CN114807078A - 一种生物合成nmn的方法 - Google Patents
一种生物合成nmn的方法 Download PDFInfo
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- CN114807078A CN114807078A CN202210408487.1A CN202210408487A CN114807078A CN 114807078 A CN114807078 A CN 114807078A CN 202210408487 A CN202210408487 A CN 202210408487A CN 114807078 A CN114807078 A CN 114807078A
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- gene
- nmn
- nicotinamide
- escherichia coli
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Abstract
本发明属于生物工程技术领域，具体涉及一种重组大肠杆菌生物合成NMN的方法。所述工程菌是在大肠杆菌宿主中，通过缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadD、nadR的表达的同时，过表达NAD+合酶编码基因nadE；表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、核糖磷酸二磷酸激酶编码基因Prs以及核糖激酶编码基因RbsK获得的；本发明同时对NadV进行了定向进化，使催化效率达到最佳。本发明采用上述重组菌全细胞为催化剂催化转化，方法简单；反应底物为烟酰胺和核糖，生产成本较低；建了依赖烟酰胺和核糖的NMN生物合成途径，使相关酶能够高效表达，使NMN产量达到2.88g/L。
Description
技术领域：
本发明属于生物工程技术领域，具体涉及一种重组大肠杆菌生物合成NMN的方法。
背景技术：
作为生命体内200多种还原反应的辅酶，NAD是3类NAD消耗酶的底物，在多种细胞的生理过程中起着至关重要的作用，NAD的生物能量状态甚至决定了细胞的生死存亡。同时，NAD的代谢在健康和疾病状态中起着重要作用，逐渐引起人们的关注，因此参与NAD生物合成和代谢的酶也成为了疾病治疗中具有关注热度的药物靶标。
NMN(nicotinamide mononucleotide)，即烟酰胺单核苷酸，是一种自然存在的生物活性核苷酸，NMN作为细胞内参与NAD合成的重要底物，在人体细胞能量生成中扮演重要角色。NMN是人体内原本存在的物质，但随着年龄的增长而减少。在哺乳动物体内，NMN主要由烟酰胺在烟酰胺磷酸核糖转移酶(Nicotinamide phosphate ribose transferase，Nampt)的催化作用下生成，随后NMN在烟酰胺单核苷酸腺苷转移酶的催化下生成NAD+。在细胞外的NMN则需要脱去磷酸转化为烟酰胺核糖(NR)才能进入肝细胞内部，进入胞内后，烟酰胺核糖在烟酰胺核苷激酶的作用下再磷酸化生成NMN，随后NMN和ATP结合生成NAD+。
目前NMN合成主要通过化学催化与酶催化进行。传统的体外制备β-烟酰胺单核苷酸的方法为化学合成法，催化NMN时需要用到危险化学品和大量的有机溶剂，会破坏环境且操作复杂、反应步骤多、中间产物多、得率低、产品纯化困难。比如，Tanimori等人用乙酰基保护的核糖与烟酰胺在TMSOTf的催化下发生缩合反应；又比如Palmarisa等人使用硅烷化试剂对烟酰胺进行硅烷化，然后与乙酰核糖在TMSOTf的催化下进行反应。这些化学合成方法存在成本高、收率低而且化学试剂污染大等问题。
至于酶催化，所需原料ATP比较昂贵，生产NMN的成本很高。早在1994年，Jeck等利用二磷酸吡啶核苷酸为原料，在焦磷酸化酶的催化水解下生成烟酰胺单核苷酸。2016年，深圳邦泰公司利用烟酰胺、ATP和核糖为底物，在烟酰胺磷酸核糖转移酶、核糖磷酸焦磷酸激酶以及核糖激酶的催化下生成烟酰胺单核苷酸。2017年，该公司进一步改进工艺，以烟酰胺、焦磷酸或其盐和AMP为原料，在烟酰胺磷酸核糖转移酶和腺膘呤磷酸核糖转移酶的催化作用下发生反应，获得烟酰胺单核苷酸；该工艺优势在于使用磷酸核糖焦磷酸为原料，降低了成本。2018年，尚科生物报道以D-5-磷酸核糖、ATP和烟酰胺为原料，通过固定化含有磷酸核糖焦磷酸合成酶和烟酰胺磷酸核糖转移酶的活性细胞，实现了高效生物催化合成β-烟酰胺单核苷酸。固定化细胞或固定化酶可以重复多次使用，利于纯化，以降低生产成本，但是合成所需原料ATP相对比较昂贵，也增加了NMN的生产成本。
因此考虑到食品安全因素以及化学催化与酶催化的环境污染问题和局限性，目前多使用生物合成法制备NMN，即通过构建工程菌使相关酶在大肠杆菌中重组表达，再进行发酵生产。最早由Marinescu等构建基因工程菌发酵合成NMN，该团队将烟酰胺磷酸核糖转移酶和5’-磷酸核糖焦磷酸(Phosphoribosyl_pyrophosphate，PRPP)合成酶在大肠杆菌中重组表达，以尼克酰胺和乳糖为底物进行发酵生产，但最终NMN产量比较低，仅为15.4mg/L。随后该团队还开发了分子筛色谱法分离NMN的方法，但是从色谱图发现，还是有大量尼克酰胺没有转化为NMN。分析可知，NMN合成涉及细胞能量代谢，未来可以通过高密度发酵提高单位体积的产物得率。生物合成的NMN产量比较低，且合成出NMN后有较多的途径分解NMN，最终导致NMN在微生物体内无法保存较高的含量，因此常规的生物合成手段无法解决高效发酵生产NMN的问题。
发明内容：
为了解决上述技术问题，本发明通过降低或敲除了大肠杆菌菌体内降解NMN的功能基因，使产生的NMN不容易被降解。利用基因编辑技术对这些途径的基因进行敲除，并通过启动子温敏突变体的替换从而构建出了一株可以在菌体内高效保留NMN的工程菌株。另外本发明在该菌株中构建了一条依赖于烟酰胺、核糖的NMN的生物合成途径。然后通过易错PCR筛选技术，筛选出了一个NadV的突变体，该突变体可以在该菌株体系中更高效的生成NMN。
本发明提供的技术方案之一，是一株可以大量合成并积累NMN(nicotinamidemononucleotide，即烟酰胺单核苷酸)的大肠杆菌基因工程菌，所述工程菌是在大肠杆菌宿主中，缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadD、nadR的表达的同时，过表达NAD+合酶编码基因nadE来提高NMN的保留量；表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、核糖磷酸二磷酸激酶编码基因Prs以及核糖激酶编码基因RbsK来构建依赖烟酰胺和核糖的NMN生物合成途径；
进一步地，pncC、nadR基因缺失表达的方式为基因敲除；
进一步地，nadD基因缺失表达的方式为通过CIts蛋白及PR/PL启动子温敏控制nadD基因的表达；
进一步地，nadE基因通过pET-28a载体进行表达；
进一步地，NadVmu、Prs、RbsK基因通过质粒过表达；
优选地，采用pGEX4T3载体对NadVmu、Prs、RbsK基因进行过表达；
进一步地，野生型NadV基因的核苷酸序列如序列表SEQ ID NO：1所示；
进一步地，pncC基因的核苷酸序列如序列表SEQ ID NO：2所示；
进一步地，nadD基因的核苷酸序列如序列表SEQ ID NO：3所示；
进一步地，nadR基因的核苷酸序列如序列表SEQ ID NO：4所示；
进一步地，nadE基因的核苷酸序列如序列表SEQ ID NO：5所示；
进一步地，Prs基因的核苷酸序列如序列表SEQ ID NO：6所示；
进一步地，RbsK基因的核苷酸序列如序列表SEQ ID NO：7所示；
进一步地，烟酸磷酸核糖转移酶突变体编码基因NadVmu的核苷酸序列如序列表SEQID NO：11所示；烟酸磷酸核糖转移酶突变体的氨基酸序列如SEQ ID NO：12所示；
进一步地，所述大肠杆菌宿主选自JM109、BL21(DE3)、Top 10、DH5α、Rosetta、Rosetta-gami pLysS等；
优选地，所述大肠杆菌宿主为大肠杆菌JM109。
本发明提供的技术方案之二，是上述基因工程菌的构建方法，具体如下：
(1)构建高NMN保留量的工程菌株
利用大肠杆菌基因编辑载体pCas9对大肠杆菌宿主细胞基因组进行基因编辑，构建pncC、nadR基因的敲除菌株(JM109ΔpncCΔnadR)，因nadD途径无法敲除，所以通过ts温敏表达对nadD的表达进行控制，构建温敏表达nadD的工程菌株(JM109ΔpncCΔnadR nadD(ts))；继续在基因组构建受到IPTG诱导的nadE基因；该菌株作为底盘细胞可以高效的保存大肠杆菌合成的NMN不被降解；
(2)构建依赖烟酰胺和核糖的NMN生物合成途径
利用pGEX4T3载体为骨架，构建可在IPTG诱导情况下高效表达NadVmu、Prs以及RbsK的表达载体，并将所述表达载体在步骤(1)所得菌株中表达。
本发明提供的技术方案之三，是上述工程菌在生产NMN中的应用；
进一步地，采用上述工程菌发酵生产NMN的方法如下：
反应体系包括：烟酰胺5-20mmol/L、核糖10-20mmol/L、ATP 5-10mmol/L、NAD+ 5-10mmol/L、Na2HPO4/NaH2PO4 50mmol/L、醋酸钠10mmol/L、氯化钙1mmol/L，其余为水pH5-8.5；加入生产菌使菌体浓度达到5-100g/L，在15-22℃，50-200rpm，避光下反应10-25h；
10-25h后，NMN产量达到1.31-2.88g/L；
进一步地，菌体培养方法如下：
将工程菌种子液按1％的接种量接种于PYA8培养基中，37℃、200rpm培养6h(OD600为0.7左右)，自来水冷却10min，加1mM IPTG后，22℃、200rpm条件下诱导表达24h；离心收集菌体；
更进一步地，所述PYA8培养基组成为(w/v)：0.1％大豆蛋白胨，1％葡萄糖，1.61％磷酸氢二钠，0.136％磷酸二氢钾，0.05％氯化钠，0.5％酵母浸膏，1％醋酸钠，其余为水，pH7.0～7.2；
优选地，NMN生物转化条件为：菌体浓度为50g/L，烟酰胺15mmol/L、核糖20mmol/L、ATP 5mmol/L、NAD+ 10mmol/L，pH8.0，20℃，100rpm，避光下反应20h；NMN产量达到8.36mmol/L。
有益效果：
与现有技术相比，本发明具有以下优点：
1、本发明采用特定的重组大肠杆菌全细胞为催化剂催化转化，方法简单；反应底物为烟酰胺和核糖，生产成本较低；本发明构建了依赖烟酰胺和核糖的NMN生物合成途径，使相关酶能够高效表达，提高了NMN产量。
2、采用本发明方法进行生物转化，敲除了大肠杆菌菌体内降解NMN的功能基因，使产生的NMN不容易被降解，高效保留NMN，提高了产量，NMN产量达到2.88g/L。
3、本发明对NadV即编码合成烟酸磷酸核糖转移酶的基因进行了定向进化，使该酶的催化效率达到最佳，可将NMN产量由1.88g/L提高到2.88g/L。
附图说明：
图1基因编辑载体pCas9质粒图谱；
图2 pTargetF质粒图谱；
图3 pncC基因敲除验证；
图4 nadR基因敲除验证；
图5温敏表达nadD基因验证；
图6 pGEX4T3-NadV-Prs-RbsK质粒图谱。
具体实施方式：
为了使本专利的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本专利进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本专利，并不用于限定本发明。
实施例1 NMN生产基因工程菌株的构建
(1)构建高NMN保留量的工程菌株
利用大肠杆菌基因编辑载体pCas9对大肠杆菌宿主细胞JM109基因组进行基因编辑，构建pncC、nadR基因的敲除菌株(JM109ΔpncCΔnadR)，因nadD途径无法敲除，所以再构建温敏表达nadD的工程菌株(JM109ΔpncCΔnadR nadD(ts))；继续在基因组构建受到IPTG诱导的nadE基因；该菌株作为底盘细胞可以高效的保存大肠杆菌合成的NMN不被降解；
具体的构建步骤如下：
①底盘构建
如附图1所示，将大肠杆菌基因编辑载体pCas9转化入JM109感受态细胞，获得稳定表达CAS9蛋白的底盘。
②pncC基因进行敲除
采用pTargetF为载体，在其中插入sgRNA序列1和供体DNA片段，得到重组质粒，将重组质粒转化至表达CAS9蛋白的JM109感受态细胞，得到包含质粒的敲除菌株，经筛选得到敲除菌株JM109ΔpncC。
其中，pncC的核苷酸序列如SEQ ID NO.2所示，蛋白ID：AAN81705.1，通过NCBI数据库获取得到。pTargetF质粒如图2所示。
首先，利用CRISPR网站(http://crispr.dbcls.jp/)进行分析，选取评分较高且靠近起始密码子的序列为基因敲除靶点，设计靶点序列引物，通过PCR方式将敲除靶点序列引入至pTargetF质粒上的启动子和gRNA scaffold序列之间，使靶点序列和gRNA scaffold序列连接形成sgRNA序列1，获得pTargetF-1质粒。进一步根据pncC上下游各300bp左右的序列设计pncC上、下游同源臂引物，分别引入Xho I和PshA I酶切位点。通过PCR获得上下游同源臂，再用F3和R2引物，通过融合PCR方式获得供体DNA片段，序列如SEQ ID NO.8所示。
然后将pTargetF-1质粒和供体DNA进行双酶切，回收片段之后连接，转化步骤①获得的表达CAS9蛋白的JM109感受态细胞，再涂布氯霉素/壮观霉素双抗平板，以筛选引物对长出的单克隆进行菌落PCR检测，结果如图3所示(原长度1312bp(阴性)，敲除部分472bp，成功敲除后840bp(阳性))，筛选出成功敲除pncC的阳性克隆，再用氯霉素单抗性培养基进行连续传代培养，得到去除pTargetF质粒的JM109ΔpncC菌株。
相关引物或序列见表1记载：
表1.pncC基因敲除相关序列
③nadR基因敲除
采用pTargetF为载体，在其中插入sgRNA序列2和供体DNA片段，得到重组质粒，将重组质粒转化至JM109ΔpncC感受态细胞，得到包含质粒的敲除菌株，经筛选得到敲除菌株JM109ΔpncCΔnadR。
其中，nadR的核苷酸序列如SEQ ID NO.4所示，蛋白ID：ACI72736.1，通过NCBI数据库获取得到。
与步骤②相同，同样的选择基因敲除靶点，设计引物将靶点序列插入pTargetF载体，形成sgRNA序列2，获得pTargetF-2质粒。设计nadR基因上、下游同源臂引物，引入Xho I和PshA I酶切位点，获取两段同源臂后，通过融合PCR获得供体DNA片段，序列如SEQ IDNO.9所示。然后将pTargetF-2质粒和供体DNA进行双酶切，回收片段之后连接，转化步骤②获得的JM109ΔpncC的感受态细胞，再涂布氯霉素/壮观霉素双抗平板。以筛选引物对长出的单克隆进行菌落PCR检测，结果如图4所示(原长度1723bp(阴性)，敲除部分1098bp，成功敲除后625bp(阳性))，筛选出成功敲除nadR的阳性单克隆，再用氯霉素单抗性培养基进行连续传代培养，得到去除pTargetF质粒的JM109ΔpncCΔnadR菌株。
相关引物或序列见表2记载：
表2.nadR基因敲除相关序列
④温敏表达nadD基因的菌株构建
采用pTargetF为载体，在其中插入sgRNA序列3和供体DNA片段，得到重组质粒，将重组质粒转化至JM109ΔpncCΔnadR感受态细胞，经筛选得到菌株JM109ΔpncCΔnadRnadD(ts)。
nadD的核苷酸序列如SEQ ID NO.3所示，蛋白ID：CTV94997.1，来源于E.coli。
根据nadD上游区域序列分析基因插入靶点，设计引物将靶点序列插入pTargetF载体，形成sgRNA序列3，获得pTargetF-3质粒。选择nadD基因的上游部分序列和基因前段部分序列为两段同源臂，中间插入CIts蛋白及PR/PL启动子序列(SEQ ID NO.10)，两端引入XhoI和PshAI酶切位点，共约2.2kb进行全合成，得到供体DNA片段。
将pTargetF-3质粒和供体DNA进行双酶切，回收片段之后连接，转化步骤③获得的JM109ΔpncCΔnadR感受态细胞，再涂布氯霉素/壮观霉素双抗平板。以筛选引物对长出的单克隆进行菌落PCR，结果如图5所示(成功插入DNA的单克隆可以通过PCR检测到1kb左右的条带(阳性)，未成功插入的无法检测到条带(阴性))，筛选出改造成功的阳性单克隆，再用氯霉素单抗性培养基进行连续传代培养，得到去除pTargetF质粒的JM109ΔpncCΔnadRnadD(ts)，使nadD成为温敏表达型蛋白。
相关引物或序列见表3记载：
表3.温敏表达nadD基因的菌株构建相关序列
⑤IPTG诱导nadE基因的菌株构建
nadE的核苷酸序列如SEQ ID NO.5所示，蛋白ID：WP_003037081.1，来源于弗朗西斯氏菌(Francisella tularensis)。
全合成nadE基因序列，5’端引入EcoR I酶切位点，3’端引入Nco I酶切位点，即：5’-CCGGAATTC-nadE-CCATGGATG-3’，共765bp。用pET-28a载体构建重组质粒，基因片段和pET-28a质粒均进行Nco I/EcoR I双酶切，回收酶切片段，用T4连接酶进行连接。转化入步骤④获得的JM109ΔpncCΔnadR nadD(ts)感受态细胞，在含有Kan+的LB平板上涂布，挑取转化子进行验证，得到JM109ΔpncCΔnadR nadD(ts)-nadE菌株。
(2)构建依赖烟酰胺和核糖的NMN生物合成途径
利用pGEX-4T-3载体为骨架以及tac启动子控制元件，构建可在IPTG诱导情况下高效表达NadV、Prs以及RbsK的表达载体，并将所述表达载体在步骤(1)所得菌株中表达。
用pGEX-4T-3载体构建重组质粒，分别从NCBI数据库获取NadV、Prs以及RbsK的CDS序列(SEQ ID NO.1、6、7)，中间插入lac操纵子及tac启动子元件，两端分别设计BamHII和NotI酶切位点，共3330bp的序列进行全合成。然后将合成序列与pGEX-4T-3质粒分别进行双酶切，回收酶切片段，进行连接，构建pGEX4T3-NadV-Prs-RbsK质粒，如图6所示。将连接产物转化入JM109ΔpncCΔnadR nadD(ts)-nadE感受态细胞，在含有Kan+和Amp+双抗的LB平板上涂布，挑取转化子进行验证，获得重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX4T3-NadV-Prs-RbsK。
实施例2 NadV定向进化
NadV(烟酸磷酸核糖转移酶)是决定NMN合成的关键基因，该基因的催化效率以及催化的最适环境是配合其它两个酶(核糖磷酸二磷酸激酶编码基因Prs以及核糖激酶编码基因RbsK)催化的关键因素，而野生型NadV的催化效率达不到配适需求。
本发明利用易错PCR技术对NadV进行了进化筛选，选取在LB培养基中生长速度下降的菌株为筛选目标。
具体为：以实施例1构建的pGEX4T3-NadV-Prs-RbsK质粒为模板，设计NadV两端的引物，分别引入BamHI和SgrAI酶切位点，用易错PCR方式进行突变。将PCR产物和模板质粒进行BamHI/SgrAI双酶切，回收片段进行连接，再转化JM109ΔpncCΔnadR nadD(ts)-nadE感受态细胞。
以在LB培养基中生长速度下降的菌株为筛选目标，筛选出菌株后，接种在96孔板的LB培养基中，37℃震荡培养4h，降温至22℃,加入IPTG诱导表达24h，得到菌液；然后在96孔板中加入反应体系进行反应，所述反应体系为4mM NR(烟酰胺核糖)，2mM ATP，2mM氯化钙，与菌液按体积比1:1混合，20℃反应1h，离心得到上清液。
取上清液，用荧光酶标仪进行荧光检测(入射光382nm，发射光445nm)，荧光越强表明产NMN能力越强，最终筛选出6株荧光较强的突变体，对这些菌株测序发现其有2个共同的突变位点：Q54L和D453G。
以pGEX4T3-NadV-Prs-RbsK质粒为模板，设计引物在这两处进行定点突变，将突变后的质粒转化回JM109ΔpncCΔnadR nadD(ts)-nadE感受态细胞，挑选单克隆验证，经测序后确认构建出工程菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX4T3-NadVmu-Prs-RbsK。
点突变引物：
点突变后获得的烟酸磷酸核糖转移酶突变体编码基因NadVmu的核苷酸序列如序列表SEQ ID NO：11所示，烟酸磷酸核糖转移酶突变体氨基酸序列如SEQ ID NO：12所示。
实施例3NMN生物合成
(1)菌株培养及表达。
实验菌株：
实施例2构建的重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX-NadVmu-Prs-RbsK；
实施例1构建的重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX-NadV-Prs-RbsK。
挑取重组大肠杆菌接种于20mL含卡那霉素50μg/mL、氨苄100μg/mL的LB培养，37℃、200rpm培养16h，然后按1％接种于PYA8培养基(0.1％大豆蛋白胨，1％葡萄糖，1.61％磷酸氢二钠；0.136％磷酸二氢钾；0.05％氯化钠；0.5％酵母浸膏；1％醋酸钠，其余为水，pH7.0～7.2)培养基中(500ml三角瓶)，37℃、200rpm培养6h(OD600(～0.7))，自来水冷却10min，加1mM IPTG后，22℃、200rpm条件下诱导表达24h。然后离心(4℃，10000g，离心5min)收集菌体。
(2)NMN生物转化
使用上述收集菌体为催化剂进行生物转化，按表1称取原料配置反应液，溶解后加入菌体，搅拌均匀，在对应条件下避光反应得到产物。
(3)产物测定
反应结束后，向反应液中加入1/2倍体积的双蒸水，置10℃，10min。在4℃、12000g条件下离心10min，收集上清，在向上清中加入5倍体积的丙酮，置4℃过夜，在4℃、12000g条件下离心10min，收集沉淀，得到初步纯化的反应物。再采用去离子水溶解沉淀，对反应产物进行高效液相色谱法(HPLC法)。
色谱条件如下：
检测前样品处理——用70％甲醇水溶液将样品稀释50倍，然后用孔径为0.22μm的微孔滤器过滤后装到样品瓶中；
仪器设备——美国安捷伦公司1260高效液相色谱仪；
色谱柱：Phenomenex Luna C18色谱柱(4.6mm×250mm 5μm)；
柱温：35℃；
流动相：流动相为流动相A为0.25％磷酸二氢钠和0.2‰磷酸的水溶液；流动相B为甲醇，流动相A：流动相B＝1：1；
进样量：5μL。
流速：0.3mL/min。
表1 重组大肠杆菌催化体系
上述结果说明，由本发明构建的重组菌JM109ΔpncCΔnadR nadD(ts)-nadE，pGEX-NadVmu-Prs-RbsK，生产NMN的产量能够达到8.26mmol/L(约为2.88g/L)，且经过改造后的烟酸磷酸核糖转移酶突变体能够进一步提高NMN的产量。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。
SEQUENCE LISTING
<110> 四川盈嘉合生科技有限公司
<120> 一种生物合成NMN的方法
<130> 1
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 1488
<212> DNA
<213> 湿热淋病杆菌（Haemophilusducreyi）
<400> 1
atggacaacc tgctgaacta tagcagccgt gcgagcgcga tcccgagcct gctgtgcgat 60
ttctacaaga ccagccaccg tattatgtat ccggagtgca gccagatcat ttacagcacc 120
tttaccccgc gtagcaacga acaggcgccg tacctgaccc aagtggttag cttcggtttt 180
caagcgttca tcatcaagta cctgatccac tacttcaacg acaacttctt tagccgtgac 240
aagcacgatg tggttaccga gtatagcgcg ttcatcgaaa aaaccctgca actggaggat 300
accggtgaac acattgcgaa gctgcacgag ctgggctacc tgccgatccg tattaaagcg 360
atcccggaag gcaagaccgt ggcgatcaaa gtgccggtta tgaccattga gaacacccac 420
agcgacttct tttggctgac caactatctg gaaaccctga ttaacgttag cctgtggcaa 480
ccgatgacca gcgcgagcat cgcgttcgcg taccgtaccg cgctgattaa gtttgcgaac 540
gagacctgcg acaaccagga acacgtgccg ttccaaagcc acgattttag catgcgtggt 600
atgagcagcc tggagagcgc ggaaaccagc ggtgcgggtc acctgaccag cttcctgggc 660
accgacacca tcccggcgct gagctttgtt gaggcgtact atggtagcag cagcctgatc 720
ggcaccagca ttccggcgag cgagcacagc gtgatgagca gccacggtgt tgatgaactg 780
agcaccttcc gttatctgat ggcgaagttt ccgcacaaca tgctgagcat cgtgagcgac 840
accaccgatt tctggcacaa cattaccgtt aacctgccgc tgctgaaaca ggagatcatt 900
gcgcgtccgg aaaacgcgcg tctggtgatc cgtccggaca gcggtaactt ctttgcgatc 960
atttgcggcg acccgaccgc ggataccgag cacgagcgta agggtctgat cgaatgcctg 1020
tgggacattt tcggtggcac cgtgaaccag aagggttata aagttattaa cccgcacatc 1080
ggcgcgattt acggtgatgg cgttacctat gagaagatgt ttaaaatcct ggaaggtctg 1140
caagcgaaag gcttcgcgag cagcaacatt gtgtttggtg ttggcgcgca gacctaccaa 1200
cgtaacaccc gtgacaccct gggtttcgcg ctgaaggcga ccagcatcac cattaacggc 1260
gaggaaaaag cgatcttcaa gaacccgaaa accgacgatg gttttaagaa aagccagaag 1320
ggccgtgtga aagttctgag ccgtgacacc tacgtggatg gtctgaccag cgcggacgat 1380
ttcagcgacg atctgctgga gctgctgttt gaagatggca aactgctgcg tcagaccgac 1440
tttgatgaga tccgtcaaaa cctgctggtt agccgtacca ccctgtaa 1488
<210> 2
<211> 498
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 2
atgactgaca gtgaactgat gcagttaagt gaacaggttg ggcaggcgct gaaagcccgt 60
ggcgcaaccg taacaactgc cgagtcttgt accggtggtt gggtagcgaa agtgattacc 120
gatattgccg gtagctccgc ctggtttgaa cgcggatttg tcacctacag taacgaagcc 180
aaagcgcaga tgatcggcgt acgcgaagag acgctggcgc agcatggcgc ggtgagtgaa 240
cccgtcgtgg tggaaatggc gataggcgca ctgaaagcgg ctcgtgctga ttatgccgtg 300
tctattagtg gtatcgccgg gccggatggc ggcagtgaag agaagcctgt cggcaccgtc 360
tggtttgctt ttgccactgc ccgcggtgaa ggcattaccc ggcgggaatg cttcagcggc 420
gaccgtgatg cggtgcgtcg tcaggctact gcgtatgcat tgcagacctt gtggcaacaa 480
tttctacaaa acacttga 498
<210> 3
<211> 642
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 3
atgaaatctt tacaggctct gtttggcggc acctttgatc cggtgcacta tggtcatcta 60
aaacccgtgg aaacgctggc gaatttgatt ggtctgacgc gggtcacaat catccctaat 120
aatgttcctc cgcatcgtcc ccagccggaa gcgaacagcg tgcagcgtaa acacatgctt 180
gaactggcga ttgccgacaa gccattattt actcttgatg aacgcgagct aaagcgcaat 240
gccccctctt acactgcgca aacactgaaa gagtggcggc aggaacaagg accggacgtg 300
ccgctggcgt ttattattgg tcaggattca ctgctgacct ttccgacctg gtacgaatac 360
gaaacgatac tcgacaatgc acatttgatc gtctgtcggc gtccaggtta cccacttgaa 420
atggcgcaac cgcaatacca gcaatggctg gaagatcatt tgacacataa cccggaagat 480
cttcaccttc agcctgccgg taaaatttat ctggctgaaa cgccgtggtt taacatctcg 540
gcgaccatca tccgcgaacg tttgcaaaac ggtgaatcat gtgaggattt attgccggaa 600
ccggtactga cttacattaa ccaacaaggc ttgtatcgct ga 642
<210> 4
<211> 1233
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 4
atgtcgtcat ttgattacct gaaaactgcc atcaagcaac agggctgcac gctacagcag 60
gtagctgatg ccagcggtat gaccaaaggg tatttaagcc agttactgaa tgccaaaatc 120
aaaagcccca gcgcgcaaaa gctggaggcg ttgcaccgtt ttttggggct tgagtttccc 180
cggcagaaga aaacgatcgg tgtcgtattc ggtaagttct acccactgca taccggacat 240
atctacctta tccagcgcgc ctgtagccag gttgacgagc tgcatatcat tatgggtttt 300
gacgataccc gtgaccgcgc gttgttcgaa gacagtgcca tgtcgcagca gccgaccgtg 360
ccggatcgtc tgcgttggtt attgcaaact tttaaatatc agaaaaatat tcgcattcat 420
gctttcaacg aagagggcat ggagccgtat ccgcacggct gggatgtgtg gagcaacggc 480
atcaaaaagt ttatggctga aaaagggatc cagccggatc tgatctacac ctcggaagaa 540
gccgatgcgc cacagtatat ggaacatctg gggatcgaga cggtgctggt cgatccgaaa 600
cgtaccttta tgagtatcag cggtgcgcag atccgcgaaa acccgttccg ctactgggaa 660
tatattccta ccgaagtgaa gccgtttttt gtgcgtaccg tggcgatcct tggcggcgag 720
tcgagcggta aatccaccct ggtaaacaaa cttgccaata tcttcaacac caccagtgcg 780
tgggaatatg gccgcgatta tgtcttttca cacctcggcg gtgatgagat cgcattgcag 840
tattctgact acgataaaat cgcgctgggc cacgctcaat acattgattt tgcggtgaaa 900
tatgccaata aagtggcatt tatcgatacc gattttgtca ccactcaggc gttctgcaaa 960
aagtacgaag ggcgggaaca tccgttcgtg caggcgctga ttgatgaata ccgtttcgat 1020
ctggtgatcc tgctggagaa caacacgccg tgggtggcgg atggtttacg cagcctcggc 1080
agttcggtgg atcgcaaaga gttccagaac ttgctggtgg agatgctcga agagaacaat 1140
atcgaatttg tgcgggttga agaggaagat tacgacagtc gtttcctgcg ctgcgtggaa 1200
ctggtgcggg agatgatggg ggagcagaga taa 1233
<210> 5
<211> 750
<212> DNA
<213> 土拉热弗朗西斯杆菌（Francisellatularensis）
<400> 5
atgaagattg tgaaagattt cagcccgaaa gaatacagcc agaaactggt gaactggctg 60
agcgatagct gcatgaacta tccggcggaa ggctttgtga ttggcctgag cggcggcatt 120
gatagcgcgg tggcggcgtc attagcggtg aaaaccggtt taccgaccac cgcgctgatt 180
ctgccgagcg ataataacca gcatcaggat atgcaggatg cgctggatct gattgaaatg 240
ctgaacattg aacattacac catcagcatt cagccggcgt atgaagcgtt tctggcgagc 300
acccagcgct ttaccaacct gcagaacaac cgccagctgg tgattaaagg caacgcgcag 360
gcgcgtctgc gcatgatgta tctgtatgcg tatgcgcagc agtataaccg cattgtgatt 420
ggcaccgata acgcgtgcga atggtatatg ggctatttta ccaaatttgg cgatggcgcg 480
gcggatattc tgccgctggt taacttgaag aaaagccagg tgtttgagct gggcaaatat 540
ctggatgtgc cgaaaaacat tctggataaa gcgccgagcg cgggcctgtg gcaaggtcag 600
accgatgaag atgaaatggg cgtgacctat caggaaattg atgattttct ggatggcaaa 660
caggtgagcg cgaaagcgct ggaacgcatt aacttttggc ataaccgcag ccatcataaa 720
cgcaaactgg cgctgacccc gaacttttaa 750
<210> 6
<211> 954
<212> DNA
<213> 人工序列
<400> 6
atgagcaacg agtacggcga caagaacctg aaaattttca gcctgaacag caacccggag 60
ctggcgaagg aaatcgcgga taacgtgggt gttcagctgg gcaaatgcag cgtgacccgt 120
tttagcgacg gcgaagttca aatcaacatt gaggaaagca tccgtggttg cgattgctac 180
atcattcaga gcaccagcgc gccggtgaac gagcacatta tggaactgct gatcatggtt 240
gacgcgctga agcgtgcgag cgcgaaaacc atcaacattg tgatcccgta ctatggttat 300
gcgcgtcaag atcgtaaggc gcgtagccgt gagccgatca ccgcgaaact gtttgcgagc 360
ctgctggaaa ccgcgggcgc gacccgtgtg attgcgctgg acatccacgc gccgcagatt 420
caaggtttct ttgacattcc gatcgatcac ctgatgggtg ttccgatcct gggccactat 480
tttgagggca aggacctgaa agatattgtg atcgttagcc cggatcatgg tggcgttacc 540
cgtgcgcgta agctggcgga ccgtctgaaa gcgccgattg cgatcattga taagcgtcgt 600
ccgcgtccga acgaggtgga agttatgaac atcgtgggta acgttgaagg caaaaccgcg 660
attctgatcg acgacatcat tgataccgcg ggcaccatca ccctggcggc gaacgcgctg 720
gttgagaacg gtgcggcgga agtttacgcg tgctgcaccc acccggtgct gagcggtccg 780
gcggttgagc gtattaacaa cagcaagatc aaagaactgg tggttaccaa cagcattaag 840
ctgccggagg aaaagaaaat cgagcgtttc aaacaactga gcgtgggtcc gctgctggcg 900
gaggcgatca ttcgtgtgca cgaaaagcaa agcgttagct acctgttcag ctaa 954
<210> 7
<211> 930
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 7
atgcaaaacg caggcagcct cgttgttctt ggcagcatta atgctgacca cattcttaat 60
cttcaatctt ttcctactcc aggcgaaacc gtaaccggta accactatca ggttgcattt 120
ggcggcaaag gcgcgaatca ggctgtggct gctgggcgta gcggtgcgaa tatcgcgttt 180
attgcctgta cgggtgatga cagcattggt gagagcgttc gccagcagct cgccactgat 240
aacattgata ttactccggt cagcgtgatc aaaggcgaat caacaggtgt ggcgctgatt 300
tttgttaatg gcgaaggtga gaatgtcatc ggtattcatg ccggcgctaa tgctgccctt 360
tccccggcgc tggtggaagc gcaacgtgag cgtattgcca acgcgtcagc attattaatg 420
cagctggaat caccactcga aagtgtgatg gcagcggcga aaatcgccca tcaaaataag 480
actatcgttg cgcttaaccc ggctccggct cgcgaacttc ctgacgaact gctggcgctg 540
gtggacatta ttacgccaaa cgaaacggaa gcagaaaagc tcaccggtat tcgtgttgaa 600
aatgatgaag atgcagcgaa ggcggcgcag gtactgcatg aaaaaggtat ccgtactgta 660
ctgattactt taggaagtcg tggtgtatgg gctagcgtga atggtgaagg tcagcgcgtt 720
cctggattcc gggtgcaggc tgtcgatacc attgctgccg gagatacctt taacggtgcg 780
ttaatcacgg cattgctgga agaaaaacca ttgccagagg cgattcgttt tgcccatgct 840
gccgctgcga ttgccgtaac acgtaaaggc gcacaacctt ccgtaccgtg gcgtgaagag 900
atcgacgcat ttttagacag gcagaggtga 930
<210> 8
<211> 726
<212> DNA
<213> 人工序列
<400> 8
ctactcgaga acgccacagc agccgctggg caaccatcaa caagccagcc tgctgcgtct 60
ggatgttggc accggctacc agtactggta cggtctgccg aacttctaca ccatcacccg 120
ttacaaccac agcacccatt acgcaatggc ggtctggcag ttaggacaag ccgtggcgct 180
ggcgcgagta cagtagctat ttacaagagg ggggcgtgag cttccctctg gctaatcagc 240
ttttttctga atcctcctcg taaaattgca acgccaacac catcttcctg acgaaagtgc 300
tatcttgtcc ggcataaatt tttgactgac agaggttgtg atgactgaca gtgacaaaac 360
acttgatact gtatgagcat acagtataat tacttcaaca gaacatattg actatccggt 420
attacccggc atgacaggag taaaaatggc tatcgacgaa aacaaacaga aagcgttggc 480
ggcagcactg ggccagattg agaaacaatt tggtaaaggc tccatcatgc gcctgggtga 540
agaccgttcc atggatgtgg aaaccatctc taccggttcg ctttcactgg atatcgcgct 600
tggggcaggt ggtctgccga tgggccgtat cgtcgaaatc tacggaccgg aatcttccgg 660
taaaaccacg ctgacgttgc aggtgatcgc cgcagcgcag cgtgaaggac tgagactgat 720
gtccta 726
<210> 9
<211> 529
<212> DNA
<213> 人工序列
<400> 9
ctactcgagc gtcgcatcag gcattgagca ccgaatgccg gatacggcgt gaacgccttt 60
tccggcctac aacagcctta gtatccgcaa tttcgtagcg ggtcacccgt gcaggttttt 120
tcaaaccttg tttaatctcg acagcctctt tcagagaggt tttcaactca tcaaaaaact 180
tattttatac ctcgcctttg agccgtttca tcgcggttat ctctgctccc ccatcatctc 240
ccgcaccagc tccacgcagc gcaggaaacg gctgtcataa tcgtcctctt caacccgcac 300
gaattcgata ttgttctcga tggcagtttt caggtaatca aatgacgaca tatctccctc 360
cgtatctctc attataagtc gtcgaacacg ctaagcgcgt cggagagttt tttaacgcca 420
aaaatctgca tcccttccgg cgcttttttc ggtacgttag ccgccggaac aatcgcccgg 480
cgaaaaccgt gtttcgccgc ttctgaaatt cgttccgact gatgtcatc 529
<210> 10
<211> 1141
<212> DNA
<213> 人工序列
<400> 10
tcagccaaac gtctcttcag gccactgact agcgataact ttccccacaa cggaacaact 60
ctcattgcat gggatcattg ggtactgtgg gtttagtggt tgtaaaaaca cctgaccgct 120
atccctgatc agtttcttga aggtaaactc atcaccccca agtctggcta tgcagaaatc 180
acctggctca acagcctgct cagggtcaac gagaattaac attccgtcag gaaagcttgg 240
cttggagcct gttggtgcgg tcatggaatt accttcaacc tcaagccaga atgcagaatc 300
actggctttt ttggttgtgc ttacccatct ctccgcatca cctttggtaa aggttctaag 360
ctcaggtgag aacatccctg cctgaacatg agaaaaaaca gggtactcat actcacttct 420
aagtgacggc tgcatactaa ccgcttcata catctcgtag atttctctgg cgattgaagg 480
gctaaattct tcaacgctaa ctttgagaat ttttgcaagc aatgcggcgt tataagcatt 540
taatgcattg atgccattaa ataaagcacc aacgcctgac tgccccatcc ccatcttgtc 600
tgcgacagat tcctgggata agccaagttc atttttcttt ttttcataaa ttgctttaag 660
gcgacgtgcg tcctcaagct gctcttgtgt taatggtttc ttttttgtgc tcatacgtta 720
aatctatcac cgcaagggat aaatatctaa caccgtgcgt gttgactatt ttacctctgg 780
cggtgataat ggttgcatgt actaaggagg ttgtatggaa caacgcataa ccctgaaaga 840
ttatgcaatg cgctttgggc aaaccaagac agctaaaaga tctctcacct accaaacaat 900
gcccccctgc aaaaaataaa ttcatataaa aaacatacag ataaccatct gcggtgataa 960
attatctctg gcggtgttga cataaatacc actggcggtg atactgagca catcagcagg 1020
acgcactgac caccatgaag gtgacgctct taaaaattaa gccctgaaga agggcagcat 1080
tcaaagcaga aggctttggg gtgtgtgata cgaaacgaag cattggttaa aaattaagga 1140
g 1141
<210> 11
<211> 1488
<212> DNA
<213> 人工序列
<400> 11
atggacaacc tgctgaacta tagcagccgt gcgagcgcga tcccgagcct gctgtgcgat 60
ttctacaaga ccagccaccg tattatgtat ccggagtgca gccagatcat ttacagcacc 120
tttaccccgc gtagcaacga acaggcgccg tacctgaccc tagtggttag cttcggtttt 180
caagcgttca tcatcaagta cctgatccac tacttcaacg acaacttctt tagccgtgac 240
aagcacgatg tggttaccga gtatagcgcg ttcatcgaaa aaaccctgca actggaggat 300
accggtgaac acattgcgaa gctgcacgag ctgggctacc tgccgatccg tattaaagcg 360
atcccggaag gcaagaccgt ggcgatcaaa gtgccggtta tgaccattga gaacacccac 420
agcgacttct tttggctgac caactatctg gaaaccctga ttaacgttag cctgtggcaa 480
ccgatgacca gcgcgagcat cgcgttcgcg taccgtaccg cgctgattaa gtttgcgaac 540
gagacctgcg acaaccagga acacgtgccg ttccaaagcc acgattttag catgcgtggt 600
atgagcagcc tggagagcgc ggaaaccagc ggtgcgggtc acctgaccag cttcctgggc 660
accgacacca tcccggcgct gagctttgtt gaggcgtact atggtagcag cagcctgatc 720
ggcaccagca ttccggcgag cgagcacagc gtgatgagca gccacggtgt tgatgaactg 780
agcaccttcc gttatctgat ggcgaagttt ccgcacaaca tgctgagcat cgtgagcgac 840
accaccgatt tctggcacaa cattaccgtt aacctgccgc tgctgaaaca ggagatcatt 900
gcgcgtccgg aaaacgcgcg tctggtgatc cgtccggaca gcggtaactt ctttgcgatc 960
atttgcggcg acccgaccgc ggataccgag cacgagcgta agggtctgat cgaatgcctg 1020
tgggacattt tcggtggcac cgtgaaccag aagggttata aagttattaa cccgcacatc 1080
ggcgcgattt acggtgatgg cgttacctat gagaagatgt ttaaaatcct ggaaggtctg 1140
caagcgaaag gcttcgcgag cagcaacatt gtgtttggtg ttggcgcgca gacctaccaa 1200
cgtaacaccc gtgacaccct gggtttcgcg ctgaaggcga ccagcatcac cattaacggc 1260
gaggaaaaag cgatcttcaa gaacccgaaa accgacgatg gttttaagaa aagccagaag 1320
ggccgtgtga aagttctgag ccgtgacacc tacgtgggtg gtctgaccag cgcggacgat 1380
ttcagcgacg atctgctgga gctgctgttt gaagatggca aactgctgcg tcagaccgac 1440
tttgatgaga tccgtcaaaa cctgctggtt agccgtacca ccctgtaa 1488
<210> 12
<211> 495
<212> PRT
<213> 人工序列
<400> 12
Met Asp Asn Leu Leu Asn Tyr Ser Ser Arg Ala Ser Ala Ile Pro Ser
1 5 10 15
Leu Leu Cys Asp Phe Tyr Lys Thr Ser His Arg Ile Met Tyr Pro Glu
20 25 30
Cys Ser Gln Ile Ile Tyr Ser Thr Phe Thr Pro Arg Ser Asn Glu Gln
35 40 45
Ala Pro Tyr Leu Thr Leu Val Val Ser Phe Gly Phe Gln Ala Phe Ile
50 55 60
Ile Lys Tyr Leu Ile His Tyr Phe Asn Asp Asn Phe Phe Ser Arg Asp
65 70 75 80
Lys His Asp Val Val Thr Glu Tyr Ser Ala Phe Ile Glu Lys Thr Leu
85 90 95
Gln Leu Glu Asp Thr Gly Glu His Ile Ala Lys Leu His Glu Leu Gly
100 105 110
Tyr Leu Pro Ile Arg Ile Lys Ala Ile Pro Glu Gly Lys Thr Val Ala
115 120 125
Ile Lys Val Pro Val Met Thr Ile Glu Asn Thr His Ser Asp Phe Phe
130 135 140
Trp Leu Thr Asn Tyr Leu Glu Thr Leu Ile Asn Val Ser Leu Trp Gln
145 150 155 160
Pro Met Thr Ser Ala Ser Ile Ala Phe Ala Tyr Arg Thr Ala Leu Ile
165 170 175
Lys Phe Ala Asn Glu Thr Cys Asp Asn Gln Glu His Val Pro Phe Gln
180 185 190
Ser His Asp Phe Ser Met Arg Gly Met Ser Ser Leu Glu Ser Ala Glu
195 200 205
Thr Ser Gly Ala Gly His Leu Thr Ser Phe Leu Gly Thr Asp Thr Ile
210 215 220
Pro Ala Leu Ser Phe Val Glu Ala Tyr Tyr Gly Ser Ser Ser Leu Ile
225 230 235 240
Gly Thr Ser Ile Pro Ala Ser Glu His Ser Val Met Ser Ser His Gly
245 250 255
Val Asp Glu Leu Ser Thr Phe Arg Tyr Leu Met Ala Lys Phe Pro His
260 265 270
Asn Met Leu Ser Ile Val Ser Asp Thr Thr Asp Phe Trp His Asn Ile
275 280 285
Thr Val Asn Leu Pro Leu Leu Lys Gln Glu Ile Ile Ala Arg Pro Glu
290 295 300
Asn Ala Arg Leu Val Ile Arg Pro Asp Ser Gly Asn Phe Phe Ala Ile
305 310 315 320
Ile Cys Gly Asp Pro Thr Ala Asp Thr Glu His Glu Arg Lys Gly Leu
325 330 335
Ile Glu Cys Leu Trp Asp Ile Phe Gly Gly Thr Val Asn Gln Lys Gly
340 345 350
Tyr Lys Val Ile Asn Pro His Ile Gly Ala Ile Tyr Gly Asp Gly Val
355 360 365
Thr Tyr Glu Lys Met Phe Lys Ile Leu Glu Gly Leu Gln Ala Lys Gly
370 375 380
Phe Ala Ser Ser Asn Ile Val Phe Gly Val Gly Ala Gln Thr Tyr Gln
385 390 395 400
Arg Asn Thr Arg Asp Thr Leu Gly Phe Ala Leu Lys Ala Thr Ser Ile
405 410 415
Thr Ile Asn Gly Glu Glu Lys Ala Ile Phe Lys Asn Pro Lys Thr Asp
420 425 430
Asp Gly Phe Lys Lys Ser Gln Lys Gly Arg Val Lys Val Leu Ser Arg
435 440 445
Asp Thr Tyr Val Gly Gly Leu Thr Ser Ala Asp Asp Phe Ser Asp Asp
450 455 460
Leu Leu Glu Leu Leu Phe Glu Asp Gly Lys Leu Leu Arg Gln Thr Asp
465 470 475 480
Phe Asp Glu Ile Arg Gln Asn Leu Leu Val Ser Arg Thr Thr Leu
485 490 495
Claims (10)
1.一种烟酸磷酸核糖转移酶突变体，其特征在于，所述突变体氨基酸序列如序列表SEQID NO:12所示。
2.权利要求1所述烟酸磷酸核糖转移酶突变体的编码基因。
3.一株生产NMN的大肠杆菌基因工程菌，其特征在于，所述工程菌是在大肠杆菌宿主中，通过缺失对烟酰胺核苷酸酰胺酶编码基因pncC、烟酰胺单核苷酸腺苷转移酶基因nadD、nadR的表达的同时，过表达NAD+合酶编码基因nadE；表达烟酸磷酸核糖转移酶突变体编码基因NadVmu、核糖磷酸二磷酸激酶编码基因Prs以及核糖激酶编码基因RbsK获得的。
4.如权利要求3一株生产NMN的大肠杆菌基因工程菌，其特征在于，pncC、nadR基因缺失表达的方式为基因敲除；nadD基因缺失表达的方式为通过CIts蛋白及PR/PL启动子温敏控制表达。
5.如权利要求3一株生产NMN的大肠杆菌基因工程菌，其特征在于，pncC基因的核苷酸序列如序列表SEQ ID NO：2所示；nadD基因的核苷酸序列如序列表SEQ ID NO：3所示；nadR基因的核苷酸序列如序列表SEQ ID NO：4所示；nadE基因的核苷酸序列如序列表SEQ IDNO：5所示；Prs基因的核苷酸序列如序列表SEQ ID NO：6所示；RbsK基因的核苷酸序列如序列表SEQ ID NO：7所示；烟酸磷酸核糖转移酶突变体编码基因NadVmu的核苷酸序列如序列表SEQ ID NO：11所示。
6.如权利要求3一株生产NMN的大肠杆菌基因工程菌，其特征在于，所述大肠杆菌宿主选自JM109、BL21(DE3)、Top 10、DH5α、Rosetta、或Rosetta-gami pLysS。
7.如权利要求3一株生产NMN的大肠杆菌基因工程菌，其特征在于，是以大肠杆菌JM109为宿主，通过敲除pncC、nadR基因，CIts蛋白及PR/PL启动子温敏控制nadD基因表达，采用pET-28a载体对nadE基因进行过表达，采用pGEX4T3载体对NadV、Prs、RbsK基因进行过表达获得。
8.权利要求3-8任一所述大肠杆菌基因工程菌在生产NMN中的应用。
9.如权利要求8所述的应用，其特征在于，采用上述工程菌发酵生产NMN的方法如下：
反应体系包括：烟酰胺5-20mmol/L、核糖10-20mmol/L、ATP 5-10mmol/L、NAD+5-10mmol/L、Na2HPO4/NaH2PO4 50mmol/L、醋酸钠10mmol/L、氯化钙1mmol/L，其余为水pH5-8.5；加入生产菌使菌体浓度达到5-100g/L，在15-22℃，50-200rpm，避光下反应10-25h。
10.如权利要求9所述的应用，其特征在于，菌体培养方法如下：
将工程菌种子液接种于培养基中，37℃、200rpm培养至OD600为0.7，自来水冷却10min，加1mM IPTG后，22℃、200rpm条件下诱导表达24h；离心收集菌体。
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