CN118166033A - 产生烟酰胺单核苷酸的方法及由此获得的细胞及其应用 - Google Patents
产生烟酰胺单核苷酸的方法及由此获得的细胞及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,更具体而言,本发明涉及一种产生烟酰胺单核苷酸(NMN)的方法、产生NMN的细胞以及其应用。通过本发明提供的产生NMN的方法,可以在不额外添加相关原料的情况下,简单、高效且低成本的大规模生产NMN。
Description
技术领域
本发明属于生物技术领域,更具体而言,本发明涉及一种产生烟酰胺单核苷酸(NMN)的方法。
背景技术
烟酰胺单核苷酸(NMN)是哺乳动物体内辅酶I-NAD+的重要前体,研究表明,烟酰胺单核苷酸在延缓衰老、治疗帕金森等老年病、调节胰岛素分泌、调控mRNA的表达等方面具有显著效果。近年的研究进一步发现,人为补充NMN可以修复脑损伤、改善胰岛功能、保护心脏免于缺血再灌注损伤、修复脑线粒体呼吸缺陷,对老年退行性疾病、视网膜退行性疾病等均具有一定治疗作用,因此NMN在药物、保健品领域具有广阔的应用前景。
NMN的现有化学合成主要以烟酰胺、三苯甲酰基-β-D-核糖、四乙酰核糖等为原料,通过糖基化、磷酸化、氨解等关键步骤合成。具体可以分为溴代乙酰核糖法、TMSOTF催化缩合法、AMP酸水解催化法、缩酮化保护合成法和烟酸乙酯为原料的合成方法。但化学合成法中往往存在成本较高且产生手性化合物的问题。此外,化学合成法的反应步骤较多、NMN得率低、产品纯度低,而且需要用到大量的有机溶剂,对环境破坏严重。因此,化学法合成NMN在实际应用中存在很大局限。
相较化学合成法,生物合成法具有价格低廉、绿色环保无公害、适宜大规模工业化生产等优点。NMN的生物合成技术路径包括酶法合成及发酵合成。NMN酶法合成主要路线主要是模仿生物体内NAD+和NMN的反应路径。现有技术中在大肠杆菌和酵母中对NMN的发酵合成进行了探索研究。Marinescu等人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-1’-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产量仅为15.4mg/L。2020年,Black等人研究设计大肠杆菌全细胞的从头生物合成途径,虽然不需要额外辅助因子添加,但NMN产量也仅有1.5mmol/L。2021年,Shoji等构建了可以摄入葡萄糖和烟酰胺的基因工程重组大肠杆菌,含有不同物种来源的烟酸转运蛋白、烟酰胺核苷转运蛋白以及NAMPT酶,最终可使NMN产量达到6.79g/L。
烟碱又名尼古丁(Nicotine),为烟草中最主要的生物碱占总生物碱含量的90%左右。烟碱的骨架结构由吡啶环(Pyridine)和吡咯烷(Pyrrolidine)组合而成。吡啶环的最初前体为烟酸(Nicotinic acid),由烟酰胺腺嘌呤二核苷酸(nicotinamide adeninedinucleotide,NAD)合成通路生成,而吡咯烷(Pyrrolidine)的最初前体为N-甲基吡咯啉鎓(N-methylpyrrolinium)阳离子,由多胺合成通路生成。然而,自烟酸和N-甲基吡咯啉鎓阳离子后的详细反应步骤目前仍不清晰,已知由异黄酮还原酶和小檗碱桥接酶两种酶参与催化反应最终生成烟碱(图1)。而烟酸和目标产物NMN均参与NAD代谢通路。
目前NMN的主要生物合成方法为以烟酰胺、ATP和核糖或是烟酰胺和PRPP作为底物进行体外催化(图2)。此类方法需要首先在重组大肠杆菌或其他重组底盘细胞中表达催化酶并进行纯化,步骤繁琐并且所用的底物价格较高且来源受限。若采用Black等人的方法直接利用重组大肠杆菌进行从头生物合成,虽然不需要添加额外物料于培养液中,但NMN产量很低。Shoji等人构建的可以摄入烟酰胺的重组大肠杆菌虽然可以提高最终NMN产量,但依然需要在培养液中额外添加烟酰胺,从而增加了生产成本。
因此,本领域亟需一种产生NMN的方法,不需要在大肠杆菌或其他模式微生物中表达及纯化NMN相关合成酶,也不需要额外添加昂贵底物,从而以简便且高效同时又能降低生产成本的方式大规模地生产NMN。
发明内容
如上所述,现有的产生NMN的方法仍存在工艺繁琐、成本高昂等多种不足。因此,本领域亟需一种产生NMN的方法,可以简便、高效、低成本的大规模生产NMN。
有鉴于此,在第一方面,本发明提供了一种产生烟酰胺单核苷酸(NMN)的方法,所述方法通过阻断积累烟碱的植物或细胞内的烟碱合成通路,使得所述烟碱合成通路中的烟酸合成相关代谢产物回流至烟酰胺腺嘌呤二核苷酸(NAD)代谢通路从而积累产生NMN。
在一些实施方案中,所述积累烟碱的植物包括茄科烟草属的烟草;所述积累烟碱的细胞包括烟草细胞系。
在一些实施方案中,可以通过基因编辑、育种、T-DNA插入和/或使用突变剂来阻断烟碱合成通路。
在一些实施方案中,可以通过敲除烟碱合成通路中一个或多个酶的编码基因来阻断烟碱合成通路。
在一个优选的实施方案中,可以通过敲除烟碱合成通路中A622酶和A622-Like酶的编码基因来阻断烟碱合成通路。在另一个优选的实施方案中,可以进一步敲除烟碱合成通路中的小檗碱桥接酶(BBL)的编码基因。
在一些实施方案中,敲除烟碱合成通路中所述酶的编码基因具体包括以下步骤:
1)针对所述酶的编码基因设计靶序列,接着合成与靶序列对应的正向寡核苷酸序列和反向寡核苷酸序列,再经退火处理获得双链DNA片段作为插入片段,将所述插入片段克隆至载体中,获得烟碱合成通路中所述酶基因编辑载体;
2)用所述酶基因编辑载体转化所述积累烟碱的植物或细胞。
在一些实施方案中,所述编码基因为SEQ ID NO:1和SEQ IDNO:2所示的A622基因和A622L基因,针对所述编码基因的靶序列为共同靶向A622基因和A622L基因的SEQ ID NO:3所示的序列。
在第二方面,本发明提供了一种产生NMN的细胞,其中,所述细胞内的烟碱合成通路被阻断,使得所述烟碱合成通路中的烟酸合成相关代谢产物回流至NAD代谢通路从而积累产生NMN。
在一些实施方案中,所述细胞包括植物细胞。
本发明的有益效果如下:
1)简单、高效且生产成本低,无需要额外添加相关原料;
2)可以利用植物或植物细胞作为底盘进行大规模生产;
3)可以利用已有的植物栽培的成熟体系,无需对生产体系进行生产优化。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的实施方案。
图1为烟草中烟碱合成通路的示意图。
图2为现有技术中NMN的主要合成方法的示意图。
图3为本发明中通过阻断烟碱合成通路使得代谢产物回流从而积累产生NMN的示意图。
图4为中间载体CRR-BBLa(图4A)和CRR-A/L(图4B)以及最终载体AtU6-A3N3-Cas9(图4C)图谱的示意图。
图5为烟草的转化、分化和生长过程的照片,其中,图5A为烟草无菌苗的叶片,图5B为经转化的烟草出现的丛生芽,图5C和图5D为于生根培养基中培养2周后的烟草苗。
图6为经基因编辑烟草的Sanger测序结果。
图7为单基因编辑的烟草植株(HD-353-A3-13)与双基因编辑烟草植株(HD-355-A3-5)中烟碱及NMN含量的HPLC检测结果。
具体实施方式
下面将结合本发明的实施方案和附图,对本发明进行清楚、完整的描述。显然,所描述的实施方案仅仅是本发明的一部分实施方案,而不是全部的实施方案。基于本发明中的实施方案,本领域普通技术人员可以获得的所有其他实施方案,都属于本发明保护的范围。
如上所述现有的NMN的生物合成法都需要在合成过程中添加相关底物或在微生物中表达合成酶,使得合成步骤和成本增加,最终获得的产量也有限。因此,本发明的目的在于提供一种新的产生NMN的方法。由于一些植物或细胞中同时存在烟碱合成通路和NAD代谢通路,而烟酸既是合成烟碱过程中的吡啶环前体,又参与产生目标产物NMN的NAD代谢通路。发明人通过实验研究发现,可以利用植物或细胞中原有的烟碱合成通路,通过阻断烟碱合成相关通路上的代谢产物回流至NAD代谢通路从而累计产生NMN,从而完成了本发明。
因此,在第一方面,本发明提供了一种产生烟酰胺单核苷酸(NMN)的方法,所述方法通过阻断积累烟碱的植物或细胞内的烟碱合成通路,使得所述烟碱合成通路中的相关代谢产物回流至烟酰胺腺嘌呤二核苷酸(NAD)代谢通路从而积累产生NMN。
虽然目前仍不清楚烟碱合成通路中烟酸和N-甲基吡咯啉鎓阳离子之后的详细反应过程,但可以看出烟酸等中间代谢产物同时参与了烟碱合成通路和NAD代谢通路。因此同时具备两者的植物或细胞即可适用于本发明的方法。由于烟碱为烟草中最主要的生物碱,占其总生物碱含量的90%左右,因此可以选择烟草作为本发明的方法中的植物底盘。因此,在一些实施方案中,所述积累烟碱的植物包括茄科烟草属的烟草。此外,多个品种的烟草都可以用于本发明的方法,在一些实施方案中,所述积累烟碱的植物为烟草K326或红花大金元。除了植物之外,可以产生烟碱和NMN的细胞也可以用作本发明方法中的生物反应器。在一个实施方案中,所述积累烟碱的细胞可以为烟草细胞。在一个优选的实施方案中,所述积累烟碱的细胞可以为烟草BY-2细胞。
可以理解的是,在本发明的方法中,可以通过基因编辑、育种例如杂交育种、T-DNA插入和/或使用突变剂来阻断烟碱合成通路,但不限于于此。任何其他可以阻断积累烟碱的植物或细胞内烟碱合成通路从而使得所述烟碱合成通路中的相关代谢产物回流至NAD代谢通路以积累产生NMN的方法和技术均在本发明考虑范围内。
在一个实施方案中,通过敲除烟碱合成通路中一个或多个酶的编码基因来阻断烟碱合成通路。本发明中所述的“烟碱合成通路中的酶”是指在烟碱合成通路中参与催化反应以及烟碱最终生成的关键酶,并且通过敲除所述关键酶可以阻断烟碱合成通路,从而导致烟酸合成相关代谢产物回流至烟酰胺腺嘌呤二核苷酸(NAD)代谢通路从而积累产生NMN。从背景技术可知,异黄酮还原酶和小檗碱桥接酶(BBL)参与催化烟酸和N-甲基吡咯啉鎓阳离子的后续反应以生成烟碱(参见图1)。而在已知的烟草品种中A622酶(SEQ ID NO:4)和A622-Like酶(SEQ ID NO:5)都同时参与烟碱的合成。
因此,在一个优选的实施方案中,通过敲除烟碱合成通路中A622酶和A622-Like酶的编码基因来阻断烟碱合成通路。相对于异黄酮还原酶,小檗碱桥接酶(SEQ ID NO:6)为处于合成通路更下游的合成酶,因此在另一个优选的实施方案中,还可以进一步敲除小檗碱桥接酶的编码基因(即,同时敲除A622酶、A622-Like酶和小檗碱桥接酶的编码基因)。
为了达到阻断烟碱合成通路的目的,本发明可以采用以下方法进行烟碱合成通路中所述酶的编码基因的基因敲除:
1)针对所述酶的编码基因设计靶序列,接着合成与靶序列对应的正向寡核苷酸序列和反向寡核苷酸序列,再经退火处理获得双链DNA片段作为插入片段,将所述插入片段克隆至载体中,获得烟碱合成通路中所述酶基因编辑载体;
2)用所述酶基因编辑载体转化所述积累烟碱的植物或细胞。
当选择A622酶和A622-Like酶作为目标酶时,由于其编码基因A622(SEQ ID NO:1)和A622L(SEQ ID NO:2)基因是两个DNA序列相似性达到84.8%的同源基因,在筛选sgRNA靶序列时,选择可以用于共同靶向两个基因(A622和A622L基因)的同一靶序列,所述靶序列可以为如SEQ ID NO:3所示的序列(5’-GTGGTGATTTCCACTGTCGG-3’)。
在第二方面,本发明提供了一种产生NMN的细胞,其中,所述细胞内的烟碱合成通路被阻断,使得所述烟碱合成通路中的烟酸合成相关代谢产物回流至NAD代谢通路从而积累产生NMN。
在本发明的方法中,可以采用具有烟碱合成通路同时产生NMN的细胞作为生物合成的反应器来制备NMN。在一些实施方案中,所述细胞可以为植物细胞,例如烟草细胞系。在一个优选实施方案中,所述细胞可以为BY-2细胞。需要注意的是,在本发明的方法中,植物细胞其本身可以作为产生NMN生物合成反应器,而不需要转化为完整植株,也不作为发育成完整植株的繁殖材料。
本领域技术人员可以理解,上文中与本发明第一方面中通过阻断烟碱合成通路从而积累产生NMN相关的描述均适用于本发明的第二方面,因此在此不再赘述。
在第三方面,本发明提供了本发明第二方面所述的细胞在制备NMN中的应用。
下面结合实施例对本发明进行更为具体和详细的描述。下述实施例中的试验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规化试剂商店购买所得。应注意,上文的发明内容部分以及下文的详细描述仅为具体阐释本发明之目的,无意于以任何方式对本发明进行限制。
实施例
实施例1.通过阻断普通烟草(Nicotiana tabacum)中的烟碱合成通路从而合成并积累NMN。
一、BBLa基因sgRNA以及A622&A622L基因sgRNA基因编辑载体构建
1.BBLa基因的sgRNA靶序列选择
针对SEQ ID NO:7所示的BBLa基因,遵循下列原则对sgRNA靶序列进行选择:
(1)在BBLa基因外显子编码区寻找符合5′-N(20)NGG-3′规则的靶序列,其中N(20)表示20个连续的碱基,其中每个N表示A或T或C或G,符合上述规则的靶序列位于正义链或反义链;
(2)利用在线序列分析工具(http://skl.scau.edu.cn/targetdesign/,targetDesign)分析上述靶序列在烟草基因组中的同源情况,舍弃存在显着同源的靶序列,根据评分进一步挑选在BBLa基因上唯一的靶序列(SEQ ID NO:8)进行合成与载体构建。
2.A622&A622L基因的sgRNA靶序列选择
(1)A622和A622L基因sgRNA靶序列的选择标准与上述BBLa基因靶序列的选择标准相同。
(2)A622和A622L基因是两个同源基因,DNA序列相似性达到84.8%,挑选能够共同靶向两个基因(A622&A622L基因)的同一sgRNA靶序列(SEQ ID NO:3),所述sgRNA靶序列符合5′-N(20)NGG-3′的规则,N(20)中20个连续的碱基在两个基因中完全相同。
3.构建BBLa基因以及A622&A622L基因的sgRNA表达载体
(1)靶序列寡核苷酸合成
发明人研究了BBLa基因的靶序列和A622&A622L双基因共同靶序列的多敲除效果,根据上述设计合成的BBLa基因的靶序列对应的正向和反向寡核苷酸序列如下:
BBLa-N3-sgRNA-F:5’-GATTGCTTGGTAGGATAATGAAGGT-3’
(SEQ ID NO:9)
BBLa-N3-gRNA-R:5’-AAACACCTTCATTATCCTACCAAGC-3’
(SEQ ID NO:10)
A622&A622L基因靶序列对应的正向寡核苷酸序列和反向寡核苷酸序列如下:
A/L-A3-sgRNA-F:5’-GATTGGTGGTGATTTCCACTGTCGG-3’
(SEQ ID NO:11)
A/L-A3-gRNA-R:5’-AAACCCGACAGTGGAAATCACCACC-3’
(SEQ ID NO:12)
(2)构建AtU6-A3N3-Cas9烟草编辑载体
将上述BBLa及A622/A622L基因靶序列对应的正向和反向寡核苷酸进行退火、复性,形成具有粘性末端的双链DNA片段,分别与AtU6-sgRNA-CRR载体BsaI酶切片段连接,构建CRR-BBLa和CRR-A/L中间载体(图4A,4B)。利用KpnI、XbaI限制性内切酶酶切CRR-BBLa中间载体,并利用EcoRI、XbaI限制性内切酶酶切CRR-A/L中间载体,然后分别回收小片段一起与KpnI、EcoRI限制性内切酶酶切的终载体pCambia1300 35S::Cas9大片段连接,构建获得AtU6-A3N3-Cas9烟草编辑载体(图4C)。
二、基因编辑烟草的获得
1.烟草无菌苗的获得
栽培烟草K326和红花大金元种子消毒后接种于1/2MS固体培养基上萌发,每个培养皿约10粒,置于28℃的光照培养室(16h光/8h暗)培养一周;长出幼苗后,转入新鲜的1/2MS固体培养基,每瓶种1株烟草小苗,于28℃光照培养室(16h光/8h暗)继续培养约5周,获得适龄烟草无菌苗。(1/2MS固体培养基:1/2MS,0.8%琼脂,pH 5.8)2.农杆菌介导的叶盘法烟草转化
(1)农杆菌菌液制备
将构建好的基因编辑载体通过化学转化法转化农杆菌感受态LBA4404,挑取含潮霉素抗性目的质粒的农杆菌菌株LBA4404单菌落,接种到4mL LB液体培养基(含卡那霉素(Kan)50mg/L,利福平(Rif)30mg/L),于28℃,250rpm振荡过夜,之后取上述的小摇菌液400μL加入到40mL LB液体培养基(含Kan 50mg/L,Rif 30mg/L)于三角瓶中在28℃,250rpm摇床震荡的条件下培养约14h,在OD 600=0.5左右时作为侵染菌液使用。(LB液体培养基:1%胰蛋白胨,0.5%酵母提取物,1%氯化钠,1.5%琼脂)
(2)农杆菌侵染烟草
从培养5周的烟草无菌苗上剪下较大的叶片(绿色无花纹叶片),放在含有湿润灭菌滤纸的玻璃培养皿中;用刀去掉叶片的叶缘和主叶脉,并切成直径5至10mm的方形小叶片,并转移到另一个含有湿润灭菌滤纸的玻璃培养皿中(图5A)。
收集待用菌液,于3500rpm离心,倒掉上层液体。用含100μm/L的乙酰丁香酮的液体MS重悬菌体;将重悬好的侵染菌液倒入装有切好叶片的培养皿中,充分浸泡10min;吸干烟草叶片上的多余菌液,将烟草小叶片接种到共培养基上,叶面朝上,每个培养皿中约10块叶片;倒置培养皿,于28℃暗培养3天。(共培养基:MS,100μm/L乙酰丁香酮,0.8%琼脂,pH=5.8)
3.转化烟草的筛选分化与生根
将经共培养的叶盘转移到筛选分化培养基上,每个培养皿10块,叶面朝上,于28℃光照培养2周;每2周继代一次,大约4周出现丛生芽(图5B)。(分化培养基:MS,0.5mg/L NAA,2.0mg/L 6-BA,200mg/L羧苄青霉素,20mg/L潮霉素,0.8%琼脂,pH 5.8)
当再生芽长至约1至2cm时,切下芽接种到生根培养基,每瓶1株烟草苗;于28℃光照培养室培养2周;除去幼苗基部的小叶子,转移到新鲜的生根培养基,每瓶1株烟草苗,壮苗培养2周(图5C和图5D)。(生根培养基:MS,200mg/L羧苄青霉素,20mg/L潮霉素,0.8%琼脂,pH5.8)
三、基因编辑烟草材料的基因型鉴定
1.烟草T0代叶片DNA抽提(CTAB法)
挑取目的植株的组织;将其放入2mL(含1个小钢珠)的离心管中,冻干;用研磨仪磨碎冻干样品,加入预热的CTAB 0.5mL;于65℃水浴30min,每5min摇匀一次;随后于12000rpm离心5min;吸取350μL上清,加入等体积氯仿/异戊醇(24:1),上下轻柔颠倒数次,于12000rpm离心5min;吸取300μL上清于新EP管中,加等体积的异丙醇,混匀,室温放置10min,于12000rpm离心10min;去上清,加适量75%酒精浸洗两次,干燥;加50μLddH2O溶解,置于-20℃保存。
2.基因特异性鉴定引物如下:
BBLa-F:5’-CTACTGGAGCTGTTACAAA-3’(SEQ ID NO:13)
BBLa-R:5’-ATTGCCCAAAACACGTCTTC-3’(SEQ ID NO:14)
A622-F:5’-ATTTATGATGGACGGTTACCTG-3’(SEQ ID NO:15)
A622-R:5’-CCTTCACATATATTGCTGAAATAG-3’(SEQ ID NO:16)
A622L-F:5’-CTAACTTGCTGATAACTAGGGAG-3’(SEQ ID NO:17)
A622L-R:5’-TCACATATATTGCTAATTACATTG-3’(SEQ ID NO:18)3.基因编辑烟草材料的T7EI酶切鉴定:
PCR扩增后,将经基因编辑个体的PCR产物与野生型的PCR产物混合,退火(95℃,5min;95℃-85℃,以2℃/sec缓慢降温;85℃-25℃,以0.1℃/sec缓慢降温;4℃,保持温度),进行T7EI孵育酶切,然后通过琼脂糖凝胶电泳检测。
4.基因编辑烟草材料的Sanger测序结果与统计
将T7内切酶切鉴定条带正确的编辑样品的编辑样品DNA的A622、A622-like、以及BBLa特异PCR扩增产物进行Sanger测序,验证靶向序列旁侧的核苷酸插入及缺失情况,结果如图6所示,获得了HD-35S-A3-32-13单基因突变体与HD-35S-A3-32-5双基因突变体。HD-35S-A3-32-5双基因突变体中A622的基因型为+A纯合,读码框改变导致蛋白翻译提前终止(图6A,6C,6E);同时A622-Like的基因型为+T纯合,读码框改变导致蛋白翻译提前终止(图6B,6D,6E)。另外也获得了K326-A3N3-21(K326-A3N3-21-5,K326-A3N3-21-7,K326-A3N3-21-17)三基因突变体,其中A622的基因型为+A,A622-Like的基因型为+T纯合或双等位突变,BBLa的基因型为+A杂合体(图6F)。
5.基因编辑烟草纯合材料的NMN和烟碱含量测定
取生长1月苗龄的野生型与A622和A622-like双基因编辑导致蛋白翻译提前终止的HD-35S-A3-32-5植株以及A622单基因编辑的HD-35S-A3-32-13单基因突变植株的叶片,将0.1g剪碎的新鲜叶片置于2mL EP管中,加入2颗小钢珠以及1mL于-20℃预冷的70%甲醇水溶液,用组织研磨机(220rpm,4min)进行破碎,于4℃超声10min,随后于-20℃静置1h,最后于17000rpm离心15min,取上清液过膜,上机进行烟碱和NMN含量的HPLC检测(色谱柱:Agilent ZORBAX SB-C18,5μm,250x 4.6mm;流动相:乙腈+5mM三氟乙酸=5%+95%;流速:0.5mL/min;进样量:30μL;柱温:20℃)。
HPLC检测结果显示,与野生型植株(对照)相比,单基因突变植株(HD-35S-A3-32-13)的NMN含量没有明显变化;而A622和A622-like双基因编辑植株(HD-35S-A3-32-5)的烟碱含量降低至约10%,NMN含量升高至4.5倍,达到0.3955mg/g的鲜重(图7A和7B),与此类似,A622,A622-like和BBLa三基因编辑植株中NMN的含量也比野生型植株(对照)中提高了5-7倍(图7C)。上述实验结果表明通过敲除烟碱合成通路中关键酶的编码基因(例如:A622和A622-like,或者A622、A622-like和BBLa)都可以阻断烟碱合成通路,并使吡啶环前体逆转至NAD代谢通路,促进了NMN的合成,为开发生产NMN的生物反应器提供了新的合成技术方案。
表1.相关序列信息
Claims (10)
1.一种产生烟酰胺单核苷酸(NMN)的方法,所述方法通过阻断积累烟碱的植物或细胞内的烟碱合成通路,使得所述烟碱合成通路中的烟酸合成相关代谢产物回流至烟酰胺腺嘌呤二核苷酸(NAD)代谢通路从而积累产生NMN。
2.根据权利要求1所述的方法,其中,所述积累烟碱的植物包括茄科烟草属的烟草,例如K326或红花大金元等;所述积累烟碱的细胞包括烟草细胞系,如BY-2细胞。
3.根据权利要求1或2所述的方法,其中,通过基因编辑、育种例如杂交育种、T-DNA插入和/或使用突变剂来阻断烟碱合成通路。
4.根据权利要求1-3所述的方法,其中,通过敲除烟碱合成通路中一个或多个酶例如异黄酮还原酶、小檗碱桥接酶(BBL)的编码基因来阻断烟碱合成通路。
5.根据权利要求4所述的方法,其中,通过敲除烟碱合成通路中A622酶和A622-Like酶的编码基因来阻断烟碱合成通路。
6.根据权利要求5所述的方法,其中,进一步敲除烟碱合成通路中的小檗碱桥接酶的编码基因。
7.根据权利要求4-6所述的方法,其中,敲除烟碱合成通路中所述酶的编码基因具体包括以下步骤:
1)针对所述酶的编码基因设计靶序列,接着合成与靶序列对应的正向寡核苷酸序列和反向寡核苷酸序列,再经退火处理获得双链DNA片段作为插入片段,将所述插入片段克隆至载体中,获得烟碱合成通路中所述酶基因编辑载体;
2)用所述酶基因编辑载体转化所述积累烟碱的植物或细胞。
8.根据权利要求7所述的方法,其中,所述编码基因为SEQ IDNO:1和SEQ ID NO:2所示的A622基因和A622L基因,针对所述编码基因的靶序列为共同靶向A622基因和A622L基因的SEQ ID NO:3所示的序列。
9.一种产生NMN的细胞,其中,所述细胞内的烟碱合成通路被阻断,使得所述烟碱合成通路中的烟酸合成相关代谢产物回流至NAD代谢通路从而积累产生NMN。
10.根据权利要求9所述的细胞,其中,所述细胞包括植物细胞,例如烟草细胞系,优选为BY-2细胞。
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