CN113667627A - 一种提高l-谷氨酸生产效率的谷氨酸棒杆菌的构建及应用 - Google Patents
一种提高l-谷氨酸生产效率的谷氨酸棒杆菌的构建及应用 Download PDFInfo
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- CN113667627A CN113667627A CN202110992844.9A CN202110992844A CN113667627A CN 113667627 A CN113667627 A CN 113667627A CN 202110992844 A CN202110992844 A CN 202110992844A CN 113667627 A CN113667627 A CN 113667627A
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Abstract
本发明公开了一种提高L‑谷氨酸生产效率的谷氨酸棒杆菌的构建及应用,属于基因工程和发酵工程领域。本发明在谷氨酸棒杆菌中敲除谷氨酸棒杆菌基因组上分枝菌酸酮酰基还原酶基因cmrA,使获得的C.glutamicum△cmrA在发酵培养基中L‑谷氨酸产量为13.14g/L,是出发菌株C.glutamicum ATCC 13869产量的10.77倍。本发明的方法提供了一种提高谷氨酸棒杆菌中氨基酸产量的新策略,在氨基酸的工业生产中有巨大的应用前景。
Description
技术领域
本发明涉及一种提高L-谷氨酸生产效率的谷氨酸棒杆菌的构建及应用,属于基因工程和发酵工程领域。
背景技术
L-谷氨酸是生物机体内氮代谢的基本氨基酸之一,可用于治疗肝性昏迷。另外,L-谷氨酸广泛应用于味精、香料、动物饲料和保健品的生产。
谷氨酸棒杆菌是重要的工业微生物,在L-谷氨酸生产方面具有天然优势。它的细胞壁由共价连接的肽聚糖(PG),阿拉伯半乳聚糖(AG),分枝菌酸(MA)以及糖脂组成的外层松散结构组成。位于外层的分枝菌酸层破坏后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。
用于破坏分枝菌酸层诱导L-谷氨酸外排的传统方式是生物素限制,添加表面活性剂吐温40或者亚致死量的抗生素青霉素等,这些方法耗费大量的人力物力且有操作复杂容易引入污染等缺陷。因此,通过代谢工程改造获得谷氨酸棒杆菌分枝菌酸精简的L-谷氨酸高产菌株具有重大的应用价值。
发明内容
本发明提供了谷氨酸棒杆菌中敲除谷氨酸棒杆菌基因组上分支菌酸酮酰基还原酶基因cmrA得到突变菌谷氨酸棒杆菌△cmrA,谷氨酸棒杆菌△cmrA菌株在发酵培养基中,可以高效合成L-谷氨酸。
本发明提供一种谷氨酸棒杆菌,所述谷氨酸棒杆菌敲除了基因组上分支菌酸酮酰基还原酶基因cmrA。
在一种实施方式中,所述分支菌酸酮酰基还原酶基因cmrA具有SEQ ID NO.1所示的核苷酸序列。
在一种实施方式中,所述谷氨酸棒杆菌为谷氨酸棒杆菌(Corynebacteriumglutamicum)ATCC 13869。
本发明还提供了分支菌酸酮酰基还原酶基因在提高谷氨酸棒杆菌细胞通透性,或提高氨基酸合成效率方面的应用。
在一种实施方式中,所述应用是敲除或沉默谷氨酸棒杆菌基因组中的分支菌酸酮酰基还原酶基因。
本发明还提供一种提高发酵产品产量的方法,所述方法是敲除谷氨酸棒杆菌基因组上分支菌酸酮酰基还原酶基因cmrA,所述分支菌酸酮酰基还原酶基因cmrA具有SEQ IDNO.1所示的核苷序列。
在一种实施方式中,所述发酵产品包括但不限于谷氨酸棒杆菌的代谢产物。
在一种实施方式中,所述发酵产品为氨基酸或有机酸;所述氨基酸包括但不限于:丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、甲硫氨酸、色氨酸、谷氨酰胺、苏氨酸、天冬酰胺、甘氨酸、天冬氨酸、谷氨酸、赖氨酸或精氨酸;所述有机酸包括但不限于:柠檬酸、丙酮酸、乳酸、葡萄糖酸、衣康酸或苹果酸。
本发明还提供所述重组谷氨酸棒杆菌在发酵培养基中合成L-谷氨酸的应用。
在一种实施方式中,所述方法是将所述谷氨酸棒杆菌接种在发酵培养基中,于25-35℃,有氧条件下培养至少48小时。
在一种实施方式中,所述方法是将所述谷氨酸棒杆菌接种在发酵培养基中,于25-35℃,150~250rpm培养48~72小时。
在一种实施方式中,所述重组谷氨酸棒杆菌生产L-谷氨酸的方法是:菌株在平板上活化培养36小时,接种到培养基中,置于29-31℃,150-250rpm条件下培养16-18小时得到种子液,初始OD562为1接种于发酵培养基,置于25-35℃,150-250rpm条件下培养60-80小时。
在一种实施方式中,所述发酵培养基,培养基组成包括:90g/L葡萄糖,15g/L玉米浆,1g/L酵母膏,40g/L(NH4)2SO4,1g/L KH2PO4,10mg/L FeSO4·7H2O,500mg/L MgSO4,10mg/L MnSO4·H2O,1mg/L thiamine·HCl,20g/L CaCO3。
本发明还提供所述重组谷氨酸棒杆菌或上述方法在发酵领域、药物制备、食品制备领域的应用。
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分枝菌酸酮酰基还原酶基因cmrA,使获得的C.glutamicum△cmrA在发酵培养基中L-谷氨酸产量为13.14g/L,是出发菌株C.glutamicum ATCC 13869产量的10.77倍。本发明的方法提供了一种提高谷氨酸棒杆菌中氨基酸产量的新策略。
附图说明
图1:CmrA的功能(A)和C.glutamicum△cmrA的构建流程示意图(B)。
图2:C.glutamicum ATCC 13869和C.glutamicum△cmrA的(A)生长曲线,(B)葡萄糖含量和(C)L-谷氨酸的生产情况。
图3:以C.glutamicum ATCC 13869为对照,分析C.glutamicum△cmrA中与谷氨酸合成相关基因的转录水平。
具体实施方式
(1)基因的敲除方法
采用Cre/LoxP敲除系统对谷氨酸棒杆菌进行基因敲除。首先构建含同源臂片段和loxL-kan-loxR筛选标记的质粒电转入谷氨酸棒杆菌(Corynebacterium glutamicum)。涂布于卡那霉素抗性平板,30℃培养36小时后,菌落PCR筛选突变菌株。将正确的突变菌株制备感受态细胞电转化含Cre基因的质粒pDTW109用于去除kan抗性基因以获得正确的谷氨酸棒杆菌敲除菌株。
(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。
(3)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。
(4)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×4.0mmODS-2HYPERSIL C18色谱柱。
(5)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。
L-谷氨酸种子培养基(g/L):25g/L葡萄糖,30g/L玉米浆,1.25g/L尿素,500mg/L(NH4)2SO4,1g/L KH2PO4,500mg/L MgSO4,pH:7.2。
L-谷氨酸发酵培养基(g/L):90g/L葡萄糖,15g/L玉米浆,1g/L酵母膏,40g/L(NH4)2SO4,1g/L KH2PO4,10mg/L FeSO4·7H2O,500mg/L MgSO4,10mg/L MnSO4·H2O,1mg/Lthiamine·HCl,20g/L CaCO3,pH:7.2。
实施例1谷氨酸棒杆菌突变株的构建
具体构建过程如下:
(1)谷氨酸棒杆菌感受态细胞制备:
将C.glutamicum ATCC 13869接种于LBHIS液体培养基中,30℃,200rpm过夜培养。转接至100mL EPO液体培养基,使接种后的初始OD562达0.3,30℃,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,将菌体沉淀用预冷的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。
(2)敲除质粒pHDW-1构建:
以C.glutamicum ATCC 13869的基因组DNA作为模板,使用引物对cmrA-UF和cmrA-UR扩增左侧同源臂。使用引物对cmrA-DF和cmrA-DR扩增右侧同源臂。以pDTW202作为模板,使用引物对cmrA-kan-F和cmrA-kan-R扩增loxL-kan-loxR片段。然后将扩增获得的三个片段左侧同源臂、loxL-kan-loxR和右侧同源臂片段作为模板,使用引物对cmrA-U-F和cmrA-D-R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BamHI和EcoRI进行双酶切,使用T4连接酶连接两个片段以构建质粒pHDW-1;
cmrA-UF:TATAGGATCCATCCACAGCCCCATTATTC;
cmrA-UR:CGCCCTATAGTGAGTCGTATTCTAGCTGGGGTAGTG;
cmrA-DF:CTTTAGTGAGGGTTAATTGCGCGCTGTACTCTCCCCTGTTATG;
cmrA-DR:TATAGAATTCAAAAGTCGTGGAAGAAACCG;
cmrA-kan-F:CGTTACCACTACCCAGCAAGAATACGACTCACTATAGGGCG;
cmrA-kan-R:CATAACAGGGGAGAGTACAGCGCGCAATTAACCCTCACTAAAG。
(3)Cre/LoxP构建突变株:
使用Cre-loxP系统进行cmrA基因的敲除。将步骤(1)构建的质粒pHDW-1电转到C.glutamicum ATCC 13869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cmrA-YF和cmrA-YR进一步筛选loxL-kan-loxR正确插入C.glutamicum ATCC 13869基因组中的克隆;
cmrA-YF:GGCGGTGATGGTGGTGTTC;
cmrA-YR:TGGGTCATATCATGGATTCCTC。
将携带消除卡那霉素抗性的Cre基因的pTDW109电转到C.glutamicum△cmrA kan感受态细胞中,通过菌落PCR使用引物对cmrA-YF/cmrA-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为C.glutamicumΔcmrA。
实施例2 C.glutamicumΔcmrA合成L-谷氨酸的应用
将实施例1构建获得的菌株C.glutamicumΔcmrA在活化平板上培养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,接种于50mL发酵培养基中使初始OD562=1,置于30℃,200rpm条件下培养72小时。测定每12小时的细胞生长、葡萄糖消耗和氨基酸浓度(图2)。
对数早期时,C.glutamicum△cmrA比C.glutamicum ATCC 13869生长慢,但在平稳期达到更高的OD值(图2A)。对应于生长速率,C.glutamicum△cmrA在对数早期消耗葡萄糖的速度也比C.glutamicum ATCC 13869慢,但两者都在60小时后消耗了所有葡萄糖(图2B)。培养72小时后,C.glutamicum△cmrA细胞产生13.14g/L L-谷氨酸,而C.glutamicum ATCC13869细胞仅产生1.22g/L L-谷氨酸(图2C)。结果表明,C.glutamicum△cmrA中的L-谷氨酸产量显著提高。
实施例3 C.glutamicumΔcmrA中与氨基酸合成相关基因的表达量变化
将C.glutamicum在发酵培养基培养至对数期,5000g,4℃离心10分钟收获细胞后用预冷的PBS洗涤两次,并迅速在液氮中冷冻。RNA的提取,建库以及转录组的测定与分析由苏州金唯智生物科技有限公司完成,分析机械敏感型通道蛋白合成基因msccG,msccG2,mscL和酮戊二酸脱氢酶复合物的elo亚基编码基因odhA以及精氨酸合成基因的表达量变化。
转录组结果显示C.glutamicum△cmrA中与谷氨酸外排相关的机械敏感型通道蛋白合成基因msccG,msccG2,mscL表达均为上调,使谷氨酸外排增加。酮戊二酸脱氢酶复合物的elo亚基编码基因odhA下调,使更多的碳流转向谷氨酸的生成。谷氨酸下游合成氨基酸精氨酸合成的相关基因均为下调,使谷氨酸的消耗减少。
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。
SEQUENCE LISTING
<110> 江南大学
<120> 一种提高L-谷氨酸生产效率的谷氨酸棒杆菌的构建及应用
<130> BAA211102A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 801
<212> DNA
<213> Corynebacterium glutamicum
<400> 1
atggcgttac cactacccag caagagcgct cgagcacttg ttactggggc aagccaaggc 60
attggcctcg ccatcgccaa agatttggcg cggtatgggc acaacctcat tttggttgct 120
cgccgcgagg atgtcctcaa ggagatcgcc gcggatttgg agaagaagca cggtgtgatc 180
gttgaggtcc gcccggtgga tttgagtgat gagcaagccc gcaaggtgtt gatcgatgag 240
atcaagacaa gggaaatcaa catcatcatt aactctgctg gcatcgcaag ctttgggccg 300
ttcaaggacc aggattggtc ttatgagacg gcccagttct cacttaatgc cactgccgtt 360
tttgagctca cccacgcggt gttaggcgga atgattgacc gtggcacggg cgctatttgc 420
aatgtgggat ctgcggctgg caatgtgcca atccccaaca acgccacgta tgtgctcacc 480
aaggctggcg tgaacgcctt caccgaggca ctgcactacg agctgcgcgg gaccggtgtg 540
tcttgtacat tactcgcgcc ggggcctgtc cgtgaggcag agattcctga gtctgagaag 600
tcgatcgtgg acaaggttgt ccctgatttc ttgtggacca cctatgagtc ctgctctgca 660
gagaccttgc gtgcgctgtc taagaatcag cgtcgcgtgg ttccaggtcc gctgtccaag 720
gccatgaatt ttgtgtcctc tgttgctcca accgctgtac tctcccctgt tatgggctgg 780
gtttataaga agatgggtta g 801
Claims (10)
1.一种谷氨酸棒杆菌(Corynebacterium glutamicum),其特征在于,沉默或敲除了基因组上分支菌酸酮酰基还原酶基因cmrA。
2.根据权利要求1所述的谷氨酸棒杆菌,其特征在于,所述分支菌酸酮酰基还原酶基因cmrA具有SEQ ID NO.1所示的核苷酸序列。
3.根据权利要求1或2所述的谷氨酸棒杆菌,其特征在于,所述谷氨酸棒杆菌为谷氨酸棒杆菌ATCC 13869。
4.SEQ ID NO.1所示的分支菌酸酮酰基还原酶基因在提高谷氨酸棒杆菌细胞通透性和/或提高氨基酸合成效率方面的应用。
5.根据权利要求4所述的应用,其特征在于,敲除或沉默谷氨酸棒杆菌基因组中的分支菌酸酮酰基还原酶基因,以提高谷氨酸棒杆菌细胞通透性和/或提高氨基酸合成效率。
6.一种提高发酵产品产量的方法,其特征在于,敲除谷氨酸棒杆菌基因组上分支菌酸酮酰基还原酶基因cmrA,所述分支菌酸酮酰基还原酶基因cmrA具有SEQ ID NO.1所示的核苷序列;所述发酵产品包括但不限于谷氨酸棒杆菌的代谢产物。
7.根据权利要求6所述的方法,其特征在于,所述发酵产品为氨基酸或有机酸;所述氨基酸包括但不限于:丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、甲硫氨酸、色氨酸、谷氨酰胺、苏氨酸、天冬酰胺、甘氨酸、天冬氨酸、谷氨酸、赖氨酸或精氨酸;所述有机酸包括但不限于:柠檬酸、丙酮酸、乳酸、葡萄糖酸、衣康酸或苹果酸。
8.权利要求1~3任一所述的谷氨酸棒杆菌在合成L-谷氨酸的应用。
9.根据权利要求8所述的应用,其特征在于,将所述谷氨酸棒杆菌接种在发酵培养基中,于25-35℃,有氧条件下培养至少48小时。
10.权利要求1~3任一所述的谷氨酸棒杆菌或权利要求6~7任一所述的方法在发酵领域,或制备食品或药物方面的应用。
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CN110951661A (zh) * | 2019-12-26 | 2020-04-03 | 新疆梅花氨基酸有限责任公司 | 一种高产l-谷氨酰胺的谷氨酸棒杆菌及其构建方法与应用 |
Non-Patent Citations (2)
Title |
---|
LEA-SMITH ET AL.: "The Reductase That Catalyzes Mycolic Motif Synthesis Is Required for Efficient Attachment of Mycolic Acids to Arabinogalactan", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 282, no. 15, pages 3 - 4 * |
高云飞: "分枝菌酸缺失对谷氨酸棒杆菌ATCC13869的影响研究", 《中国优秀硕士学位论文全文数据库》, pages 2 - 3 * |
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