CN116426449A - 用于生产谷氨酸的大肠杆菌基因工程菌及构建方法与应用 - Google Patents
用于生产谷氨酸的大肠杆菌基因工程菌及构建方法与应用 Download PDFInfo
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- CN116426449A CN116426449A CN202310301025.4A CN202310301025A CN116426449A CN 116426449 A CN116426449 A CN 116426449A CN 202310301025 A CN202310301025 A CN 202310301025A CN 116426449 A CN116426449 A CN 116426449A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种用于生产谷氨酸的大肠杆菌基因工程菌及构建方法与应用,菌株通过代谢工程改造方法获得,使用pbba‑j23105启动子强化Icd基因表达;使用trc启动子强化谷氨酸棒杆菌来源的gdha基因过表达;使用pbba‑j23110启动子表达来源于青春双歧杆菌的xfp基因;敲除了aceA、glnA基因,使用pbba‑j23117启动子替换了sucA、sucB基因原有启动子,弱化该基因表达;菌株具有较好的稳定性,通过发酵方法生产获得谷氨酸,对发酵营养要求较低,发酵条件温和、产品分离提取简单、发酵周期短的优点,为谷氨酸工业化提供新思路,具有实际应用价值。
Description
技术领域
本发明涉及生物技术生产领域,尤其是一种用于生产谷氨酸的大肠杆菌基因工程菌及构建方法与应用。
背景技术
谷氨酸是一种构成蛋白质的α-氨基酸,大量存在于谷类蛋白质及动物脑中,在生物体代谢过程中发挥着重要作用。谷氨酸用途广泛,在食品行业中,谷氨酸与碱作用可以生成谷氨酸钠,俗称味精,是重要的调味品;在医药行业中,人体吸收的谷氨酸容易在体内形成谷氨酰胺,提高大脑的机能;在日用化妆品行业中,谷氨酸经过脱羧处理生成N-酰基谷氨酸钠,可添加于香皂、洗洁精等日用品,具有很强的发泡和洗涤能力;在农产品行业中,谷氨酸铜可以用作产品杀菌剂,可以防止农作物腐败。谷氨酸已成为了世界上最大的氨基酸产品,年产量接近300万吨,产值超过400亿,市场前景广阔,潜力巨大。
目前,谷氨酸的生产菌种为谷氨酸棒状杆菌,国内现有谷氨酸生产菌株主要类型为温度敏感型和生物素亚适量型,传统温度敏感型菌株发酵谷氨酸时,玉米浆用量高,发酵易起泡,发酵后期易染菌,提取过程谷氨酸发酵液浓缩比低;传统生物素亚适量型菌株发酵谷氨酸时,需要严格控制生物素的含量,传统发酵生物素主要来源为玉米浆,不同批次玉米浆生物素含量不一,对发酵造成很大影响,产物收率低,工序多,产酸不稳定。
综上所述,传统的谷氨酸棒状杆菌对市场上谷氨酸工业化要求严格,且分子学改造复杂不稳定,因此急需一种高效、方便、安全、稳定的工程菌株。而大肠杆菌培养方便、操作简单、成本低廉、基础生物学、分子遗传学等方面的背景知识清晰,对其基因表达调控的分子机理也比较清楚,而且经历二十年的基因工程实践,大肠杆菌已经发展为一种安全的基因工程实验体系,有多种适用的寄主菌株和载体体系,大肠杆菌易于进行工业化批量生产。发酵工艺要求较谷氨酸棒状杆菌低,成为替代目前生产菌株的首选。
发明内容
本发明所要解决的技术问题在于提供一种用于生产谷氨酸的大肠杆菌基因工程菌。
本发明所要解决的技术问题在于提供上述用于生产谷氨酸的大肠杆菌基因工程菌的构建方法。
本发明所要解决的技术问题在于提供上述大肠杆菌基因工程菌的应用。
为解决上述技术问题,本发明的技术方案是:
一种用于生产谷氨酸的大肠杆菌基因工程菌,是利用代谢工程改造野生型E.coliMG1655得到的:敲除aceA基因,并在该位点整合了使用Pj23105启动子控制表达的Icd基因;和/或敲除了glnA基因,并在该位点整合了使用Ptrc启动子控制表达的gdha基因;和/或敲除了rph假基因位点,并在该位点整合了使用Pj23110启动子控制表达的xfp基因;和/或敲除了sucA、sucB基因原有的启动子,使用Pj23117启动子控制sucA、sucB基因共同表达。
即上述用于生产谷氨酸的大肠杆菌基因工程菌,是在出发菌株野生型E.coliMG1655基础上进行进一步改造获得的,具体为:
在aceA基因位点使用Pj23105启动子控制Icd基因过表达得到菌株Glu01;
以菌株Glu01为出发菌株,在glnA基因位点使用Ptrc启动子控制gdhA基因过表达,得到菌株Glu02;
以菌株Glu02为出发菌株,在rph假基因位点使用PJ23110启动子控制xfp基因表达得到菌株Glu03;
以菌株Glu03为出发菌株,敲除了串联基因sucA、sucB的原有启动子,使用Pj23117启动子控制sucA、sucB基因共同表达得到菌株Glu04。
上述菌株Glu04为改造后的最优目的菌。
优选的,上述用于生产谷氨酸的大肠杆菌基因工程菌,所述gdhA基因来源于谷氨酸棒杆菌;所述xfp基因来源于青春双歧杆菌;所述Icd、aceA、glnA、sucA、sucB、rph基因均为大肠杆菌自身来源基因。
上述谷氨酸生产菌株的定向改造方法,其中:
Pj23105启动子具有序列表SEQ ID NO.1所示核苷酸序列;
Pj23110启动子具有序列表SEQ ID NO.2所示核苷酸序列;
Pj23117启动子具有序列表SEQ ID NO.3所示核苷酸序列;
Ptrc启动子具有序列表SEQ ID NO.4所示核苷酸序列;
Icd基因具有序列表SEQ ID NO.5所示核苷酸序列;
gdhA基因具有序列表SEQ ID NO.6所示核苷酸序列;
xfp基因具有序列表SEQ ID NO.7所示核苷酸序列。
aceA基因具有序列表SEQ ID NO.8所示核苷酸序列。
glnA基因具有序列表SEQ ID NO.9所示核苷酸序列。
sucA基因具有序列表SEQ ID NO.10所示核苷酸序列。
sucB基因具有序列表SEQ ID NO.11所示核苷酸序列。
上述用于生产谷氨酸的大肠杆菌基因工程菌的构建方法,通过定向改造方法得到生产菌株,具体为:利用代谢工程改造方法调整Icd、aceA、glnA、sucA、sucB基因的表达强度,并引入了外源基因gdhA、xfp,其中:
(1)在aceA基因位点使用Pj23105启动子控制lcd基因过表达;
(2)在glnA基因位点使用Ptrc启动子控制gdhA基因过表达;
(3)在rph假基因位点使用PJ23110启动子控制xfp基因表达;
(4)敲除了串联基因sucA、sucB的原有启动子,使用Pj23117启动子控制sucA、sucB基因共同表达。
优选的,上述用于生产谷氨酸的大肠杆菌基因工程菌的构建方法,所述代谢工程改造方法为CRISPR-Cas9基因编辑技术。
上述大肠杆菌基因工程菌在生产谷氨酸方面的应用。
优选的,上述大肠杆菌基因工程菌的应用,通过发酵培养的方法生产谷氨酸,具体步骤如下:
(1)菌种活化:将基因工程菌使用接种环从甘油保菌管种接取3环菌液,在试管斜面固体培养基上均匀划开,于37℃培养箱中培养12h;
(2)种子培养:待菌种活化好以后,使用无菌生理盐水将菌体重悬,将所得菌液接种至5L搅拌式生物反应器中,培养温度为37℃,通过自动流加25%氨水溶液维持培养pH在7.0±0.2,通过调整搅拌转速或通风量维持培养溶氧值为45%,当OD600nm为25时达到接种要求;
(3)发酵培养:使用5L机械搅拌式发酵罐,接种量为20%,培养温度为37℃,通过自动流加25%氨水溶液维持培养pH在7.0±0.2,通过调整搅拌转速或通风量维持培养溶氧值为50%,通过流加80%葡萄糖溶液将罐内葡萄糖浓度控制在≤3g/L,发酵周期≤28h。
优选的,上述大肠杆菌基因工程菌的应用,所述步骤(1)中固体培养基为LB固体培养基。
优选的,上述大肠杆菌基因工程菌的应用,所述步骤(2)种子培养中所用的培养基组分为:葡萄糖25g/L,酵母2g/L,蛋白胨2g/L,,K2HPO4·3H2O 2g/L,MgSO4·7H2O 1g/L,MnSO4·H2O 5mg/L,生物素1mg/L,其余为水。
优选的,上述大肠杆菌基因工程菌的应用,所述步骤(3)发酵培养中所用的培养基组分为:葡萄糖20g/L,酵母粉4g/L,丝肽粉1g/L,(NH4)2SO4 3g/L,FeSO4.7H2O 10mg/L,MnSO4.H2O 15mg/L,MgSO4.7H2O 2g/L,KH2PO4 7g/L,Na2HPO4.12H2O 4g/L,生物素10μg/L,氯化胆碱0.4g/L;甜菜碱0.2g/L,VB1 2mg/L,VB5 2mg/L,CoCl2.6H2O10mg/L,其余为水。
有益效果:
上述用于生产谷氨酸的大肠杆菌基因工程菌,过定向改造方法获得,具有较好的稳定性,发酵生产谷氨酸过程中对发酵营养要求较低,发酵条件温和、产品分离提取简单、发酵周期短,与传统依赖谷氨酸棒杆菌生产谷氨酸的方法相比,所述大肠杆菌基因工程菌生产谷氨酸避免使用玉米浆、豆浓等芽孢含量高的复杂氮源,降低了染菌风险,同时对生物素亚适量的要求降低,产物外排能力强,单位时间生产效率提升明显,为谷氨酸的工业化生产提供了新的思路,具有实际应用意义。
附图说明
图1为制备谷氨酸工程菌的构建方法图解。
具体实施方式
为了使本领域的技术人员更好的理解本发明的技术方案,下面结合具体实施方式对本发明所述技术方案作进一步的详细说明。
实施例中涉及到的百分号“%”,若未特别说明,指质量百分比,溶液的百分比指100mL中含有溶质的克数,液体之间的百分比,是指在25℃时溶液的体积比例。
实施例中所涉及的基因序列如下:
Pj23105启动子具有序列表SEQ ID NO.1所示核苷酸序列;
Pj23110启动子具有序列表SEQ ID NO.2所示核苷酸序列;
Pj23117启动子具有序列表SEQ ID NO.3所示核苷酸序列;
Ptrc启动子具有序列表SEQ ID NO.4所示核苷酸序列;
lcd基因具有序列表SEQ ID NO.5所示核苷酸序列;
gdhA基因具有序列表SEQ ID NO.6所示核苷酸序列;
xfp基因具有序列表SEQ ID NO.7所示核苷酸序列;
aceA基因具有序列表SEQ ID NO.8所示核苷酸序列;
glnA基因具有序列表SEQ ID NO.9所示核苷酸序列;
sucA基因具有序列表SEQ ID NO.10所示核苷酸序列;
sucB基因具有序列表SEQ ID NO.11所示核苷酸序列。
实施例1
1.基因编辑的方法
采用的基因编辑方法参照文献(Li Y,Lin Z,Huang C,et al.Metabolicengineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting.Metabolic Engineering,2015,31:13-21.)。该方法涉及的工程质粒pREDCas9、pGRB,其中pREDCas9携带gRNA表达质粒pGRB的消除系统、λ噬菌体的Red重组系统、Cas9蛋白表达系统以及奇霉素抗性(工作浓度:100mg/L);pGRB以pUC18为骨架,包括gRNA-Cas9结合区域序列和终止子序列以及氨节青霉素抗性(工作浓度:100mg/L)。下列实施例2-4中涉及到的专业名词均可在该文章中解释。
2.菌株构建过程中用到的引物见表1。
表1菌株构建过程中所涉及的引物
实施例2
本实施例旨在解释说明基因敲除以及整合的步骤,以在aceA基因位点整合使用Pj23105启动子控制lcd基因为例,本发明所有分子改造操作步骤均可借鉴本实施例。如图1所示,具体步骤如下:
①以E.coliMG1655基因组为模板,分别以aceA-Up-s、aceA-Up-A、aceA-DN-S、aceA-DN-A与lcd-S、lcd-A为引物,通过HS酶PCR扩增获得到上游同源臂、下游同源臂、与目的基因片段,再以其为模板,通过HS酶重叠PCR获得Pj23105-lcd(aceA)基因整合片段,所述基因整合片段由aceA上游同源臂、Pj23105-lcd目的基因和aceA下游同源臂组成。
②以pGRB-aceA-S和pGRB-aceA-A为引物,通过PCR退火程序构建PGRB-aceA使用的含靶序列的DNA片段,并将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pGRB-aceA。
③将步骤②、③中得到的Pj23105-Icd(aceA)基因整合片段与pGRB-aceA质粒电转进入野生型E.coliMG1655菌株中,经过筛选获得阳性转化子,并命名为Glu01。
④以E.coliMG1655基因组为模板,分别以glnA-Up-s、glnA-Up-A、glnA-DN-S、glnA-DN-A与gdhA-S、gdhA-A为引物,通过HS酶PCR扩增获得到上游同源臂、下游同源臂、与目的基因片段,再以其为模板,通过HS酶重叠PCR获得Ptrc-gdhA(glnA)基因整合片段,所述基因整合片段由glnA上游同源臂、Ptrc-gdhA目的基因和glnA下游同源臂组成。
⑤以pGRB-glnA-S和pGRB-glnA-A为引物,通过PCR退火程序构建PGRB-glnA使用的含靶序列的DNA片段,并将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pGRB-glnA。
⑥将步骤④、⑤中得到的Ptrc-gdhA(glnA)基因整合片段与pGRB-glnA质粒电转进入野生型E.coliMG1655菌株中,经过筛选获得阳性转化子,并命名为Glu02。
⑦以E.coliMG1655基因组为模板,分别以rph-Up-s、rph-Up-A、rph-DN-S、rph-DN-A与Xfp-S、Xfp-A为引物,通过HS酶PCR扩增获得到上游同源臂、下游同源臂、与目的基因片段,再以其为模板,通过HS酶重叠PCR获得Pj23110-Xfp(rph)基因整合片段,所述基因整合片段由rph上游同源臂、Pj23110-Xfp目的基因和rph下游同源臂组成。
⑧以pGRB-rph-S和pGRB-rph-A为引物,通过PCR退火程序构建PGRB-rph使用的含靶序列的DNA片段,并将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pGRB-rph。
⑨将步骤⑦、⑧中得到的Pj23110-Xfp(rph)基因整合片段与pGRB-rph质粒电转进入野生型E.coliMG1655菌株中,经过筛选获得阳性转化子,并命名为Glu03。
⑩以E.coliMG1655基因组为模板,分别以suc-Up-s、suc-Up-A、suc-DN-S、suc-DN-A为引物,通过HS酶PCR扩增获得到上游同源臂、下游同源臂,再以其为模板,通过HS酶重叠PCR获得Pj23117-suc基因整合片段,所述基因整合片段由suc上游同源臂和suc下游同源臂组成。
以pGRB-suc-S和pGRB-suc-A为引物,通过PCR退火程序构建PGRB-suc使用的含靶序列的DNA片段,并将其化转至DH5α化转感受态细胞中,筛选获得阳性转化子,提取质粒pGRB-suc。
实施例3
本实施例旨在主要解释说明菌株Glu04的应用方法,即利用菌株Glu04发酵生产谷氨酸的操作步骤,实际上,该方法同样适用于本发明所述的所有菌株。具体为:
①菌种活化:使用接种环从甘油保菌管种接取3环Glu04菌株菌液,在试管斜面固体培养基上均匀划开,于37℃培养箱中培养12h。
②种子培养:待菌种活化好以后,使用无菌生理盐水将菌体重悬,将所得菌液接种至5L搅拌式生物反应器中,培养温度为37℃,通过自动流加25%氨水溶液维持培养pH在7.0±0.2,通过调整搅拌转速或通风量维持培养溶氧值为45%,当OD600nm为25时达到接种要求。
(3)发酵培养:将种子培养步骤得到的成熟种子液接入预先配置好的发酵培养基中,使用5L机械搅拌式发酵罐,接种量为20%,发酵培养温度为37℃,通过自动流加25%氨水溶液维持培养pH在7.0±0.2,通过调整搅拌转速或通风量维持培养溶氧值为50%,通过流加80%葡萄糖溶液将罐内葡萄糖浓度控制在≤3g/L,发酵周期≤28h。
其中,所述试管斜面固体培养基为LB固体培养基,即:酵母粉5g/L,蛋白胨10g/L,NaCl 5g/L,琼脂粉25g/L,其余为水。
所述种子培养所用培养基组分为:葡萄糖25g/L,酵母2g/L,蛋白胨2g/L,,K2HPO4·3H2O 2g/L,MgSO4·7H2O 1g/L,MnSO4·H2O 5mg/L,生物素1mg/L,其余为水。
所述发酵培养所用培养基组分为:葡萄糖20g/L,酵母粉4g/L,丝肽粉1g/L,(NH4)2SO4 3g/L,FeSO4.7H2O 10mg/L,MnSO4.H2O 15mg/L,MgSO4.7H2O 2g/L,KH2PO4 7g/L,Na2HPO4.12H2O 4g/L,生物素10μg/L,氯化胆碱0.4g/L;甜菜碱0.2g/L,VB12mg/L,VB5 2mg/L,CoCl2.6H2O 10mg/L,其余为水。
实施例4
本实施例旨在展示各菌株的谷氨酸生产能力,将实施例2中得到的各菌株以实施例3中的应用方法进行3次发酵。
结果显示,菌株Glu04经过28h发酵培养,能够积累52g/L谷氨酸,生产强度为1.85g/L/h。
其他各菌株Glu01、Glu02和Glu03对应发酵结果如下:
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,本发明菌株的构建步骤不分先后顺序,本技术领域技术人员以本发明的方法或以本方法为基础进行的菌种改造等改进和润饰均视为本发明的保护范围。
Claims (10)
1.一种用于生产谷氨酸的大肠杆菌基因工程菌,其特征在于:是利用代谢工程改造野生型E.col i MG1655得到的:敲除aceA基因,并在该位点整合了使用Pj23105启动子控制表达的Icd基因;和/或敲除了glnA基因,并在该位点整合了使用Ptrc启动子控制表达的gdha基因;和/或敲除了rph假基因位点,并在该位点整合了使用Pj23110启动子控制表达的xfp基因;和/或敲除了sucA、sucB基因原有的启动子,使用Pj23117启动子控制sucA、sucB基因共同表达。
2.根据权利要求1所述的用于生产谷氨酸的大肠杆菌基因工程菌,其特征在于:所述gdhA基因来源于谷氨酸棒杆菌;所述xfp基因来源于青春双歧杆菌;所述Icd、aceA、glnA、sucA、sucB、rph基因均为大肠杆菌自身来源基因。
3.根据权利要求1所述的用于生产谷氨酸的大肠杆菌基因工程菌,其特征在于:
Pj23105启动子具有序列表SEQ ID NO.1所示核苷酸序列;
Pj23110启动子具有序列表SEQ ID NO.2所示核苷酸序列;
Pj23117启动子具有序列表SEQ ID NO.3所示核苷酸序列;
Ptrc启动子具有序列表SEQ ID NO.4所示核苷酸序列;
Icd基因具有序列表SEQ ID NO.5所示核苷酸序列;
gdhA基因具有序列表SEQ ID NO.6所示核苷酸序列;
xfp基因具有序列表SEQ ID NO.7所示核苷酸序列。
aceA基因具有序列表SEQ ID NO.8所示核苷酸序列。
glnA基因具有序列表SEQ ID NO.9所示核苷酸序列。
sucA基因具有序列表SEQ ID NO.10所示核苷酸序列。
sucB基因具有序列表SEQ ID NO.11所示核苷酸序列。
4.权利要求1所述用于生产谷氨酸的大肠杆菌基因工程菌的构建方法,其特征在于:通过定向改造方法得到生产菌株,具体为:利用代谢工程改造方法调整Icd、aceA、glnA、sucA、sucB基因的表达强度,并引入了外源基因gdhA、xfp,其中:
(1)在aceA基因位点使用Pj23105启动子控制lcd基因过表达;
(2)在glnA基因位点使用Ptrc启动子控制gdhA基因过表达;
(3)在rph假基因位点使用PJ23110启动子控制xfp基因表达;
(4)敲除了串联基因sucA、sucB的原有启动子,使用Pj23117启动子控制sucA、sucB基因共同表达。
5.根据权利要求4所述的用于生产谷氨酸的大肠杆菌基因工程菌的构建方法,其特征在于:所述代谢工程改造方法为CRISPR-Cas9基因编辑技术。
6.权利要求1所述大肠杆菌基因工程菌在生产谷氨酸方面的应用。
7.根据权利要求6所述的大肠杆菌基因工程菌的应用,其特征在于:通过发酵培养的方法生产谷氨酸,具体步骤如下:
(1)菌种活化:将基因工程菌使用接种环从甘油保菌管种接取3环菌液,在试管斜面固体培养基上均匀划开,于37℃培养箱中培养12h;
(2)种子培养:待菌种活化好以后,使用无菌生理盐水将菌体重悬,将所得菌液接种至5L搅拌式生物反应器中,培养温度为37℃,通过自动流加25%氨水溶液维持培养pH在7.0±0.2,通过调整搅拌转速或通风量维持培养溶氧值为45%,当OD600nm为25时达到接种要求;
(3)发酵培养:使用5L机械搅拌式发酵罐,接种量为20%,培养温度为37℃,通过自动流加25%氨水溶液维持培养pH在7.0±0.2,通过调整搅拌转速或通风量维持培养溶氧值为50%,通过流加80%葡萄糖溶液将罐内葡萄糖浓度控制在≤3g/L,发酵周期≤28h。
8.根据权利要求7所述的大肠杆菌基因工程菌的应用,其特征在于:所述步骤(1)中固体培养基为LB固体培养基。
9.根据权利要求7所述的大肠杆菌基因工程菌的应用,其特征在于:所述步骤(2)种子培养中所用的培养基组分为:葡萄糖25g/L,酵母2g/L,蛋白胨2g/L,,K2HPO4·3H2O 2g/L,MgSO4·7H2O 1g/L,MnSO4·H2O 5mg/L,生物素1mg/L,其余为水。
10.根据权利要求7所述的大肠杆菌基因工程菌的应用,其特征在于:所述步骤(3)发酵培养中所用的培养基组分为:葡萄糖20g/L,酵母粉4g/L,丝肽粉1g/L,(NH4)2SO4 3g/L,FeSO4.7H2O 10mg/L,MnSO4.H2O 15mg/L,MgSO4.7H2O 2g/L,KH2PO4 7g/L,Na2HPO4.12H2O 4g/L,生物素10μg/L,氯化胆碱0.4g/L;甜菜碱0.2g/L,VB1 2mg/L,VB52mg/L,CoCl2.6H2O 10mg/L,其余为水。
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