盘菌株包括枯草芽孢杆菌、大肠杆菌、酿酒酵母、毕赤酵母菌。
3.根据权利要求2所述的高产β-烟酰胺单核苷酸的基因工程菌株,其特征在于:所述枯草芽孢杆菌包括B.subtilis 168、B.subtilis WB600、B.subtilis WB800;
或者,所述大肠杆菌包括E.coli BL21(DE3)、E.coli BL21(DE3)pLysS、E.coliRosetta(DE3)、E.coli JM109(DE3);
或者,构建时使用的过表达载体质粒包括pMA5、pWB980、pHT43、pHT01和pET22b、pET28a、pET30a、pUC57。
4.根据权利要求1所述的高产β-烟酰胺单核苷酸的基因工程菌株,其特征在于:过表达的NAD+合成酶基因NadE的氨基酸序列与序列SEQ ID No.1具有至少95%的一致性;
或者,过表达的烟酸磷酸糖基转移酶基因PncB的氨基酸序列与序列SEQ ID No.2具有至少95%的一致性;
或者,过表达的转运蛋白基因PnuC的氨基酸序列与序列SEQ ID No.3具有至少95%的一致性;
或者,过表达NAD+合成酶基因NadE的氨基酸序列与序列SEQ ID No.4具有95%的一致性;
或者,过表达的烟酸磷酸糖基转移酶基因PncB的氨基酸序列与序列SEQ ID No.5具有至少95%的一致性。
5.根据权利要求1所述的高产β-烟酰胺单核苷酸的基因工程菌株,其特征在于:所述基因工程菌株包括BacillussubtilisNadE(B.S.N)或Bacillus subtilis 168NadE1(B.S168.N1)、Bacillus subtilis PncB1(B.S.P1)或Bacillus subtilis PncB2(B.S.P2)或Bacillus subtilis WB800PncB(B.S800.P3)或Bacillus subtilis WB600PncB(B.S800.P4)、Bacillus subtilis NadE-PncB1(B.S.PN1)或Bacillus subtilis NadE-PncB2(B.S.PN2)或Bacillus subtilis PncB2-PnuC(B.S.PP)或E.coli BL21(DE3)NadE(E.N)或E.coli BL21(DE3)pLysS NadE2(E.N2)或E.coli Rosetta(DE3)NadE3(E.N3)或E.coli BL21(DE3)PncB(E.P)或E.coli JM109(DE3)PncB(E.P2)或E.coli BL21(DE3)NadE-PncB(E.NP)。
6.如权利要求1所述的基因工程的构建方法,其特征在于:步骤如下:
一、重组质粒的构建:
⑴目的片段基因的获得:根据目的基因设计引物扩增,或者根据基因序列直接合成;根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF1/NadE-RR1,在基因两端分别引入酶切位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF1/PncB-RR1,在基因两端分别引入酶切位点;PCR分别扩增Bacillus subtilis 168中NadE、PncB,Bacillus mycoides中PnuC基因根据基因序列直接合成;
根据NCBI查询得到的NadE酶基因设计引物序列NadE-FF2/NadE-RR2,在基因两端分别引入酶切位点;根据NCBI查询得到的PncB酶基因设计引物序列PncB-FF2/PncB-RR2,在基因两端分别引入酶切位点;PCR分别扩增大肠杆菌BL21中的NadE、PncB目的基因;
⑵重组质粒的构建:将扩增的B.subtilis168中的NadE基因片段纯化回收后与单酶切后的质粒pMA5质粒使用非连接酶依赖型单片段快速克隆试剂盒进行同源重组,得到重组产物质粒pMA5-NadE;将扩增的B.subtilis168中PncB基因片段纯化回收后与酶切后pMA5质粒进行同源重组,得到重组质粒pMA5-PncB;设计引物pP-FF1/pP-RR1,以重组质粒pMA5-PncB为模板,扩增HpaⅡpromoter和PncB序列,将扩增的HpaⅡpromoter和PncB基因片段纯化回收后与单酶切后的质粒pMA5-NadE质粒进行同源重组,得到重组产物质粒pMA5-NadE-PncB;用单酶切质粒pMA5-PncB,将合成的Bacillus mycoides中的PnuC片段与酶切后的质粒pMA5-PncB同源重组,得到重组质粒pMA5-PncB-PnuC;
用内切酶单酶切载体质粒pET22b,将扩增的E.coli BL21中NadE基因片段纯化回收后与单酶切后的pET22b质粒进行同源重组,得到重组质粒pET22b-NadE,用内切酶单酶切质粒pET22b-NadE,将扩增的E.coli BL21中PncB基因片段纯化回收后与单酶切后的质粒pET22b-NadE质粒进行同源重组,得到重组质粒pET22b-NadE-PncB;
二、过表达基因工程菌株的构建:
将构建的重组质粒分别转化到大肠杆菌DH5α感受态细胞中,通过菌落PCR验证,筛选大肠杆菌DH5α阳性转化子,于含有抗生素的LB液体培养基中,30~37℃恒温震荡过夜培养,-80℃保存菌种备用;提取大肠杆菌DH5α阳性转化子中的重组质粒,将提取得到的重组质粒转化至B.subtilis或E.coli底盘菌株中,涂布于含有抗生素的抗性平板中,进行菌落PCR验证,挑选B.subtilis或E.coli菌株中的阳性转化子于含有抗生素的液体培养基中,37℃恒温震荡过夜培养,-80℃保存菌种备用,得到高产β-烟酰胺单核苷酸基因工程菌株。
7.一种重组载体,其特征在于:所述载体含有如权利要求1至6任一项所述的基因工程菌株的氨基酸序列。
8.如权利要求1至6任一项所述的基因工程菌株在β-烟酰胺单核苷酸生产方面中的应用。
9.利用如权利要求1至6任一项所述的基因工程菌株生产β-烟酰胺单核苷酸的方法,其特征在于:步骤如下:
通过发酵培养所述高产β-烟酰胺单核苷酸基因工程菌株,从发酵液中提取β-烟酰胺单核苷酸。
10.根据权利要求9所述的方法,其特征在于:发酵培养时的发酵培养基碳源为甘油或葡萄糖,氮源为蛋白胨或酵母提取物,培养温度为30-37℃;
或者,提取β-烟酰胺单核苷酸使用反相高效液相色谱制备柱纯化,固定相为十八烷基硅烷键合硅胶。
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