CN104531597B - 一株产l‑苯丙氨酸的重组谷氨酸棒状杆菌及其构建与应用 - Google Patents
一株产l‑苯丙氨酸的重组谷氨酸棒状杆菌及其构建与应用 Download PDFInfo
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Abstract
本发明公开了一株产L‑苯丙氨酸的重组谷氨酸棒状杆菌及其构建与应用,属于代谢工程领域。本发明在谷氨酸棒杆菌模式菌株Corynebacterium glutamcium ATCC 13032中,使用谷氨酸棒杆菌与大肠杆菌的两个穿梭表达载体pEC‑XK99E和pXMJ19表达L‑Phe合成途径中八个关键酶基因:aroFfbr,tktA,ppsA,aroL,pheAfbr,aroE,aroA,tyrB,并通过使用两个不同强度的启动子Ptac和Plac对八个基因进行组合表达提高L‑Phe产量,L‑Phe产量最高达到5.59±0.11g/L,莽草酸积累为0.31±0.11g/L。本发明提供了一种通过过量表达L‑Phe合成途径关键酶基因,提高谷氨酸棒杆菌发酵生产L‑Phe的方法。
Description
技术领域
本发明涉及一株产L-苯丙氨酸的重组谷氨酸棒状杆菌及其构建与应用,属于代谢工程领域。
背景技术
苯丙氨酸(Phenylalanine,Phe),即D.L-α-氨基-β-苯基丙酸,是一种芳香族杂环、非极性、电中性的氨基酸,有外消旋DL-型、L-型和D-型三种,具有生物活性的光学异构体为L-苯丙氨酸(L-Phe),比旋光度为-35.1°。L-Phe在生物体内可被辅酶四氢生物喋呤不可逆地转化为L-酪氨酸(L-Tyrosine,L-Tyr),后继续分解,经转氨基生成少量苯丙酮酸。L-Phe广泛存在于自然界中,是人体和动物所必须的8种氨基酸之一,而D-Phe在自然界中并不存在,只有通过合成的方法获得。
L-Phe的生产技术主要包括天然蛋白质水解法、化学合成法、酶法和微生物发酵法。其中天然蛋白质水解法由于其生产工艺相对复杂,产品质量不稳定,难以用于工业化生产。化学合成法因其生产路线长、副产物多且产物为消旋不宜推广使用等缺点,因而逐渐被淘汰。由于酶法具有生产工艺简单、产物浓度高、纯化步骤简单等特点是目前工业化L-Phe的主要生产方式之一,微生物发酵法具有原料廉价易得、环境污染较小,产物浓度高等优点成为国内外工业化生产L-Phe的主要方法。
谷氨酸棒杆菌中L-Phe合成途径主要分为三个部分:1、中心碳源代谢途径提供两个前体物质,来源于糖酵解途径的磷酸烯醇式丙酮酸(Phophoenol pyruvate,PEP)以及来源于磷酸戊糖途径的4-磷酸-赤藓糖(Erythrose-4-phosphate,E4P);2、莽草酸途径是芳香族氨基酸合成的共同代谢途径;3、分支酸路径,以分支酸(Chorismate)为节点通过不同的酶作用分别流向三种不同的芳香族氨基酸L-Phe,L-酪氨酸和L-色氨酸。
虽然大肠杆菌发酵生产L-苯丙氨酸能到很高的水平,但大肠杆菌由于其自身的特点而在大工业化生产中受到了限制,尤其在制备食品级的大工业化生产中。这是因为大肠杆菌本身是一种条件致病菌,所生产的重组蛋白、有机酸等产物中残留有大肠杆菌的相关抗原,从而易引起人或其他动物的免疫反应,这是公众卫生所不能接受的;此外,大肠杆菌是一种很好的噬菌体宿主菌,在大工业化发酵生产中极易遭受噬菌体的感染。
谷氨酸棒杆菌是一种食品级的微生物,本发明旨在提供一种L-苯丙氨酸产量提高的重组谷氨酸棒状杆菌。
发明内容
本发明要解决的第一个技术问题是提供一株产L-苯丙氨酸的重组谷氨酸棒状杆菌,是在谷氨酸棒状杆菌(Corynebacterium glutamicum)中过量表达了八个L-Phe合成途径的八个关键酶基因:aroFfbr(3-脱氧-D***庚酮糖-7磷酸合酶),aroE(莽草酸脱氢酶),ppsA(磷酸烯醇式丙酮酸合成酶)、tktA(转酮酶)、pheAfbr(双功能酶分支酸变位酶/预苯酸脱水酶),aroA(5-烯醇丙酮酸莽草酸-3-磷酸合酶),tyrB(氨基转移酶)和aroL(莽草酸激酶)。
在本发明的一种实施方式中,所述谷氨酸棒状杆菌是谷氨酸棒状杆菌ATCC13032。
在本发明的一种实施方式中,aroFfbr的核苷酸序列如SEQ ID NO.1所示,aroE的核苷酸序列如Gene ID:3343183所示,ppsA的核苷酸序列如Gene ID:14791674所示,tktA的核苷酸序列如Gene ID:3343601所示,pheAfbr的核苷酸序列如SEQ ID NO.2所示,aroA的核苷酸序列如Gene ID:3345010,tyrB的核苷酸序列如Gene ID:12933673所示,aroL的核苷酸序列如Gene ID:12930837所示。
在本发明的一种实施方式中,aroFfbr,aroE,ppsA和tktA与pEC-XK99E连接后转化谷氨酸棒状杆菌;pheAfbr,aroA,tyrB和aroL与pXMJ19连接后转化谷氨酸棒状杆菌。
在本发明的另一种实施方式中,aroFfbr与aroE融合,ppsA与tktA融合,连接表达载体pEC-XK99E后,在aroFfbr-aroE、ppsA-tktA前分别***启动子Ptac和Plac构建重组质粒pEC-XK99E-Ptac-aroFfbr-aroE-Plac-ppsA-tktA,简写为pSUTL;pheAfbr与aroA融合,tyrB与aroL融合,连接表达载体pXMJ19后,在pheAfbr-aroA、tyrB-aroL前分别***启动子Ptac和Plac构建重组质粒pXMJ19-Ptac-pheAfbr-aroA-Plac-tyrB-aroL,简写为pSDTL。
所述启动子Ptac由Trp启动子和Plac启动子杂合而成,受IPTG诱导,具有更高的转录效率。
所述启动子Plac受IPTG诱导,其强度约为Ptac启动子强度的1/11。
本发明要解决的第二个技术问题是提供一种构建所述产L-苯丙氨酸的谷氨酸棒状杆菌的方法,是将aroFfbr、aroE、ppsA、tktA连接到一个表达载体,将pheAfbr、aroA、tyrB和aroL连接到一个表达载体,共同转化谷氨酸棒状杆菌。
在本发明的一种实施方式中,aroFfbr,aroE,ppsA和tktA与pEC-XK99E连接后转化谷氨酸棒状杆菌;pheAfbr,aroA,tyrB和aroL与pXMJ19连接后转化谷氨酸棒状杆菌。
在本发明的另一种实施方式中,aroFfbr与aroE融合,ppsA与tktA融合,连接表达载体pEC-XK99E后,在aroFfbr-aroE、ppsA-tktA前分别***启动子Ptac和Plac构建重组质粒pEC-XK99E-Ptac-aroFfbr-aroE-Plac-ppsA-tktA,简写为pSUTL;pheAfbr与aroA融合,tyrB与aroL融合,连接表达载体pXMJ19后,在pheAfbr-aroA、tyrB-aroL前分别***启动子Ptac和Plac构建重组质粒pXMJ19-Ptac-pheAfbr-aroA-Plac-tyrB-aroL,简写为pSDTL。
本发明要解决的第三个技术问题是提供一种应用所述产L-苯丙氨酸的重组谷氨酸棒状杆菌发酵生产L-Phe的方法,是将种子培养基接入发酵培养基中,同时加入1.0mMIPTG诱导质粒表达重组酶,置于巡回式摇床(200-300r/min)上,30℃发酵培养60-80h。
种子活化培养基(LBG)(g/L):蛋白胨10.0,酵母膏5.0,氯化钠10.0,葡萄糖5.0,装液量20mL/250mL。
种子活化培养基(LBG固体)(g/L):葡萄糖5.0,酵母粉5.0,蛋白胨10.0,氯化钠10.0,营养琼脂15.0~20.0。
发酵种子培养基(g/L):葡萄糖25.0,玉米浆干粉17.5,硫酸铵5.0,硫酸镁0.5,磷酸二氢钾1.0,尿素2.0,pH 6.8-7.0。装液量20mL/250mL。
发酵培养基(g/L):葡萄糖100.0,玉米浆干粉6.0,硫酸铵25.0,硫酸镁0.5,磷酸二氢钾1.0,柠檬酸钠2.0,碳酸钙20.0,pH 6.8-7.0。装液量20mL/250mL。
谷氨酸棒杆菌培养基根据需要添加对应的抗生素:氯霉素(17mg/L);硫酸卡那霉素(25mg/L),IPTG添加量终浓度为1.0mM,发酵诱导时间为0h诱导。
种子培养:接种一环LBG平板种子于发酵种子培养基,置于巡回式摇床(200r/min)上,30℃培养18h。
发酵培养:按10%接种量将种子培养基接入发酵培养基中,同时加入1.0mM IPTG诱导质粒表达重组酶,置于巡回式摇床(200r/min)上,30℃发酵培养72h。
本发明的有益效果:本发明在谷氨酸棒杆菌模式菌株C.glutamiucm ATCC 13032中,采用两个质粒pEC-XK99E和pXMJ19对L-Phe合成途径不同来源的八个关键酶基因(aroFfbr,tktA,ppsA,aroL,pheAfbr,aroE,aroA,tyrB)结合两个启动子Ptac和Plac进行调控表达,通过理性的改造提高谷氨酸棒状杆菌模式菌株ATCC 13032中L-Phe产量,获得了一株重组谷氨酸棒状杆菌C.glutamicum(pSUTL,pSDTL),最终通过诱导表达C.glutamicum(pSUTL,pSDTL)L-Phe产量最高为5.59±0.11g/L,莽草酸积累为0.31±0.11g/L,出发菌株ATCC 13032L-Phe产量为0.16±0.05g/L,莽草酸为0.29±0.02g/L,结果表明可以通过代谢工程的手段过量表达L-Phe合成代谢途径中的关键酶基因并结合调控表达的策略,构建出L-Phe的产生菌株,同时可以应用于提高L-Phe产生菌株L-Phe的产量。
附图说明
图1:调控表达载体pSUTL和pSDTL构建流程示意图。
图2:PCR以及融合PCR获取莽草酸上游基础质粒pSU组合基因
1:DL 1000DNA marker;2:DL 1000DNA marker;3:ppsA-tktA;4:tktA;5:ppsA;6:aroFfbr-aroE;7:aroE;8:aroFfbr
图3:莽草酸上游基础质粒pSU酶切验证
1:pEC-XK99E-aroFfbr-aroE-ppsA-tktA酶切aroFfbr-aroE;
2:pEC-XK99E-ppsA-tktA酶切ppsA-tktA;3:DL 10000DNAmarker
图4:PCR以及融合PCR获取莽草酸下游基础质粒pSD组合基因
1:tyrB-aroL;2:aroL;3:tyrB;4:pheAfbr-aroA;5:aroA;6:pheAfbr;7:DL2000DNAmarker
图5:莽草酸下游基础质粒pSD酶切验证
1:pSD pXMJ19-pheAfbr-aroA-tyrB-aroL双切pheAfbr-aroA;
2:pSD pXMJ19-pheAfbr-aroA-tyrB-aroL双切tyrB-aroL和双切tyrB-aroL与pheAfbr-aroA;
3:DL1000DNAmarker
图6:重组谷氨酸棒状杆菌C.glutamicum(pSUTL,pSDTL)与C.glutamicum ATCC13032发酵L-Phe以及莽草酸(Shikimate)积累量
A:菌株C.glutamicum ATCC 13032;B:菌株C.glutamicum(pSUTL,pSDTL)
具体实施方式
L-Phe的测定:高效液相色谱(HPLC)。
仪器:Agilent 1200高效液相色谱仪(配紫外可见检测器和工作站)。
色谱柱:Thermo ODS-2HYPERSIL 4.6*250mm。
流动相:A(1L):无水醋酸钠5.0g,5mL四氢呋喃,200μL三乙胺,pH 7.2。B:无水醋酸钠5.0g(定容200mL,调节pH 7.2)抽虑后加入400mL乙腈和400mL甲醇。
色谱条件:流速,1mL/min;柱温,40℃;进样量,10μL;紫外检测器波长,338nm;在线衍生化进样,梯度洗脱程序如下表所示:
氨基酸样品分析处理:
取待分析的样品,在10,000rpm常温离心10min,取上清液使用5%三氯乙酸稀释到合适的浓度,经0.45μm滤膜过滤转移到1.5mL离线管中,10,000rpm常温离心10min供氨基酸液相分析用。
实施例1 重组质粒pSUTL和pSDTL构建,以及重组菌株C.glutamicum(pSUTL,pSDTL)构建。
以pEC-XK99E(GenBank:AY219683.1)过量表达四个基因aroFfbr,aroE,ppsA和tktA,同时结合两个启动子Ptac和Plac构建重组质粒pECXK99E-Ptac-aroFfbr-aroE-Plac-ppsA-tktA简写为pSUTL。以pXMJ19(Jakoby M,Ngouoto-Nkili CE,BurkovskiA.Construction and application of new Corynebacterium glutamicum vectors[J].Biotechnology techniques.1999.13(6):437-441)过量表达四个基因pheAfbr、aroA、tyrB和aroL,同时结合两个启动子Ptac和Plac构建重组质粒pXMJ19-Ptac-pheAfbr-aroA-Plac-tyrB-aroL简写为pSDTL。
根据构建调控表达载体pSUTL和pSDTL构建流程示意图1所示,aroE、ppsA、tktA和aroA来源于C.glutamicum ATCC 13032基因组;tyrB和aroL来源于Escherichia coliW3110基因组,以对应的模板分别单独获取八个关键酶基因(aroFfbr和pheAfbr基因序列见序列表,aroE(Gene ID:3343183)、ppsA(Gene ID:14791674)、tktA(Gene ID:3343601)、aroA(Gene ID:3345010)、tyrB(Gene ID:12933673)、aroL(Gene ID:12930837),PCR反应条件为:预变性94℃5min;变性94℃30s,退火Tm(根据实际设计的引物而定)30s,延伸72℃(时间根据PCR获取基因的长度而定)(返回到变性步骤重复30个循环);后延伸72℃10min,并以1%琼脂糖凝胶电泳验证并回收PCR扩增产物,结果为扩增得到基因片段与设计的一致见图2和图3所示,进一步构建融合基因aroFfbr-aroE,ppsA-tktA,pheAfbr-aroA和tyrB-aroL(图2和图3)。将aroFfbr-aroE、ppsA-tktA与pEC-XK99E酶切连接,将pheAfbr-aroA、tyrB-aroL与pXMJ19酶切连接,最终获取八个基因的基础调控质粒pSU和pSD,酶切验证图分别见图3和图5,进一步对基础质粒pSU和pSD按照图1所示进行酶切两次,在aroFfbr-aroE、ppsA-tktA前分别***启动子Ptac(SEQ ID NO.3)和Plac(SEQ ID NO.4),在pheAfbr-aroA、tyrB-aroL前分别***启动子Ptac和Plac,构建获得到重组质粒pSUTL和pSDTL。获取的两个重组质粒pSUTL和pSDTL,通过电击转化到出发菌株C.glutamicum ATCC 13032中,通过菌落PCR验证,获得阳性重组子重组菌株C.glutamicum(pSUTL,pSDTL)。
谷氨酸棒状杆菌电击转化:
(1)-80℃保存的谷氨酸棒状杆菌感受态(Xu D,Tan Y,Huan X,Hu X,WangX.Construction of a novel shuttle vector for use in Brevibacterium flavum,anindustrial amino acid producer[J].Journal ofmicrobiological methods.2010.80(1):86-92),冰浴中融化。
(2)加入1-5μL预冷质粒混匀(DNA总量约为1μg),冰浴上放置5-10min。
(3)加入于预冷的0.1cm电击杯中,1.8KV 5ms电击2次。
(4)迅速加入预热的恢复用培养基(LBHIS)1mL混匀并转移到新的1.5mL无菌离心管中,46℃水浴6min,后放入冰浴中。
(5)将菌体置于巡回式摇床(100r/min)上,30℃后培养2h。
(6)6,000rpm,常温离心1min,涂布到加入对应抗性的转化子检出平板中,于30℃恒温培养箱,培养2-3天。
(7)感受态效率的验证:阴性对照加入5μL无菌的ddH2O,无菌落形成,阳性对照加入0.1μL的质粒pXMJ19或者pEC-XK99E,长出大量菌落。
谷氨酸棒状杆菌重组子验证:
将检出平板中的转化子,使用白色无菌枪头挑选单菌落,点种到新的相同抗生素的LBG平板中,置于30℃恒温培养箱培养12h,同时点种到菌落PCR体系中,进行PCR验证之后进行电泳,挑选正确的重组子,对平板中对应的重组子进行转接到LBG液体培养基中,培养12h,提取重组质粒,使用限制性内切酶酶切,DNA琼脂糖凝胶电泳鉴定。
实施例2 重组谷氨酸棒状杆菌C.glutamicum(pSUTL,pSDTL)过量表达L-Phe合成途径八个关键酶基因对L-Phe发酵的影响
种子活化培养基(LBG)(g/L):蛋白胨10.0,酵母膏5.0,氯化钠10.0,葡萄糖5.0。装液量20mL/250mL。
种子活化培养基(LBG固体)(g/L):葡萄糖5.0,酵母粉5.0,蛋白胨10.0,氯化钠10.0,营养琼脂15.0~20.0。
发酵种子培养基(g/L):葡萄糖25.0,玉米浆干粉17.5,硫酸铵5.0,硫酸镁0.5,磷酸二氢钾1.0,尿素2.0,pH 6.8-7.0。装液量20mL/250mL。
发酵培养基(g/L):葡萄糖100.0,玉米浆干粉6.0,硫酸铵25.0,硫酸镁0.5,磷酸二氢钾1.0,柠檬酸钠2.0,碳酸钙20.0,pH 6.8-7.0。装液量20mL/250mL。
谷氨酸棒杆菌培养基根据需要添加对应的抗生素:氯霉素(17mg/L);硫酸卡那霉素(25mg/L),IPTG添加量终浓度为1.0mM,发酵诱导时间为0h诱导。
种子培养:接种一环LBG平板种子于发酵种子培养基,置于巡回式摇床(200r/min)上,30℃培养18h。
发酵培养:按10%接种量将种子培养基接入发酵培养基中,同时加入1.0mM IPTG诱导质粒表达重组酶,置于巡回式摇床(200r/min)上,30℃发酵培养72h。
重组谷氨酸棒状杆菌C.glutamicum(pSUTL,pSDTL)与出发菌株C.glutamicumATCC13032进行发酵实验对比。重组菌C.glutamicum(pSUTL,pSDTL)进行诱导发酵72h L-Phe最高产量达到最大值5.59±0.11g/L,莽草酸积累为0.31±0.11g/L,出发菌株C.glutamicum ATCC13032L-Phe产量为0.16±0.05g/L莽草酸为0.29±0.02g/L(图6)。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (8)
1.一株产L-苯丙氨酸的重组谷氨酸棒状杆菌,其特征在于,是在谷氨酸棒状杆菌(Corynebacterium glutamicum)中过量表达了编码3-脱氧-D***庚酮糖-7磷酸合酶的aroFfbr、编码莽草酸脱氢酶的aroE,编码磷酸烯醇式丙酮酸合成酶的ppsA、编码转酮酶的tktA、编码分支酸变位酶/预苯酸脱水酶双功能酶的pheAfbr、编码5-烯醇丙酮酸莽草酸-3-磷酸合酶的aroA、编码氨基转移酶的tyrB和编码莽草酸激酶的aroL;aroFfbr的核苷酸序列如SEQ ID NO.1所示,aroE的核苷酸序列如Gene ID:3343183所示,ppsA的核苷酸序列如Gene ID:14791674所示,tktA的核苷酸序列如Gene ID:3343601所示,pheAfbr的核苷酸序列如SEQ ID NO.2所示,aroA的核苷酸序列如Gene ID:3345010,tyrB的核苷酸序列如GeneID:12933673所示,aroL的核苷酸序列如Gene ID:12930837所示;aroFfbr、aroE、ppsA和tktA与pEC-XK99E连接后转化谷氨酸棒状杆菌;pheAfbr、aroA、tyrB和aroL与pXMJ19连接后转化谷氨酸棒状杆菌。
2.根据权利要求1所述的重组谷氨酸棒状杆菌,其特征在于,以谷氨酸棒状杆菌ATCC13032为出发菌株。
3.根据权利要求1所述的重组谷氨酸棒状杆菌,其特征在于,aroFfbr与aroE融合,ppsA与tktA融合,连接表达载体pEC-XK99E后,在aroFfbr-aroE、ppsA-tktA前分别***启动子Ptac和Plac构建重组质粒pEC-XK99E-Ptac-aroFfbr-aroE-Plac-ppsA-tktA;pheAfbr与aroA融合,tyrB与aroL融合,连接表达载体pXMJ19后,在pheAfbr-aroA、tyrB-aroL前分别***启动子Ptac和Plac构建重组质粒pXMJ19-Ptac-pheAfbr-aroA-Plac-tyrB-aroL;将重组质粒转化宿主,筛选阳性克隆。
4.一种构建权利要求1所述重组谷氨酸棒状杆菌的方法,是将aroFfbr与aroE融合,ppsA与tktA融合,连接表达载体pEC-XK99E后,在aroFfbr-aroE、ppsA-tktA前分别***启动子Ptac和Plac构建重组质粒pEC-XK99E-Ptac-aroFfbr-aroE-Plac-ppsA-tktA;将pheAfbr与aroA融合,tyrB与aroL融合,连接表达载体pXMJ19后,在pheAfbr-aroA、tyrB-aroL前分别***启动子Ptac和Plac构建重组质粒pXMJ19-Ptac-pheAfbr-aroA-Plac-tyrB-aroL;将两重组质粒转化谷氨酸棒状杆菌,筛选阳性克隆。
5.一种应用权利要求1所述重组谷氨酸棒状杆菌生产L-Phe的方法,是将活化的种子接入发酵培养基中,同时加入IPTG诱导质粒表达重组酶,通风发酵培养60-80h。
6.根据权利要求5所述的方法,其特征在于,所述发酵培养基按g/L计含有:葡萄糖100.0,玉米浆干粉6.0,硫酸铵25.0,硫酸镁0.5,磷酸二氢钾1.0,柠檬酸钠2.0,碳酸钙20.0;pH 6.8-7.0。
7.根据权利要求6所述的方法,其特征在于,IPTG添加量终浓度为1.0mM。
8.权利要求1所述重组谷氨酸棒状杆菌在生产L-苯丙氨酸中的应用。
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