CN116355924A - 一种l-谷氨酸产量提高的谷氨酸棒杆菌的构建与应用 - Google Patents
一种l-谷氨酸产量提高的谷氨酸棒杆菌的构建与应用 Download PDFInfo
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Abstract
本发明公开了一种L‑谷氨酸产量提高的谷氨酸棒杆菌的构建与应用,属于基因工程和发酵工程领域。本发明敲除谷氨酸棒杆菌基因组上海藻糖合成基因treS,treY和otsA基因,得到了L‑谷氨酸产量提升的重组菌株C.glutamicum△SYA,该菌株在发酵培养基中发酵60h的L‑谷氨酸产量为14.75g/L,是出发菌株C.glutamicum ATCC13869的12.5倍,有助于实现L‑谷氨酸的工业化生产。
Description
技术领域
本发明涉及一种L-谷氨酸产量提高的谷氨酸棒杆菌的构建与应用,属于基因工程和发酵工程领域。
背景技术
L-谷氨酸,化学名称为α-氨基戊二酸,化学式为C5H9NO4,分子量为147.13,是一种酸性氨基酸。L-谷氨酸及其钠盐广泛应用于味精、香料、动物饲料和保健品的生产。
目前生产L-谷氨酸的主要方法是发酵法,用到的生产菌株主要是谷氨酸棒杆菌。谷氨酸棒杆菌具有独特的细胞壁结构,除了共价连接的肽聚糖和阿拉伯半乳聚糖外,还具有分枝菌酸层,该层结构与阿拉伯半乳聚糖层共价连接。分支菌酸层含有游离分支菌酸和分支菌酸糖脂,目前已知的能够与分支菌酸连接的糖有葡萄糖,海藻糖。分枝菌酸糖脂的减少会显著改变细胞的通透性,有利于氨基酸和其它高附加值的生物合成产物的外排。通过代谢工程改造获得谷氨酸棒杆菌分枝菌酸层精简的L-谷氨酸高产菌株具有重大的应用价值。
发明内容
为了解决上述存在的技术问题,本发明在谷氨酸棒杆菌中敲除谷氨酸棒杆菌基因组上海藻糖合成基因treS,treY,otsA得到突变菌谷氨酸棒杆菌△SYA,谷氨酸棒杆菌△SYA菌株在发酵培养基中,可以高效合成L-谷氨酸。
本发明的第一个目的是提供一种L-谷氨酸生产能力提高的重组谷氨酸棒杆菌,敲除或缺失了基因组上的海藻糖合成酶基因treS,麦芽寡糖基海藻糖合酶基因treY,海藻糖-6-磷酸合成酶基因otsA中的至少一个基因。
在一种实施方式中,所述海藻糖合成酶基因treS的核苷酸序列如SEQ ID NO.1所示;所述麦芽寡糖基海藻糖合酶基因treY的核苷酸序列如SEQ ID NO.2所示;所述海藻糖-6-磷酸合成酶基因otsA的核苷酸序列如SEQ ID NO.3所示。
在一种实施方式中,所述重组谷氨酸棒杆菌的出发菌株为谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13869。
本发明还提供了一种提高谷氨酸棒杆菌L-谷氨酸生产能力的方法,所述方法是抑制谷氨酸棒杆菌基因组上treS,treY,otsA中至少一个基因的表达。
在一种实施方式中,所述方法是敲除treS,treY和otsA基因。
本发明还提供所述重组谷氨酸棒杆菌在合成L-谷氨酸中的应用。
在一种实施方式中,所述应用是将所述重组谷氨酸棒杆菌在培养基中发酵生产L-谷氨酸。
在一种实施方式中,用于发酵的培养基含有:葡萄糖、玉米浆、酵母膏、(NH4)2SO4、KH2PO4、FeSO4、MgSO4、维生素B1、CaCO3。
在一种实施方式中,所述发酵是将所述重组谷氨酸棒杆菌在28~35℃,150~250rpm下发酵至少60h。
在一种实施方式中,所述重组谷氨酸棒杆菌生产L-谷氨酸的方法具体为:将所述重组谷氨酸棒杆菌在活化培养基中培养36小时,接种到种子培养基中,于29~35℃,150~250rpm培养12-18小时得到种子液,再将种子液以初始OD562为1的接种量接种于发酵培养基,置于29~35℃,150~250rpm条件下培养48~80小时。
本发明还提供所述重组谷氨酸棒杆菌,或上述方法在发酵、药物制备、材料或环保领域的应用。
有益效果:
本发明在谷氨酸棒杆菌中敲除谷氨酸棒杆菌基因组上海藻糖合成基因treS,treY,otsA,得到突变菌C.glutamicum△SYA,C.glutamicum△SYA菌株在发酵培养基中L-谷氨酸产量为14.75g/L,是C.glutamicum ATCC13869的12.5倍,该研究提供了一种提高谷氨酸棒杆菌中L-谷氨酸产量的新策略。
附图说明
图1为海藻糖合成酶基因treS,麦芽寡糖基海藻糖合酶基因treY,海藻糖-6-磷酸合成酶基因otsA的功能示意图。
图2为C.glutamicum△SYA的敲除质粒和构建过程。
图3为C.glutamicum ATCC13869和C.glutamicum△SYA的生长曲线、葡萄糖含量和L-谷氨酸的生产情况。
具体实施方式
(1)培养基:
LB培养基:5g/L酵母粉,10g/L蛋白胨,10g/L NaCl。
LBHIS培养基:2.5g/L酵母粉,5g/L蛋白胨,5g/L NaCl,18.5g/L脑心浸液。
EPO培养基:5g/L酵母粉,10g/L蛋白胨,10g/L NaCl,30g/L甘氨酸,0.1%Tween80。
活化培养基:10g/L胰蛋白胨,10g/L牛肉浸膏,5g/L酵母提取物,5g/L NaCl,5g/L葡萄糖。
种子培养基:25g/L葡萄糖,30g/L玉米浆,1.25g/L尿素,500mg/L(NH4)2SO4,1g/LKH2PO4,500mg/L MgSO4,pH:7.2。
发酵培养基:90g/L葡萄糖,15g/L玉米浆,1g/L酵母膏,40g/L(NH4)2SO4,1g/LKH2PO4,10mg/L FeSO4·7H2O,500mg/L MgSO4,10mg/L MnSO4·H2O,1mg/L thiamine HCl,20g/LCaCO3,pH:7.2。
(2)菌体浓度的测定
使用UV-1800紫外可见分光光度计测定在562nm处的吸光值。
(2)葡萄糖含量的测定
采用SBA-40生物分析仪分析发酵液中葡萄糖的含量,吸取25μL的标准液SBA进行标定,标定结束后吸取25μL稀释100倍后的发酵液进行测定。
(3)L-谷氨酸浓度分析:
L-谷氨酸浓度采用邻苯二甲醛柱前衍生法(Koros,A.,Varga,Z.,Molnar-Perl,I.2008.Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives incheese by high-performance liquid chromatography.J Chromatogr A,1203(2),146-52.)进行定量检测。使用Agilent 1200或1260系列高效液相色谱系统,配备Thermo 250mm×4.0mm ODS-2HYPERSIL C18色谱柱。
实施例1谷氨酸棒杆菌突变株C.glutamicum△S的构建
具体构建过程如下:
(1)敲除质粒pbs-treS的构建:
以C.glutamicum ATCC13869的基因组DNA作为模板,使用引物对treS-UF(TATACTGCAGCTTGGACAAAGGCTACCGT)和treS-UR(CGCCCTATAGTGAGTCGTATTACACATAGAAATCACCGTAGG)扩增左侧同源臂。使用引物对treS-DF(CTTTAGTGAGGGTTAATTGCGCTCACGGTTTGTTGCTGTCTC)和treS-DR(TAATAAGCTTTGCCAGAGGTAGTAGTCAGC)扩增右侧同源臂。将pDTW202(公开于公开号为CN106086056B的专利中)作为模板,使用引物对treS-kan-F(CCTACGGTGATTTCTATGTGTAATACGACTCACTATAGGGCG)和treS-kan-R(GAGACAGCAACAAACCGTGAGCGCAATTAACCCTCACTAAAG)扩增loxL-kan-loxR片段。然后将三个片段(左侧同源臂、loxL-kan-loxR、右侧同源臂)作为模板使用引物对treS-UF和treS-DR进行重叠延伸聚合酶链反应得到的约3.5kb的PCR产物。将该PCR产物和pBluescript II SK(+)分别用PstI和HindIII进行双酶切,再使用T4连接酶连接,以构建质粒pbs-treS。
(1)谷氨酸棒杆菌感受态细胞制备:
将C.glutamicum ATCC13869接种于LBHIS液体培养基中,30℃,200rpm过夜培养。按初始OD562为0.3转接到100mL EPO液体培养基,30℃,200rpm培养至OD562=0.8,将培养液冰浴半小时后转入预冷的50mL离心管中,4℃,4000rpm离心10分钟收集菌体,用预冷的10%甘油洗涤菌体3次,最后用1mL 10%甘油悬浮,每管100μL分装至预冷的无菌EP管中备用。
(3)Cre/LoxP构建突变株:
使用Cre-loxP系统进行treS基因的敲除。将步骤(1)构建的质粒pbs-treS电转到C.glutamicum ATCC13869感受态细胞中并筛选卡那霉素抗性克隆。通过菌落PCR使用引物对treS-YF(CGAATGGGTCCAGAAAAACAT)和treS-YR(CGTCGAAGTAGGCTGCGTAGT)进一步筛选loxL-kan-loxR正确插入C.glutamicum ATCC13869基因组中的克隆。第二步,将携带消除卡那霉素抗性的Cre基因的pDTW109电转到C.glutamicum△S::kan感受态细胞中,通过菌落PCR使用引物对treS-YF/treS-YR筛选经过双交换的同源重组克隆。将正确的克隆细胞接种到液体LBHIS培养基中在37℃下培养200rpm震荡培养以丢失pDTW109。培养物混浊后,在LBHIS板上划线并在30℃下培养。最后挑选单个菌落并在三个不同的LBHIS平板上依次划线:(1)不含抗生素;(2)卡那霉素;(3)使用氯霉素。对卡那霉素和氯霉素均敏感的菌落即为敲除了treS的菌株,命名为C.glutamicumΔS。
实施例2谷氨酸棒杆菌突变株C.glutamicum△SY的构建
具体构建过程如下:
(1)敲除质粒pbs-treY的构建:
以C.glutamicum ATCC13869的基因组DNA作为模板,使用引物对treY-UF(TATATCTAGATTAGATGAACAACCCCCGT)和treY-UR(CGCCCTATAGTGAGTCGTATTTGCCCAACTCGTGTGTAG)扩增左侧同源臂。使用引物对treY-DF(CTTTAGTGAGGGTTAATTGCGCAGCTGGTCAATCGTGTTTT)和treY-DR(ATATACTAGTCTTTCAGGCTTGACCATTCT)扩增右侧同源臂。将pDTW202作为模板,使用引物对treY-kan-F(CTACACACGAGTTGGGCAAATACGACTCACTATAGGGCG)和treY-kan-R(AAAACACGATTGACCAGCTGCGCAATTAACCCTCACTAAAG)扩增loxL-kan-loxR片段。然后将三个片段(左侧同源臂、loxL-kan-loxR、右侧同源臂)作为模板使用引物对treY-UF和treY-DR进行重叠延伸聚合酶链反应得到的约3.5kb的PCR产物。将该产物和pBluescript II SK(+)分别用XbaI和SpeI进行双酶切,使用T4连接酶连接两个片段以构建质粒pbs-treY。
(2)谷氨酸棒杆菌感受态细胞制备:
将C.glutamicumΔS接种于LBHIS液体培养基中,30℃,200rpm过夜培养。按初始OD562为0.3转接到100mL EPO液体培养基,30℃,200rpm培养至OD562=0.8,将培养液冰浴半小时后转入预冷的50mL离心管中,4℃,4000rpm离心10分钟收集菌体,沉淀用预冷的10%甘油洗涤3次,最后用1mL10%甘油悬浮,每管100μL分装至预冷的无菌EP管中备用。
(3)Cre/LoxP构建突变株:
使用Cre-loxP系统进行treY基因的敲除。将步骤(1)构建的质粒pbs-treY电转到C.glutamicum ATCC13869感受态细胞中并筛选卡那霉素抗性克隆。通过菌落PCR使用引物对treY-YF(TAAGAACGTGACTAAGAAGACC)和treY-YR(TTTCAACAGCACTTCAATGTC)进一步筛选loxL-kan-loxR正确插入实施例1构建的C.glutamicumΔS基因组中的克隆。第二步,将携带消除卡那霉素抗性的Cre基因的pDTW109电转到C.glutamicum△SY::kan感受态细胞中,通过菌落PCR使用引物对treY-YF/treY-YR筛选经过双交换的同源重组克隆。将正确的克隆细胞接种到液体LBHIS培养基中在37℃下培养200rpm震荡培养以丢失pDTW109。培养物混浊后,在LBHIS板上划线并在30℃下培养。最后挑选单个菌落并在三个不同的LBHIS平板上依次划线:(1)不含抗生素;(2)卡那霉素;(3)使用氯霉素。对卡那霉素和氯霉素均敏感的菌落即为敲除了treY的菌株,命名为C.glutamicumΔSY。
实施例3谷氨酸棒杆菌突变株C.glutamicum△SYA的构建
具体构建过程如下:
(1)敲除质粒pbs-otsA的构建:
以C.glutamicum ATCC13869的基因组DNA作为模板,使用引物对otsA-UF(TATATCTAGACAGGATTGTTGCCACCTATTC)和otsA-UR(CGCCCTATAGTGAGTCGTATTACGCTAACTCGCCTTCCG)扩增左侧同源臂。使用引物对otsA-DF(CTTTAGTGAGGGTTAATTGCGCAAACGAACAGCGAGCAGGT)和otsA-DR(TATACTCGAGTCGGAAACACGGATGAAGT)扩增右侧同源臂。将pDTW202作为模板,使用引物对otsA-kan-F(CGGAAGGCGAGTTAGCGTAATACGACTCACTATAGGGCG)和otsA-kan-R(ACCTGCTCGCTGTTCGTTTGCGCAATTAACCCTCACTAAAG)扩增loxL-kan-loxR片段。然后将三个片段(左侧同源臂、loxL-kan-loxR、右侧同源臂)作为模板使用引物对otsA-UF和otsA-DR进行重叠延伸聚合酶链反应得到的约3.5kb的PCR产物。将该产物和pBluescript II SK(+)分别用XbaI和SpeI进行双酶切,再用T4连接酶连接两个片段以构建质粒pbs-otsA。
(2)谷氨酸棒杆菌感受态细胞制备:
将C.glutamicumΔSY接种于LBHIS液体培养基中,30℃,200rpm过夜培养。按初始OD562为0.3转接到100mL EPO液体培养基,30℃,200rpm培养至OD562=0.8,将培养液冰浴半小时后转入预冷的50mL离心管中,4℃,4000rpm离心10分钟收集菌体,沉淀用预冷的10%甘油洗涤3次,最后用1mL 10%甘油悬浮,每管100μL分装至预冷的无菌EP管中备用。
(3)Cre/LoxP构建突变株:
使用Cre-loxP系统进行otsA基因的敲除。将步骤(1)构建的质粒pbs-otsA电转到C.glutamicum ATCC13869感受态细胞中并筛选卡那霉素抗性克隆。通过菌落PCR使用引物对otsA-YF(TGGTCCTCGGTGATTATTGC)和otsA-YR(GTCTACGTGTGCTGCTTGGG)进一步筛选loxL-kan-loxR正确插入C.glutamicumΔSY基因组中的克隆。第二步,将携带消除卡那霉素抗性的Cre基因的pDTW109电转到C.glutamicum△SYA::kan感受态细胞中,通过菌落PCR使用引物对otsA-YF/otsA-YR筛选经过双交换的同源重组克隆。将正确的克隆细胞接种到液体LBHIS培养基中在37℃下培养200rpm震荡培养以丢失pDTW109。培养物混浊后,在LBHIS板上划线并在30℃下培养。最后挑选单个菌落并在三个不同的LBHIS平板上依次划线:(1)不含抗生素;(2)卡那霉素;(3)使用氯霉素。对卡那霉素和氯霉素均敏感的菌落即为敲除了otsA的菌株,命名为C.glutamicumΔSYA。
实施例4C.glutamicumΔSYA合成L-谷氨酸的应用
将实施例3构建的菌株C.glutamicumΔSYA在活化培养基上培养36小时,挑取1大环菌苔接种到含有50mL种子培养基的500mL锥形瓶中,置于30℃,200rpm条件下培养18小时得到种子液,将种子液接种于50mL发酵培养基中使初始OD562=1,置于30℃,200rpm条件下培养60小时。测定每12小时的细胞生长、葡萄糖消耗和氨基酸浓度。以C.glutamicumATCC13869作为对照,按照上述相同方法培养。
结果如图3所示。对数早期时,C.glutamicum△SYA比C.glutamicum ATCC13869生长慢,但在平稳期达到更高的OD值。对应于生长速率,C.glutamicum△SYA在对数早期消耗葡萄糖的速度也比C.glutamicum ATCC13869慢,两者在60小时分别剩余3g和5.3g的葡萄糖。培养60小时后,C.glutamicum ATCC13869细胞仅产生1.18g/L L-谷氨酸,而C.glutamicum△SYA的L-谷氨酸产量显著提高,产量可达14.75g/L L-谷氨酸。
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 (10)
1.一种L-谷氨酸生产能力提高的重组谷氨酸棒杆菌,其特征在于,敲除或缺失了位于基因组上的海藻糖合成酶基因treS,麦芽寡糖基海藻糖合酶基因treY,海藻糖-6-磷酸合成酶基因otsA中的至少一个基因。
2.根据权利要求1所述的重组谷氨酸棒杆菌,其特征在于,所述海藻糖合成酶基因treS的核苷酸序列如SEQ ID NO.1所示;所述麦芽寡糖基海藻糖合酶基因treY的核苷酸序列如SEQ ID NO.2所示;所述海藻糖-6-磷酸合成酶基因otsA的核苷酸序列如SEQ ID NO.3所示。
3.根据权利要求1或2所述的重组谷氨酸棒杆菌,其特征在于,所述重组谷氨酸棒杆菌的出发菌株为谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13869。
4.一种提高谷氨酸棒杆菌L-谷氨酸生产能力的方法,其特征在于,抑制谷氨酸棒杆菌基因组上treS,treY,otsA中至少一个基因的表达。
5.根据权利要求4所述的方法,其特征在于,敲除treS,treY和otsA基因。
6.根据权利要求4或5所述的方法,其特征在于,所述谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13869。
7.一种生产L-谷氨酸的方法,其特征在于,将权利要求1~3任一所述的重组谷氨酸棒杆菌在培养基中发酵合成L-谷氨酸。
8.根据权利要求7所述的方法,其特征在于,所述发酵是将所述重组谷氨酸棒杆菌在28~35℃,150~250rpm下发酵至少60h。
9.根据权利要求7或8所述的方法,其特征在于,用于发酵的培养基含有:葡萄糖、玉米浆、酵母膏、(NH4)2SO4、KH2PO4、FeSO4、MgSO4、维生素B1和CaCO3。
10.权利要求1~3任一所述的重组谷氨酸棒杆菌,或权利要求4~9任一所述的方法在发酵、药物制备、材料或环保领域的应用。
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