CN105695383B - 重组菌株及其应用 - Google Patents
重组菌株及其应用 Download PDFInfo
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- CN105695383B CN105695383B CN201610119402.2A CN201610119402A CN105695383B CN 105695383 B CN105695383 B CN 105695383B CN 201610119402 A CN201610119402 A CN 201610119402A CN 105695383 B CN105695383 B CN 105695383B
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Abstract
本发明涉及微生物领域,特别涉及重组菌株及其应用。实验结果表明,野生型谷氨酸棒杆菌ATCC 13869在未经改造前基本不产谷氨酸,本发明提供的菌株MHZ‑0112‑1谷氨酸的产量为4.7g/L;MHZ‑0112‑2谷氨酸的产量为18.5g/L;菌株MHZ‑0112‑4和MHZ‑0112‑5谷氨酸的产量分别为23.4g/L和26.4g/L。在单独利用强启动子Psod表达gdh基因,谷氨酸产量提高约1.8倍,单独利用Ptuf启动子表达gltA基因将CS酶活增加了4倍,谷氨酸产量提高至30.8g/L;将两个基因同时进行过表达后,谷氨酸产量继续提高至35.5g/L,转化率达到约59%。
Description
技术领域
本发明涉及微生物领域,特别涉及重组菌株及其应用。
背景技术
谷氨酸(Glutamic acid),其学名为2-氨基-5-梭基戊酸,是构成蛋白质的20种常见氨基酸之一。谷氨酸是目前世界上产量最大的氨基酸品种,全球每年对谷氨酸的需求量达到2500万吨。L-谷氨酸是谷氨酸的左旋体,其具有增鲜、降低血氨、改善中枢神经***、改善儿童智力发育、扩张血管、增强血液循环、促进花穗发育等功能,并且可以生产许多重要的下游产品,如L-谷氨酸钠、L-苏氨酸、聚谷氨酸等,被广泛应用于食品、医药、人造制革、化妆品、农业等行业。
谷氨酸的制备起始于18世纪。1866年德国化学家从小麦面筋的水解物中提取得到谷氨酸,1957年日本率先从自然界分离得到谷氨酸棒杆菌(Corynebacteriumglutamicum),开启了利用糖和氨发酵生产谷氨酸的研究,因发酵法具有原料成本低、反应条件温和、可大规模生产等优点,目前仍是谷氨酸生产的主要方法。
谷氨酸棒杆菌是一种好氧、不产抱子的***,基因组大小为3282kb,它不仅能利用葡萄糖为唯一碳源进行生长,而且能利用包括乙酸和唬拍酸在内的其它有机酸和碳水化合物为唯一碳源或混合碳源进行生长。谷氨酸棒杆菌为生物素缺陷型,需要补充生物素才能生长。当培养基中的生物素过量时,菌体不向细胞外分泌产生谷氨酸;当生物素被限量添加时,菌体则大量分泌产生谷氨酸,因而野生型的谷氨酸棒杆菌可通过生物素限量添加来诱导分泌产生谷氨酸。此外,在生物素过量的情况下通过添加某种表面活性剂如Twee40或Tween60也能诱导谷氨酸棒杆菌过量合成谷氨酸。
近年来,随着谷氨酸棒杆菌模式菌株ATCC13032基因组测序的完成以及谷氨酸棒杆菌基因操作技术的不断完善,使得通过分子生物学手段对谷氨酸棒杆菌进行相关改造成为现实。谷氨酸在生物体内的合成代谢途径也已相继报道,见图1。日本味之素株式会社依据谷氨酸合成的代谢途径对谷氨酸生产菌种作了一些基因改造,如引入棒状细菌的柠檬酸合成酶基因使肠细菌生产谷氨酸的能力增强;增加丙酮酸脱氢酶基因的拷贝数而增强丙酮酸脱氢酶活性,并使棒状细菌的谷氨酸生产能力提高;过表达YHFK基因使L-谷氨酸产量提高等。
现有技术往往需要在培养基中添加过量的生物素及表面活性剂如Twee40或Tween60,增加了生产成本,且产率往往较低。
发明内容
有鉴于此,本发明提供一种重组菌株及其应用。与野生菌株相比,本发明提供的菌株显著(P<0.05)或极显著(P<0.01)提高了谷氨酸的发酵产量。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种重组菌株,其包含编码突变yggB蛋白质的突变yggB基因。
在本发明的一些具体实施方案中,所述重组菌株中所述突变yggB蛋白质为A106氨基酸处具有突变。
在本发明的一些具体实施方案中,所述重组菌株中所述突变yggB蛋白质具有A106V突变,获得的菌株命名为MHZ-0112-1。
在本发明的一些具体实施方案中,所述重组菌株还包括编码降低ODHC活性的突变odhA基因;所述突变odhA基因为对odhA基因进行失活改造。具体的,本发明提供的含有突变odhA基因的菌株命名为MHZ-0112-2。制备方法如下:
由谷氨酸合成代谢途径(图1)可知,三羧酸循环中由α-酮戊二酸脱氢酶复合体(ODHC)催化的α-酮戊二酸向琥珀酰辅酶A的转化是谷氨酸合成代谢主要的竞争途径,为了降低ODHC的活性,需将其Elo亚基的编码基因odhA进行失活改造。采用的手段是引入碱基缺失,使蛋白合成过程中出现移码突变,从而达到失活的目的。
在NCBI GenBank数据库中获得谷氨酸棒杆菌(CorynebacteriumglutamicumATCC13869)odhA基因(Gene Bank NO.NCgl1084)的核苷酸序列(SEQ ID No.9),由此核苷酸序列设计在特定位置引入碱基缺失以使odhA基因失活,基于碱基序列及所选缺失位置合成了四条引物(如SEQ ID NO.10-13所示)。利用Phusion超保真聚合酶(NewEngland BioLabs),以B1/B2,B3/B4作为引物,以谷氨酸棒杆菌ATCC 13869的基因组DNA作为模板制备odhA碱基缺失位点的上下游片段。PCR程序为,98℃变性10s,50℃复性20s,72℃延伸20s,循环30次后。所得两个片段经琼脂糖凝胶回收试剂盒(天根)纯化后,利用引物组B1/B4进行overlap PCR扩增得到产物,经琼脂糖凝胶回收试剂盒(天根)纯化后利用EcoRI/BamHI进行消化,同时将pK18-mobsacB利用EcoRI/BamHI进行消化,并用T4DNA连接酶(TransGen Biotech)将片段与载体进行连接,转化Trans1T1感受态细胞(TransGenBiotech),挑取卡那抗性克隆,EcoRI/BamHI酶切鉴定得到overlap PCR片段***pK18mobsacB的阳性克隆,进一步通过测序(Invitrogen公司)鉴定***的片段确实为odhA基因待缺失位点的上下游片段。
将所得到的质粒命名为pK18-odhAda1。将pK18-odhAda1转入MHZ-0112-1菌株中。在含有15mg/L的卡那霉素的选择培养基上选择单交换转化体,其中带有odhA碱基缺失的序列通过与內源odhA基因同源重组连同重组载体整合到基因组中。利用Fast Taq DNA聚合酶(TransGen Biotech),以B1/T2,T1/B4引物对进行菌落PCR鉴定KanR克隆,PCR参数为:94℃30s,50℃30s,72℃40s,共26个循环,两个引物对均扩增出目的片段的克隆为阳性克隆。将挑选出的阳性克隆接种入无抗BHI培养基中培养12~14h,将菌液稀释100-1000倍后涂布在含有10%蔗糖的固体BHI培养基上培养36h,将所筛选出的菌株进一步进行卡那抗性表型验证,挑选KanS的重组子利用B1/B4引物对扩增目的片段,阳性克隆的目的片段较野生型所扩增的片段小,将阳性克隆进行测序验证,得到odhA碱基缺失的菌株,命名为MHZ-0112-2。
在本发明的一些具体实施方案中,所述重组菌株还包括突变gdh基因;所述突变gdh基因为过表达gdh基因。
在本发明的一些具体实施方案中,所述重组菌株还包括突变gltA基因;所述突变gltA基因为过表达gltA基因。
在本发明的一些具体实施方案中,所述重组菌株中所述过表达通过构建过表达载体或者***启动子的方式增强所述基因的表达。
在本发明的一些具体实施方案中,所述重组菌株中所述过表达载体为pEC-gdh或pEC-gltA;所述启动子为sod或tuf。
分别根据NCBIGenBank数据库中gdh基因(Gene Bank NO:NCgl1084)的序列(SEQNO.14)和gltA基因(Gene Bank NO:NCgl0795)的序列(SEQ NO.15)设计引物,如SEQ IDNO.16~19所示。
整合质粒pK18-Psodgdh,pK18-tufgltA的构建:
以谷氨酸棒杆菌ATCC13869基因组作为模板,分别以C1/C2,D1/D2引物对扩增gdh及gltA两个基因的全长片段,经琼脂糖凝胶电泳及胶回收纯化后,将所得片段分别利用EcoRI/SalI双酶切,质粒pEC-XK99E(GI:29164935)也进行相同的双酶切,利用T4DNA连接酶进行连接,转化Trans1T1感受态细胞后筛选阳性克隆,提取质粒分别进行EcoRI/SalI双酶切鉴定,最后进行测序确认,所得质粒分别命名为pEC-gdh,pEC-gltA。制备MHZ-0112-2的感受态细胞并将对照质粒pEC-XK99E及上述所构建pEC-gdh,pEC-gltA转入MHZ-0112-2菌株中。利用Kan15BHI平板筛选出阳性克隆,并提取质粒验证,所得到的三个带有复制性质粒的菌株分别命名为MHZ-0112-3,MHZ-0112-4,MHZ-0112-5。
以谷氨酸棒杆菌ATCC13869的基因组为模板,分别以C3/C4,C5/C6,C7/C8引物对(如SEQ ID NO.20~31所示)在Phusion超保真聚合酶的作用下PCR扩增,得到gdh起始密码子的上游同源臂、sod启动子和起始密码子的下游同源臂,PCR参数为98℃10s,50℃20s,72℃20s,共30个循环。将PCR产物用琼脂糖凝胶回收试剂盒进行割胶回收。以上述所得片段混合物为模板,以C3/C8引物对进行overlap PCR扩增,并割胶回收,产物用SalI和HindIII双酶切,pK18mobsacB用同样的酶进行切割,利用T4DNA连接酶在22℃下连接1-2h,转化Trans1T1感受态细胞,挑取卡那抗性克隆,SalI/HindIII酶切鉴定得到overlap PCR片段***pK18mobsacB的阳性克隆,进一步通过测序鉴定***的片段确实为添加了Psod启动子的gdh基因上下游同源臂。所得到的质粒命名为pK18-Psodgdh。
pK18-tufgltA的构建过程与上述类似,所用引物对为D3/D4,D7/D8扩增gltA起始密码子的上下游同源臂,以D5/D6扩增启动子Ptuf,Overlap PCR所用引物对为D3/D8。
高表达菌株的制备:
制备MHZ-0112-2菌株的感受态细胞,分别将质粒pK18-Psodgdh,pK18-tufgltA进行转化。
在含有15mg/L的卡那霉素的选择培养基上选择单交换转化体,其中带有sod(tuf)启动子的序列通过与內源gdh(gltA)基因同源重组连同重组载体整合到基因组中。利用Fast Taq DNA聚合酶,以C3/T2,T1/C6引物对(或D3/T2,T1/D6引物对)进行菌落PCR鉴定KanR克隆,PCR参数同上,两个引物对均扩增出目的片段的克隆为阳性克隆。将挑选出的阳性克隆接种入无抗BHI培养基中培养12-14h,将菌液稀释100-1000倍后涂布在含有10%蔗糖的固体BHI培养基上培养36h,将所筛选出的菌株进一步进行卡那抗性表型验证,挑选KanS的重组子利用C3/C6引物对(或D3/D6引物对)扩增目的片段,阳性克隆的目的片段比野生型所扩增的片段大,将阳性克隆进行测序验证,得到sod(tuf)启动子成功整合至gdh(gltA)基因ATG之前的菌株,命名为MHZ-0112-6(MHZ-0112-7)。
同理,制备MHZ-0112-6感受态细胞,将pK18-tufgltA进行转化,经两轮重组筛选,得到gdh与gltA叠加的突变株MHZ-0112-8。
在本发明的一些具体实施方案中,所述重组菌株MHZ-0112-8的保藏编号为CGMCCNo.11941。
本发明还提供了所述重组菌株发酵生产谷氨酸中的应用。
本发明还提供了所述的重组菌株的构建方法,包括如下步骤:
以野生型谷氨酸棒杆菌ATCC 13869为出发菌株;
步骤1、在yggB中引入点突变A106V;
步骤2、对odhA基因进行失活改造;
步骤3、过表达gdh基因;
步骤4、过表达gltA基因。
在本发明的一些具体实施方案中,所述构建方法的步骤3或步骤4中所述过表达通过构建过表达载体或者***启动子的方式增强所述基因的表达;所述过表达载体为pEC-gdh或pEC-gltA;所述启动子为sod或tuf。
本发明还提供了一种发酵生产谷氨酸的方法,以所述的重组菌株为发酵菌株。
本发明提供了重组菌株及其用于发酵生产谷氨酸的用途。实验结果表明,野生型谷氨酸棒杆菌ATCC 13869在未经改造前基本不产谷氨酸,在将编码通道蛋白的yggB基因修饰后(MHZ-0112-1),培养液中有少量谷氨酸积累4.7g/L,与野生菌株相比,该菌株显著(P<0.05)提高了谷氨酸的发酵产量。在将谷氨酸合成途径的竞争途径截断之后(MHZ-0112-2),谷氨酸的产量为18.5g/L,相比MHZ-0112-1提高约4倍,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。将pEC-gdh,pEC-gltA转入MHZ-0112-2菌株中,制得菌株MHZ-0112-4和MHZ-0112-5,谷氨酸的产量分别为23.4g/L和26.4g/L,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。在单独利用强启动子Psod表达gdh基因,GDH酶活较野生型提高约2.5倍,谷氨酸产量提高约1.8倍,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。单独利用Ptuf启动子表达gltA基因将CS酶活增加了约4倍,谷氨酸产量提高至30.8g/L,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量;将两个基因同时进行过表达后,谷氨酸产量继续提高至35.5g/L,转化率达到约59%,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。
生物保藏说明
生物材料MHZ-0112-8,分类命名:谷氨酸棒杆菌,Corynebacterium glutamicum于2015年12月25日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC No.11941。
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,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示谷氨酸在生物体内的合成代谢途径;
图2示sacB筛选***原理图。
具体实施方式
本发明公开了一种菌株及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
谷氨酸棒杆菌感受态的制备:-80℃冰箱中划线转接出谷氨酸棒杆菌种ATCC13869至BHI平板上,30℃培养;挑取单菌落,转接到5ml BHI液体培养基(3.7%脑心浸粉)试管中,200rpm,30℃培养12h;按1%接种量接种至100ml BHIS液体培养基(3.7%脑心浸粉,9.1%山梨醇)中,200rpm,30℃培养至OD 600达到1.5;4℃,6000rpm离心20min,收集菌体,弃上清液;用TG Buffer(1mM Tris,10%甘油(v/v),pH7.5)悬浮后于4℃,6000rpm离心20min,弃上清液,并重复一遍;用10%的甘油悬浮后于4℃,6000rpm离心20min,弃上清液,并重复一遍;加入1ml 10%甘油悬浮菌体后分装。将感受态细胞放于-70℃冰箱保存或直接用于电击转化。
电转化:将感受态细胞从冰箱中拿出并于冰水中融化,添加10-15 μl重组质粒DNA入溶化的感受态细胞中,吹吸混匀后将其转移至1mm电击杯(BioRad公司产品)中,于1.8kv,5ms条件下在电击仪(BioRad公司产品)上电击,然后立即加入46℃预热的BHI培养液,轻轻混匀,将混合液转入15ml离心管中,46℃水浴6分钟,30℃活化培养2h,离心收集菌体,将菌体涂布于含有25mg/L的BHI固体培养基上,30℃于恒温箱中培养36h。
菌株筛选:以yggB基因改造为例,首先用卡那抗性筛选,如上所述,将电转后的菌体涂布在含卡那抗性的固体BHI培养基上,挑选抗性克隆。根据pK18mobsacB质粒MCS两端的序列设计验证引物pK18T1/pK18T2(序列见表1),利用Fast Taq DNA聚合酶(全式金公司),以A1/T2,T1/A4引物对进行菌落PCR鉴定KanR克隆,PCR参数为:94℃30s,50℃30s,72℃40s,共26个循环,两个引物对均扩增出1000bp片段的克隆为阳性克隆。将挑选出的阳性克隆接种入无抗BHI培养基中培养12-14h,让菌体在繁殖的过程中发生同源重组。将菌液稀释100-1000倍后涂布在含有10%蔗糖的固体BHI培养基上培养36h,在蔗糖存在的情况下,未发生第二次同源重组的菌株因带有sacB基因而不能存活(原理图见图2)。由于蔗糖筛选存在约20%的假阳性,需将所筛选出的菌株进一步进行卡那抗性表型验证。根据点突变设计出MAMA PCR(Mismatch Amplification Mutation Assy PCR)鉴定引物106T(序列见表1),挑选KanS的重组子利用106T/A4进行点突变验证,参数同上。将验证正确的重组子利用A1/A4扩增目的片段后进行测序验证。
发酵验证谷氨酸产量的方法:将冻存于-80℃甘油管中的野生型13869菌株、MHZ-0112-1菌株以及MHZ-0112-2接种于上述斜面培养基中进行活化,于31.5℃下培养24h后长出菌苔;从新鲜活化的斜面上挑取上述三株菌的菌苔,接种于上述种子培养基中,于31.5℃,220rpm/min下振荡培养至对数生长中后期(12h左右),制得种子液;以10%的接种量将上述种子液接种至装有20ml发酵培养基的500ml摇瓶中,在31.5℃,振荡培养。在在糖被完全消耗之后,测定培养基中积累的L-谷氨酸的浓度。结果如表4所示(OD600是稀释100倍的培养液在600nm的浊度并表示细胞量,Glu(g/L)表示积累的L-谷氨酸的量)。
GDH酶活测定方法:将培养后的细菌在4℃下以10000rpm/min离心5min,收集菌体,用0.01mol/L磷酸盐缓冲液(PBS,pH7.4)洗一次,再重悬于1/10原菌液体积的PBS中,冰浴,超声破菌(350W,持续5s,间隔10s,共20次)后,10000rpm/min离心10min,去沉淀,得到粗酶溶液,以NADP+为辅酶进行测定,反应体系为:0.5mmol/L NADP+,15mmol/L谷氨酸,25mmol/LTris-HCl,pH8.0。取样品酶液测定GDH催化辅酶NADH+还原所引起的光吸收值在340nm波长处的变化,每分钟催化1 μmol NADP+还原所需的酶量定义为一个酶活力单位(U)。酶活=ΔOD340×0.2×2/0.0486,蛋白浓度=1.45×OD280-0.74×OD260,单位酶活=酶活/蛋白浓度。所有测定都在BECKMAN DU800分光光度计上完成。以Protien Assay(Bio-Rad)测定粗酶液中的蛋白质浓度,使用牛血清蛋白为标准蛋白。
CS酶活测定方法:离心培养液以分离菌体。以含有200mmol/L谷氨酸钠的50mmol/LTris缓冲液(pH 7.5)清洗细胞,然后再以相同溶液悬浮。冰浴,超声破菌(350W,持续5s,间隔10s,共20次)后,10000rpm/min离心10min,去沉淀,得到粗酶溶液,根据MethodsEnzymol.13,3-11(1969)测定柠檬酸合成酶的活性。具体而言,将此粗酶溶液加入含有100mmo/L Tris-HCl(pH 8),0.1mmol/L DTNB,200mmol/L谷氨酸钠,0.3mmol/L乙酞基辅酶A的反应液中,然后以分光光度计在30℃,412nm测定其吸光度增加,以此最为背景值,进一步加入草醋酸使其最终浓度为0.5mmol/L。测定在412nm处吸光值的增加,从背景值推算获得柠檬酸合成酶的酶活。以Protien Assay(Bio-Rad)测定粗酶液中的蛋白质浓度,使用牛血清蛋白为标准蛋白。
本发明提供了重组菌株及其用于发酵生产谷氨酸的用途。实验结果表明,野生型谷氨酸棒杆菌ATCC 13869在未经改造前基本不产谷氨酸,在将编码通道蛋白的yggB基因修饰后(MHZ-0112-1),培养液中有少量谷氨酸积累4.7g/L,与野生菌株相比,该菌株显著(P<0.05)提高了谷氨酸的发酵产量。在将谷氨酸合成途径的竞争途径截断之后(MHZ-0112-2),谷氨酸的产量为18.5g/L,相比MHZ-0112-1提高约4倍,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。将pEC-gdh,pEC-gltA转入MHZ-0112-2菌株中,制得菌株MHZ-0112-4和MHZ-0112-5,谷氨酸的产量分别为23.4g/L和26.4g/L,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。在单独利用强启动子Psod表达gdh基因,GDH酶活较野生型提高约2.5倍,谷氨酸产量提高约1.8倍,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。单独利用Ptuf启动子表达gltA基因将CS酶活增加了约4倍,谷氨酸产量提高至30.8g/L,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量;将两个基因同时进行过表达后,谷氨酸产量继续提高至35.5g/L,转化率达到约59%,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。
本发明提供的重组菌株及其用于发酵生产谷氨酸的用途中所涉及的菌株、载体、启动子、制剂均可由市场购得。
术语解释:
PCR:聚合酶链式反应
KanR:卡那霉素抗性
KanS:卡那霉素敏感。
下面结合实施例,进一步阐述本发明:
实施例1yggB基因A106V点突变的引入
(1)用于yggBA106V点突变的载体构建
根据谷氨酸棒杆菌(Corynebacterium glutamicum ATCC13869)yggB基因(GeneBank NO.:NCgl 1221)的DNA序列(SEQ NO.1)设计引物进行PCR扩增,引物序列如表1所示。
表1引物序列
用基因组DAN提取试剂盒(天根公司)提取野生型谷氨酸棒杆菌C.glutamicumATCC13869全基因组序列。以抽提的基因组为模板,分别以A1/A2,A3/A4引物对在Phusion超保真聚合酶(New England BioLabs)的作用下PCR扩增得到yggB基因106位氨基酸上下游的同源臂,PCR参数为98℃10s,50℃20s,72℃20s,共30个循环。将PCR产物用琼脂糖凝胶回收试剂盒(天根公司)进行割胶回收。以上述所得上下游同源臂1:1混合物为模板,以A1/A4引物对进行overlap PCR扩增,并割胶回收,产物用SalI和HindIII双酶切,pK18mobsacB用同样的酶进行切割,利用T4DNA连接酶(全式金公司)22℃下连接1-2h,转化Trans1T1感受态细胞(全式金公司),挑取卡那抗性克隆,SalI/HindIII酶切鉴定得到overlap PCR片段***pK18mobsacB的阳性克隆,进一步通过测序(Invitrogen公司)鉴定***的片段确实为yggB基因106位氨基酸的上下游片段。所得到的质粒命名为pK18-yggBA106V。
测序结果如SEQ ID No.14所示。
(2)yggBA106V点突变的谷氨酸棒杆菌MHZ-0112-1的制备
带有yggBA106V点突变的菌株筛选:首先用卡那抗性筛选,如上所述,将电转后的菌体涂布在含卡那抗性的固体BHI培养基上,挑选抗性克隆。根据pK18mobsacB质粒MCS两端的序列设计验证引物pK18T1/pK18T2(序列见表1),利用Fast Taq DNA聚合酶(全式金公司),以A1/T2,T1/A4引物对进行菌落PCR鉴定KanR克隆,PCR参数为:94℃30s,50℃30s,72℃40s,共26个循环,两个引物对均扩增出1000bp片段的克隆为阳性克隆。将挑选出的阳性克隆接种入无抗BHI培养基中培养12-14h,让菌体在繁殖的过程中发生同源重组。将菌液稀释100-1000倍后涂布在含有10%蔗糖的固体BHI培养基上培养36h,在蔗糖存在的情况下,未发生第二次同源重组的菌株因带有sacB基因而不能存活(原理图见图2)。由于蔗糖筛选存在约20%的假阳性,需将所筛选出的菌株进一步进行卡那抗性表型验证。根据点突变设计出MAMA PCR(Mismatch Amplification Mutation Assy PCR)鉴定引物106T(序列见表1),挑选KanS的重组子利用106T/A4进行点突变验证,参数同上。将验证正确的重组子利用A1/A4扩增目的片段后进行测序验证。得到的菌株命名为MHZ-0112-1。测序结果如SEQ IDNo.15所示。
实施例2 13869-yggBA106V菌株中odhA基因的失活
由谷氨酸合成代谢途径(图1)可知,三羧酸循环中由α-酮戊二酸脱氢酶复合体(ODHC)催化的α-酮戊二酸向琥珀酰辅酶A的转化是谷氨酸合成代谢主要的竞争途径,为了降低ODHC的活性,需将其Elo亚基的编码基因odhA进行失活改造。采用的手段是引入碱基缺失,使蛋白合成过程中出现移码突变,从而达到失活的目的。
(1)用于odhA碱基缺失载体构建
首先,在NCBI GenBank数据库中,获得谷氨酸棒杆菌(Corynebacteriumglutamicum ATCC13869)odhA基因(Gene Bank NO.NCgl1084)的核苷酸序列(SEQ IDNo.9),由此核苷酸序列设计在特定位置引入碱基缺失以使odhA基因失活,基于碱基序列及所选缺失位置合成了四条引物(SEQ ID NO.10-13,表2)。
表2引物序列
利用Phusion超保真聚合酶(New England BioLabs),以B1/B2,B3/B4作为引物,以谷氨酸棒杆菌ATCC 13869的基因组DNA作为模板制备odhA碱基缺失位点的上下游片段。PCR程序为,98℃变性10s,50℃复性20s,72℃延伸20s,循环30次后。所得两个片段经琼脂糖凝胶回收试剂盒(天根)纯化后,利用引物组B1/B4进行overlap PCR扩增得到产物,经琼脂糖凝胶回收试剂盒(天根)纯化后利用EcoRI/BamHI进行消化,同时将pK18-mobsacB利用EcoRI/BamHI进行消化,并用T4DNA连接酶(TransGen Biotech)将片段与载体进行连接,转化Trans1T1感受态细胞(TransGen Biotech),挑取卡那抗性克隆,EcoRI/BamHI酶切鉴定得到overlap PCR片段***pK18mobsacB的阳性克隆,进一步通过测序(Invitrogen公司)鉴定***的片段确实为odhA基因待缺失位点的上下游片段。所得到的质粒命名为pK18-odhAda1。测序结果如SEQ ID No.33所示。
(2)odhA碱基缺失载体在MHZ-0112-1中的引入
利用与实施例1中所述感受态制备及转化方法将pK18-odhAda1转入MHZ-0112-1菌株中。
在含有15mg/L的卡那霉素的选择培养基上选择单交换转化体,其中带有odhA碱基缺失的序列通过与內源odhA基因同源重组连同重组载体整合到基因组中。利用Fast TaqDNA聚合酶(TransGen Biotech),以B1/T2,T1/B4引物对进行菌落PCR鉴定KanR克隆,PCR参数为:94℃30s,50℃30s,72℃40s,共26个循环,两个引物对均扩增出目的片段的克隆为阳性克隆。将挑选出的阳性克隆接种入无抗BHI培养基中培养12-14h,将菌液稀释100-1000倍后涂布在含有10%蔗糖的固体BHI培养基上培养36h,将所筛选出的菌株进一步进行卡那抗性表型验证,挑选KanS的重组子利用B1/B4引物对扩增目的片段,阳性克隆的目的片段较野生型所扩增的片段小,将阳性克隆进行测序验证,得到odhA碱基缺失的菌株,命名为MHZ-0112-2。测序结果如SEQ ID No.34所示。
表3odhA基因的部分核苷酸序列
(3)MHZ-0112-2谷氨酸生产性能检测
培养基配方如下:
斜面培养基:酵母粉5g/L,牛肉膏10g/L,蛋白胨10g/L,氯化钠10g/L,琼脂粉2.5g/L,pH 7.0-7.2,121℃0.1MPa灭菌30min;
种子培养基:葡萄糖25g/L,尿素3g/L,K2HPO4·3H2O 2.2g/L,MgSO4.7H2O.0.9g/L,玉米浆33mL/L,豆饼水解液22mL/L,pH 7.0-7.2,121℃0.1MPa灭菌15min;
发酵培养基:葡萄糖60g/L,硫酸铵15g/L,KH2PO4 1g/L,MgSO4·7H2O 0.4g/L,FeSO4·7H2O 1mg/L,MnSO4·4-5H2O 1mg/L,VB1 200 μg/L,生物素300 μg/L,0.48g/L大豆水解液,用NaOH调节至pH 7.2-7.5,121℃0.1MPa灭菌15min,后添加1.0g热灭菌的碳酸钙。
将冻存于-80℃甘油管中的野生型13869菌株、MHZ-0112-1菌株以及MHZ-0112-2接种于上述斜面培养基中进行活化,于31.5℃下培养24h后长出菌苔;从新鲜活化的斜面上挑取上述三株菌的菌苔,接种于上述种子培养基中,于31.5℃,220rpm/min下振荡培养至对数生长中后期(12h左右),制得种子液;以10%的接种量将上述种子液接种至装有20ml发酵培养基的500ml摇瓶中,在31.5℃,振荡培养。在在糖被完全消耗之后,测定培养基中积累的L-谷氨酸的浓度。结果如表4所示(OD600是稀释100倍的培养液在600nm的浊度并表示细胞量,Glu(g/L)表示积累的L-谷氨酸的量)。
发现13869菌株不积累L-谷氨酸,在将odhA进行碱基缺失使菌体中α-酮戊二酸脱氢酶失活后,培养基中有谷氨酸的积累。
表4 谷氨酸的产量
OD600(×100) | Glu(g/L) | |
ATCC 13869 | 0.625 | 0.2 |
MHZ-0112-1 | 0.597 | 4.7 |
MHZ-0112-2 | 0.453 | 18.5 |
结果表明野生型谷氨酸棒杆菌ATCC 13869在未经改造前基本不产谷氨酸,在将编码通道蛋白的yggB基因修饰后,培养液中有少量谷氨酸积累,与野生菌株相比,该菌株显著(P<0.05)提高了谷氨酸的发酵产量;在将谷氨酸合成途径的竞争途径截断之后,谷氨酸的产量提高约4倍,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。
实施例3gdh,gltA基因表达的增强
(1)过表达载体pEC-gdh,pEC-gltA的构建
分别根据NCBI GenBank数据库中gdh基因(Gene Bank NO:NCgl 1084)的序列和gltA基因(Gene Bank NO:NCgl0795)的序列设计引物,如表5所示。
表5引物序列
以谷氨酸棒杆菌ATCC13869基因组作为模板,分别以C1/C2,D1/D2引物对扩增gdh及gltA两个基因的全长片段,经琼脂糖凝胶电泳及胶回收纯化后,将所得片段分别利用EcoRI/SalI双酶切,质粒pEC-XK99E(GI:29164935)也进行相同的双酶切,利用T4DNA连接酶进行连接,转化Trans1T1感受态细胞后筛选阳性克隆,提取质粒分别进行EcoRI/SalI双酶切鉴定,最后进行测序确认,所得质粒分别命名为pEC-gdh(测序结果如SEQ ID No.35所示。),pEC-gltA(测序结果如SEQ ID No.36所示。)。
(2)带有gdh,gltA基因高表达质粒的菌株谷氨酸生产能力及酶活测定的检测
利用与实施例1中所述感受态制备方法制备MHZ-0112-2的感受态细胞并将对照质粒pEC-XK99E及上述所构建pEC-gdh,pEC-gltA转入MHZ-0112-2菌株中。利用Kan15BHI平板筛选出阳性克隆,并提取质粒验证,所得到的三个带有复制性质粒的菌株分别命名为MHZ-0112-3,MHZ-0112-4,MHZ-0112-5。
将所得到的两个菌株及13869-yggBA106V-odhAda1菌株分别接种于含有表6所示成分的培养基中,于31℃下培养至对数生长期的后期,分别对菌体内谷氨酸脱氢酶(GDH)及柠檬酸合酶(CS)的表达量的测定,两个酶活的测定方法如下:
GDH酶活测定方法:将培养后的细菌在4℃下以10000rpm/min离心5min,收集菌体,用0.01mol/L磷酸盐缓冲液(PBS,pH7.4)洗一次,再重悬于1/10原菌液体积的PBS中,冰浴,超声破菌(350W,持续5s,间隔10s,共20次)后,10000rpm/min离心10min,去沉淀,得到粗酶溶液,以NADP+为辅酶进行测定,反应体系为:0.5mmol/L NADP+,15mmol/L谷氨酸,25mmol/LTris-HCl,pH8.0。取样品酶液测定GDH催化辅酶NADH+还原所引起的光吸收值在340nm波长处的变化,每分钟催化1 μmol NADP+还原所需的酶量定义为一个酶活力单位(U)。酶活=ΔOD340×0.2×2/0.0486,蛋白浓度=1.45×OD280-0.74×OD260,单位酶活=酶活/蛋白浓度。所有测定都在BECKMAN DU800分光光度计上完成。以Protien Assay(Bio-Rad)测定粗酶液中的蛋白质浓度,使用牛血清蛋白为标准蛋白。
CS酶活测定方法:离心培养液以分离菌体。以含有200mmol/L谷氨酸钠的50mmol/LTris缓冲液(pH 7.5)清洗细胞,然后再以相同溶液悬浮。冰浴,超声破菌(350W,持续5s,间隔10s,共20次)后,10000rpm/min离心10min,去沉淀,得到粗酶溶液,根据MethodsEnzymol.13,3-11(1969)测定柠檬酸合成酶的活性。具体而言,将此粗酶溶液加入含有100mmo/L Tris-HCl(pH 8),0.1mmol/L DTNB,200mmol/L谷氨酸钠,0.3mmol/L乙酞基辅酶A的反应液中,然后以分光光度计在30℃,412nm测定其吸光度增加,以此最为背景值,进一步加入草醋酸使其最终浓度为0.5mmol/L。测定在412nm处吸光值的增加,从背景值推算获得柠檬酸合成酶的酶活。以ProtienAssay(Bio-Rad)测定粗酶液中的蛋白质浓度,使用牛血清蛋白为标准蛋白。
表6 谷氨酸生产能力及酶活测定的检测
将pEC-gdh,pEC-gltA转入MHZ-0112-2菌株中,制得菌株MHZ-0112-4和MHZ-0112-5,谷氨酸的产量分别为23.4g/L和26.4g/L,与野生菌株相比,菌株MHZ-0112-4和MHZ-0112-5极显著(P<0.01)提高了谷氨酸的发酵产量。与MHZ-0112-3相比,MHZ-0112-4比MHZ-0112-3GDH表达量提高4倍,CS表达量基本持平,谷氨酸提高41.8%;MHZ-0112-5比MHZ-0112-3GDH表达量基本持平,CS表达量提高7.4倍,谷氨酸提高60%。MHZ-0112-4和MHZ-0112-5极显著(P<0.01)提高了谷氨酸的发酵产量。
(3)整合质粒pK18-Psodgdh,pK18-tufgltA的构建
以谷氨酸棒杆菌ATCC13869的基因组为模板,分别以C3/C4,C5/C6,C7/C8引物对(序列见表7)在Phusion超保真聚合酶的作用下PCR扩增,得到gdh起始密码子的上游同源臂、sod启动子和起始密码子的下游同源臂,PCR参数为98℃10s,50℃20s,72℃20s,共30个循环。将PCR产物用琼脂糖凝胶回收试剂盒进行割胶回收。以上述所得片段混合物为模板,以C3/C8引物对进行overlap PCR扩增,并割胶回收,产物用SalI和HindIII双酶切,pK18mobsacB用同样的酶进行切割,利用T4DNA连接酶在22℃下连接1-2h,转化Trans1 T1感受态细胞,挑取卡那抗性克隆,SalI/HindIII酶切鉴定得到overlap PCR片段***pK18mobsacB的阳性克隆,进一步通过测序鉴定***的片段确实为添加了Psod启动子的gdh基因上下游同源臂。所得到的质粒命名为pK18-Psodgdh(测序结果如SEQ ID No.37所示。)。
pK18-tufgltA(测序结果如SEQ ID No.38所示。)的构建过程与上述类似,所用引物对为D3/D4,D7/D8扩增gltA起始密码子的上下游同源臂,以D5/D6扩增启动子Ptuf,Overlap PCR所用引物对为D3/D8。
表7引物序列
(4)高表达菌株的制备
同实施例1中所述,制备MHZ-0112-2菌株的感受态细胞,分别将质粒pK18-Psodgdh,pK18-tufgltA进行转化。
在含有15mg/L的卡那霉素的选择培养基上选择单交换转化体,其中带有sod(tuf)启动子的序列通过与內源gdh(gltA)基因同源重组连同重组载体整合到基因组中。利用Fast Taq DNA聚合酶,以C3/T2,T1/C6引物对(或D3/T2,T1/D6引物对)进行菌落PCR鉴定KanR克隆,PCR参数同上,两个引物对均扩增出目的片段的克隆为阳性克隆。将挑选出的阳性克隆接种入无抗BHI培养基中培养12-14h,将菌液稀释100-1000倍后涂布在含有10%蔗糖的固体BHI培养基上培养36h,将所筛选出的菌株进一步进行卡那抗性表型验证,挑选KanS的重组子利用C3/C6引物对(或D3/D6引物对)扩增目的片段,阳性克隆的目的片段比野生型所扩增的片段大,将阳性克隆进行测序验证,得到sod启动子成功整合至gdh基因ATG之前的菌株,命名为MHZ-0112-6;测序结果如SEQ ID No.39所示。
得到tuf启动子成功整合至gltA基因ATG之前的菌株,命名为MHZ-0112-7。测序结果如SEQ ID No.40所示。
同理,制备MHZ-0112-6感受态细胞,将pK18-tufgltA进行转化,经两轮重组筛选,得到gdh与gltA叠加的突变株MHZ-0112-8,测序结果如SEQ ID No.41所示。
(5)高表达菌株相应酶活测定及谷氨酸产量确定
将所得到的菌株MHZ-0112-6,MHZ-0112-7及MHZ-0112-8接入BHI培养基中,于31℃下培养至对数生长期的后期,测定GDH及CS酶表达活性;将其接于实施例2中所述的发酵培养基中培养至糖消耗完全后,测定谷氨酸的产量及OD,各结果列于表8中。
表8 酶活测定及谷氨酸产量结果
如表8所示,在单独利用强启动子Psod表达gdh基因,GDH酶活较野生型提高约2.5倍,谷氨酸产量提高约1.8倍,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。
单独利用Ptuf启动子表达gltA基因将CS酶活增加了约4倍,谷氨酸产量提高至30.8g/L,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。
MHZ-0112-8与MHZ-0112-6、MHZ-0112-7相比,谷氨酸产量提高19.2%与14.6%。
将两个基因同时进行过表达后,谷氨酸产量继续提高至35.5g/L,转化率达到约59%,与野生菌株相比,该菌株极显著(P<0.01)提高了谷氨酸的发酵产量。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种谷氨酸棒杆菌(Corynebacterium glutamicum)重组菌株,其特征在于,其保藏编号为CGMCCNo.11941。
2.根据权利要求1所述的重组菌株在发酵生产谷氨酸中的应用。
3.一种发酵生产谷氨酸的方法,其特征在于,包括以如权利要求1所述的重组菌株为发酵菌株。
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