CN117126756A - 一种用于制备烟酰胺磷酸核糖基转移酶的重组菌株的构建方法及其应用 - Google Patents
一种用于制备烟酰胺磷酸核糖基转移酶的重组菌株的构建方法及其应用 Download PDFInfo
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Abstract
本发明属于基因工程领域,涉及一种用于制备烟酰胺磷酸核糖基转移酶NAMPT的重组菌株的构建方法及其应用。本发明将烟酰胺磷酸核糖基转移酶NAMPT装载至表达载体构建质粒酵母过表达***,导入到毕赤酵母菌株中,获得含外源NAMPT基因的重组毕赤酵母菌株。进一步对NAMPT酶进行理性改造,提高NAMPT酶活,经微生物发酵生产,可在毕赤酵母发酵液中生产胞外分泌蛋白酶NAMPT,在15分钟内可催化烟酰胺生成NMN的量为0.965μM。本研究提高了NAMPT的催化效率,降低了终产物NMN的生产成本。同时,所述NAMPT具有提高D‑半乳糖诱导的衰老心肌细胞活性的生物功能。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种用于制备烟酰胺磷酸核糖基转移酶NAMPT的重组菌株的构建方法及其应用。
背景技术
烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide, NAD+)是细胞氧化还原反应的辅酶和能量代谢的中心,直接或间接影响细胞的许多关键生理功能,包括DNA修复、染色质重塑、代谢途径、免疫细胞功能和细胞衰老等。NAD+水平降低将加剧组织功能衰退和机体衰老,增加多种衰老相关疾病的发病率。
NAD+补救合成途径是哺乳动物中维持胞内NAD+正常水平最重要的途径。在补救途径中,烟酰胺磷酸核糖基转移酶(Nicotinamide phosphoribosyltransferase, NAMPT)催化烟酰胺(NAM)缩合成烟酰胺单核苷酸(β-Nicotinamide Mononucleotide,NMN),然后通过烟酰胺单核苷酸转移酶(Nicotinamide nucleotidyltransferase, NMNAT)催化生成NAD+。值得注意的是,NAMPT是补救途径的限速酶,其表达水平随细胞压力如DNA损伤、饥饿等呈现高度动态变化。另外,衰老、肥胖和高卡路里饮食可同时降低多种组织内NAMPT和NAD+水平。据文献报道,利用FK866(一种有效的NAMPT抑制剂)抑制NAMPT的酶活性,或利用小干扰RNA靶向敲除或降低NAMPT的表达,均可显著降低细胞内NAD+的含量。中年小鼠NAMPT基因的敲除,将导致肝脏NAD+降低到老年小鼠的水平,并引发肝脏组织炎症反应。另外,研究发现NAMPT的过表达,能够增加小鼠成纤维细胞中的NAD+水平。这表明NAMPT是NAD+合成补救途径的限速酶,也是增加NAD+合成的一个重要的分子靶点。
近年来,直接补充NAMPT为提高细胞内NAD+水平提供了一条有效途径。然而,纯度较高的NAMPT价格昂贵,不利于广泛应用。2021年国内学者已成功利用大肠杆菌、毕赤酵母发酵生产NAMPT。然而,当前微生物发酵生产NAMPT的效率较低,产物的生物活性也未见明确不利于大规模生产和产业化应用。
发明内容
为了克服现有技术不足,本发明提供了一株具有生产烟酰胺磷酸核糖基转移酶NAMPT酶的巴斯德毕赤酵母菌株。利用该宿主可实现高效表达NAMPT酶,具有实验操作简单、生产和使用成本低,易扩大生产等优势。本发明利用基因工程技术将NAMPT基因片段整合至酵母表达***pPIC9K,将异源片段导入Pichia pastoris。进一步对NAMPT的酶活性进行理性改造,将第219号位点天冬氨酸(D)突变为亮氨酸(L),经发酵过程持续甲醇诱导表达,得到具有较高酶活的NAMPT酶。菌体经简单离心得到胞外上清溶液即为粗酶液,所述NAMPT粗酶液经纯化后具有显著的抗衰老生物活性。
本发明首先提供一种用于制备烟酰胺磷酸核糖基转移酶NAMPT的重组菌株的构建方法,其是在受体菌株中Gene ID为10135的烟酰胺磷酸核糖基转移酶NAMPT编码基因的突变体,其编码的氨基酸序列相对于野生型的氨基酸序列的第219位的天冬氨酸突变为亮氨酸。
具体地,所述受体菌为毕赤酵母菌株,例如毕赤酵母菌株C1。
另外优选地,所述突变体编码的氨基酸序列如SEQ ID NO: 2所示。进一步,所述突变体的核苷酸序列经过密码子优化以适应毕赤酵母表达***。因而优选地,所述突变体的核苷酸序列如SEQ ID NO: 1所示。
本发明提供所述的构建方法获得的重组菌株。
本发明提供一种烟酰胺磷酸核糖基转移酶NAMPT的生产方法,其包括利用所述的重组菌株生产烟酰胺磷酸核糖基转移酶NAMPT的步骤。
具体地,通过摇瓶培养所述重组菌株,其中培养基含有甲醇、酵母抽提物、蛋白胨、磷酸二氢钾缓冲液、酵母浸粉和生物素,每培养20-28小时后补加一次0.4-0.6%的甲醇。
优选地,培养时的温度30℃,pH值4.5-6.5,转速220 rpm。
本发明提供所述的重组菌株毕赤酵母菌可以高效制备的NAMPT。在具体实施时,采用培养基为BMMY培养基,培养pH 值4.5-6.0,温度28-30℃,获得的NAMPT酶在15分钟内可催化烟酰胺生成0.965μM的NMN,这种NAMPT酶应用于调控HL-1细胞衰老,对衰老细胞的细胞活性提升了1.25倍。因而,本发明在提高衰老细胞活性方面有重要的应用价值。尤其是,在优选的实施方式中,本发明所使用的毕赤酵母菌株Pichia pastoris C1,作为真核生物,具有发酵简单、易于遗传操作、蛋白表达水平高、有利于蛋白纯化等诸多优势。毕赤酵母作为专性需氧菌,可以使用甲醇作为唯一碳源;具有醇氧化酶(AOX)启动子表达***可严格调控毕赤酵母,实现高水平表达。毕赤酵母表达***除了以上特点外,同时可实现高密度培养,进行大规模发酵,十分适合进行重组蛋白的工业放大化生产制备。
与现有重组NAMPT酶生产技术相比,本发明具有优势如下:
本发明提供了一种简单高效的方法,这可以简化NAMPT酶的生产制备工艺,提高其生产的安全性并降低制造成本;本发明的毕赤酵母具有生长速度快,甲醇菌体蛋白生产效率高的特点;本发明利用DeepSite对NAMPT进行活性位点预测,并对预测的活性位点进行理性改造,即将第219位点的天冬氨酸突变为亮氨酸(GAT- TTG),进一步提升NAMPT的酶活。
本发明提供的利用毕赤酵母菌株PC01异源表达NAMPT酶的方法,经过摇瓶初步优化后,其发酵上清液中获得的NAMPT酶在15分钟内可催化烟酰胺生成0.965μM的NMN;本发明所述毕赤酵母菌株PC01发酵生产的NAMPT通过纯化应用于衰老细胞活性提升效果显著,具体为细胞活性提高1.25倍。本发明可拓展NAMPT的微生物来源,获得具有更高酶活的NAMPT,并促进其在抗衰老领域的应用。
附图说明
图1为改造后编码NAMPT的核苷酸序列连入质粒pPIC9K的质粒图谱。
图2为改造后NAMPT基因序列成功整合到Pichia pastorisC1菌株的PCR验证电泳图。注:M,5000 K DNA Ladder;泳道1为作为对照未转入目的基因的Pichia pastoris C1菌株;2-7泳道为验证的1-6号转化子,成功转入了目的基因的PC01菌株。
图3为NAMPT酶在PC01菌株中表达的SDS电泳图。其中:左边是彩虹245 plus光谱蛋白marker;泳道标注处为成功表达NAMPT酶的PC01菌株。
图4为在15分钟内PC01菌株表达的NAMPT酶催化烟酰胺生产的NMN量图。
图5为PC01菌株表达的NAMPT酶可提高HL-1衰老细胞活性图。
具体实施方式
实施例中使用的试剂、溶液、培养基如下:
主要溶液的配制:
(1)10×YNB:3.4g YNB(不含硫酸铵和氨基酸)、10g硫酸铵加入100mL灭菌后的水中,加热至YNB完全溶解,过0.22μM滤膜除菌,4℃保存。
(2)500×B(0.02%生物素):溶解10mg生物素于50mL灭菌的水中,过0.22μM滤膜除菌,4℃保存。
(3)10×D(20%葡萄糖):20g D-葡萄糖溶解于100mL水中,50℃以下加热溶解,高温高压灭菌,于4℃保存。
(4)1mol/L磷酸钾缓冲液(pH 6.0): 13.2mL 1mol/L的K2HPO4,与86.8mL 1mol/L的KH2PO4混合,调pH至6.0,高温高压灭菌后室温保存。
(5)10×GY(10%甘油):10mL 100%甘油中加入90mL水,混匀,过滤或高压灭菌,室温保存。
(6)10×M(5%甲醇):5mL甲醇与95mL水混匀,过0.22μM滤膜除菌,4℃保存。
(7)G-418:0.1g G-418溶于1mL灭菌ddH2O中配制成100mg/mL的母液,-20℃保存。
主要培养基的配制:
(1)YPD液体培养基(Yeast Extract Peptone Dextrose Medium):溶解10g YeastExtract和20g Peptone于900mL水中,高温高压灭菌20min,使用时加入100mL10×D,室温保存。
(2)MD平板培养基(Minimal dextrose medium):15g琼脂粉溶解于800mL水中,高温高压灭菌20min。60℃下加入100mL 10×YNB、2mL 500×B、100mL 10×D混匀,平板放置于4℃。
(3)MDG平板培养基:将100mg/mL G-418加入MD平板中制成5mg/mL的MDG平板,4℃保存。
(4)BMGY培养基(Buffered Glycerol-complex Medium):10g Yeast Extract、20gPeptone溶解于700mL水中,高温高压灭菌20min,冷却至室温后加入100mL 1mol/L磷酸钾缓冲液(pH 6.0)、100mL 10×YNB、2mL 500×B和100mL 10×GY。
(5)BMMY培养基(Buffered Methanol-complex Medium):10g Yeast Extract、20gPeptone溶解于700mL水中,高温高压灭菌20min,冷却至室温后加入100mL 1mol/L磷酸钾缓冲液(pH 6.0)、100mL 10×YNB、2mL 500×B和100mL 10×M。
(6)DNEM完全培养基:450 mLDMEN基本培养基、50 mL胎牛血清、1mL抗生素Primocin,混匀待用。
基因组提取具体实施步骤如下:
(1)吸取需提基因组的菌液至新的1.5mL EP管中,12000g离心3min,弃上清。
(2)加入100μL含200mM醋酸锂,1%SDS的细胞裂解液,涡旋混匀,70℃孵育5min。
(3)孵育结束后加入300μL无水乙醇,涡旋振荡15s,12000g离心3min,弃上清。
(4)重新加入300μL 70%乙醇,洗涤并于12000g下离心3min,弃上清。
(5)60℃金属浴放置5min,挥干乙醇,加入100μL ddH2O,轻轻吹打混匀,12000g离心30s。
(6)取1μL上清作为模板进行PCR。
实施例1:NAMPT酶活性的理性改造
DeepSite预测NAMPT酶的活性位点:根据所述NAMPT的氨基酸序列(Gene ID:10135)在PDB数据库中查找NAMPT三维结构(PDB ID : 2GVG),然后在DeepSite网站上输入ID : 2GVG并提交。得到活性位点预测结果后,结合实验分析筛选出D219作为潜在的活性位点。通过定点突变技术将NAMPT氨基酸序列中第219位的天冬氨酸突变为亮氨酸,即GAT突变为TTG,得到如SEQ ID NO: 1所述的突变NAMPT核苷酸序列,以及如SEQ ID NO: 2所述的突变NAMPT氨基酸序列。
实施例2:重组NAMPT毕赤酵母PC01菌株的构建
(1)重组质粒pPIC9K-NAMPT酵母表达***的构建:本实施例选用pPIC9K质粒(AOX启动子)进行质粒修改达到目的基因的编辑,将pPIC9k质粒骨架按照相对等长原则分为两段(fragment 1、fragment 2)进行PCR,其中fragment 1包含AOX1 promoter至AOX1 3’fragment序列,fragment 2包含kanR至AOX1 terminator序列,并与添加同源臂的NAMPT片段进行Gibson组装获得融合质粒,如图1所示,经过大肠杆菌转化、质粒提取和基因测序,最终获得整合质粒pPIC9K-NAMPT。
(2)pPIC9K-NAMPT构建成功后,根据NAMPT基因内的酶切位点和质粒pPIC9K上的酶切位点,选择Sac Ⅰ酶切位点进行线性化处理,经电泳验证正确后进行纯化回收,并将回收后的线性化重组质粒pPIC9K-NAMPT通过电击转化至毕赤酵母Pichia pastoris C1感受态细胞中,涂布于MD平板培养基于30℃培养。待MD平板长出菌落后,用无菌ddH2O洗下并进行适度稀释,涂布于含5mg/mLG-418的MDG平板培养基筛选,30℃下培养2-3天,得到单菌落,挑取形态状态较佳的单菌落接种于YPD液体培养基扩大培养,同时使用甘油保存重组毕赤酵母菌株于-80℃冰箱。
(3)重组毕赤酵母单克隆验证:YPD液体培养基中扩大培养的菌液进行基因组粗提取,作为PCR模板。使用phanata高保真酶PCR体系扩增,PCR产物进行电泳和测序验证(电泳如图2所示)。泳道1为作为对照未转入目的基因的Pichia pastoris C1菌株(公开于中国专利202210611032.X等);2-7泳道,为验证的1-6号转化子,表明成功转入目的基因的PC01菌株。这表明目的基因NAMPT成功整合到出发菌株Pichia pastoris C1的基因组上,在进一步测序结果中验证到与目的基因的序列相符,表明重组毕赤酵母菌株PC01构建成功。
实施例3:甲醇诱导PC01菌株表达NAMPT酶
从-80℃冰箱中取出电泳和测序验证正确的菌株,接种至3mL YPD液体培养基中,220rpm,30℃,培养14-16h获得种子液。培养至OD600=0.1-1后离心弃上清,用BMGY培养基洗涤一次并重悬,调至统一OD600值,转接到50mL BMGY培养基中扩大培养,220 rpm,30℃,培养16-20h至OD600=1-2。BMGY培养基培养所得菌液以5000g,4℃,离心10min,弃上清,用BMMY培养基洗涤一次,并重悬于50mL的BMMY培养基中,220 rpm,30℃,进行培养。每隔24小时补加一次0.5%的甲醇,继续220 rpm,30℃培养。诱导120h后结束发酵,对所得发酵菌液进行离心,分离发酵上清和菌体,上清液即为含有重组NAMPT酶的胞外粗酶液。
利用SDS-PAGE电泳检测粗酶液中NAMPT的表达,结果如图3所示,泳道M为蛋白Marker-245kDa,泳道处为重组毕赤酵母PC01菌株,在48-75kDa之间出现条带,与目的蛋白预期相对分子质量54kDa相符,也与质谱鉴定结果一致,重组毕赤酵母PC01菌株表达的目的蛋白鉴定为NAMPT。
实施例5:菌株PC01表达的NAMPT酶的活性测定
使用荧光检测法测定NAMPT活性。在1.5mL离心管中配置若干个0-1μmol/L浓度梯度的NMN水溶液,每个69μL。每个离心管中加入27.7μL,2mol/L KOH,在加入27.7μL 20%苯乙酮,短暂涡旋混匀后,冰浴2min。加入125μL 88%甲酸,低速短暂离心,在37℃摇床中反应10min。吸取离心管中240μL液体至黑色96孔板中,用Biotek-synergy H1酶标仪在激发光382nm,发射光445nm下测定荧光。以纯水为空白对照。根据NMN浓度和检测的对应荧光值做标准曲线。
酶反应体系:在1.5 mL 离心管中加入1.5 μL稀释至一定倍数的纯酶液、3.45 μL1 mol/L Tris-HCl、1.38 μL 1 % BSA、0.83 μL 1 mol/L MgCl2、1.38 μL 0.1 mol/L ATP、0.28 μL 0.1 mol/L PRPP、1.38 μL 0.1 mol/L二硫苏糖醇、58.10 μL纯水、0.69 μL 200 μmol/LNAM。在恒温震荡仪中反应,45℃,15 min。设置纯水代替酶液的反应体系为空白对照。
中止酶反应:将离心管于95℃水浴1 min灭活酶。
产物NMN衍生物反应:往离心管中加入27.7 μL,2 mol/L KOH,再加入27.7 μL 20% 苯乙酮,短暂涡旋混匀后置于冰中,冰浴2 min。加入125 μL 88 %甲酸,低速短暂离心,在恒温震荡仪中反应,37 ℃,10 min。
测定荧光:吸取离心管中240 μL液体至黑色96孔板测定孔中,用Tecan多功能酶标仪在激发光382 nm,发射光445 nm下测定荧光。
计算:结合测定荧光值和标准曲线计算NMN的含量,酶活标记为在15分钟内产生的NMN含量。结果如图4所示,经过摇瓶初步优化后,其发酵上清液中的NAMPT酶在15分钟内催化底物烟酰胺生产NMN的量为0.965μM。
实施例6:菌株PC01表达的NAMPT酶的抗衰老活性评价
从液氮中去除冻存的小鼠心肌细胞HL-1,37℃迅速融化,1000 rpm/min离心5min,完全培养基重悬至T75细胞培养瓶,置37℃,5% CO2培养箱中。细胞连续传代三次后,当培养瓶内细胞融合度达80%-90%,即细胞处于对数生长期时,用EDTA胰酶消化,制成细胞悬液,浓度调至1×105-×106个/mL,每孔100 µL,加入96孔板,实验组别设置:正常对照组:NC;模型对照组:MC;NAMPT干预组:NAMPT)。待细胞贴壁12 h,弃去培养液,NC组更换正常培养液,MC组和NAMPT组更换200 μmol/L的D半乳糖培养液,继续培养24 h,成功建立HL-1细胞衰老模型。建模成功后,NC组和MC组更换正常培养液,NAMPT组更换为PC01菌株表达的NAMPT250ng/mL,继续培养24 h。最后,每孔加入10 µLCCK8,37℃孵育2h,酶标仪450 nm吸收光检测。结果如图5所示,相较于MC组,NAMPT处理的HL-1衰老细胞活性提高了1.25倍。
Claims (10)
1.一种用于制备烟酰胺磷酸核糖基转移酶NAMPT的重组菌株的构建方法,其特征在于,在受体菌株中Gene ID为10135的烟酰胺磷酸核糖基转移酶NAMPT编码基因的突变体,其编码的氨基酸序列相对于野生型的氨基酸序列的第219位的天冬氨酸突变为亮氨酸。
2.如权利要求1所述的构建方法,其特征在于,所述受体菌为毕赤酵母菌株。
3.如权利要求1所述的构建方法,其特征在于,所述受体菌是毕赤酵母菌株C1。
4.如权利要求1所述的构建方法,其特征在于,所述突变体编码的氨基酸序列如SEQ IDNO: 2所示。
5.如权利要求1所述的构建方法,其特征在于,所述突变体的核苷酸序列经过密码子优化以适应毕赤酵母表达***。
6.如权利要求5所述的构建方法,其特征在于,所述突变体的核苷酸序列如SEQ ID NO:1所示。
7.如权利要求1至6任一项所述的构建方法获得的重组菌株。
8.一种烟酰胺磷酸核糖基转移酶NAMPT的生产方法,其包括利用权利要求7所述的重组菌株生产烟酰胺磷酸核糖基转移酶NAMPT的步骤。
9.如权利要求8所述的生产方法,其特征在于,通过摇瓶培养所述重组菌株,其中培养基含有酵母提取物、蛋白胨、磷酸二氢钾缓冲液、酵母浸粉和生物素,以甲醇为诱导剂,每培养20-28小时后补加一次0.4-0.6%的甲醇。
10.如权利要求9所述的生产方法,其特征在于,培养时的pH 值4.5-6.0,温度28-30℃,转速200-240 rpm。
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