CN114957414A - 一种RosR突变体及其重组微生物与应用 - Google Patents
一种RosR突变体及其重组微生物与应用 Download PDFInfo
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- CN114957414A CN114957414A CN202210626445.5A CN202210626445A CN114957414A CN 114957414 A CN114957414 A CN 114957414A CN 202210626445 A CN202210626445 A CN 202210626445A CN 114957414 A CN114957414 A CN 114957414A
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- rosr
- glutamine
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- recombinant microorganism
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Abstract
本发明涉及微生物工程技术领域，具体公开了一种RosR突变体及其重组微生物与应用。本发明的RosR突变体，其以野生型RosR的氨基酸序列为参考序列，所述RosR突变体含有第34位缬氨酸被取代的突变；所述野生型RosR的氨基酸序列如SEQ IDNO.12所示。本发明通过对rosR基因进行修饰，使得含有该突变体的菌株产生L‑谷氨酰胺的能力得到增强，为提升L‑谷氨酰胺及其衍生物提供了一种新途径。
Description
技术领域
本发明涉及微生物工程技术领域，具体地说，涉及一种RosR突变体及其重组微生物与应用。
背景技术
谷氨酰胺是一种非必需氨基酸。化学名称为2-氨基-4-氨甲酰基丁酸。谷氨酰胺是蛋白质合成中的编码氨基酸，可促进蛋白质的合成，抑制蛋白质的分解，可用于治疗胃及十二指肠溃疡，在医药行业中有重要作用。
目前，谷氨酰胺最常用的生产方法为发酵法，主要以谷氨酸棒杆菌(Corynebacteriumglutamicum)为生产菌发酵生产谷氨酰胺。谷氨酸棒状杆菌是异养需氧型，为革兰氏阳性菌，具有生长速度快、非致病、对自身代谢物弱降解能力的特性。发酵法具有原料来源广泛、生产成本低、产品质量可控、产物单一等优点。谷氨酸棒杆菌自身的谷氨酰胺合酶编码基因会被阻遏蛋白或者转录调控因子抑制，导致转录水平降低，酶活急剧降低，造成底物谷氨酸不能充分利用。其中RosR(Cg1324)是过氧化氢敏感MarR型转录调控因子(2010，Michael Bott，a Hydrogen Peroxide-sensitive MarR-typeTranscriptionalRegulator of Corynebacteriumglutamicum)，RosR可以结合到谷氨酰胺合酶编码基因glnA的启动子区，抑制glnA转录，失活rosR促进谷氨酰胺的积累(2022，Xiangfei Li，MarR-type transcription factor RosR regulates glutamate metabolism network andpromotes accumulation of L-glutamate in CorynebacteriumglutamicumG01)。因此解除或减弱谷氨酰胺合酶的抑制，促进谷氨酸转化为谷氨酰胺具有重要意义，有必要对其进一步研究，以获得更好的发酵效果。
发明内容
本发明的目的在于提供一种可提高L-谷氨酰胺产量的新方法。
本发明的技术方案如下：
一种RosR突变体，其以野生型RosR的氨基酸序列为参考序列，所述RosR突变体含有第34位缬氨酸被取代的突变；所述野生型RosR的氨基酸序列如SEQ ID NO.12所示。
具体地，所述RosR突变体含有第34位缬氨酸被丙氨酸、谷氨酸、甘氨酸、色氨酸或亮氨酸取代的突变。
优选，所述RosR突变体含有第34位缬氨酸被色氨酸取代的突变。
本发明提供了一种谷氨酸棒杆菌，其细胞内过氧化氢敏感MarR型转录调控因子RosR的第34位氨基酸由缬氨酸(V)突变为丙氨酸(A)、谷氨酸(E)、甘氨酸(G)、色氨酸(W)或亮氨酸(L)。
本发明意外发现，通过对谷氨酸棒杆菌的rosR基因进行特定修饰，可使得所述菌株产生L-谷氨酰胺的能力增强，且效果优于现有文献已报道的ΔrosR菌株。
本发明还提供一种DNA分子，其以SEQ ID NO.10为参考序列，所述DNA分子含有第500-502位碱基由SEQ ID NO.10中的GTG突变为GCG、GAG、GGG、TGG或CTG的突变。
本发明上述DNA分子中的各突变分别可对应获得RosR的第34位氨基酸由缬氨酸(V)突变为丙氨酸(A)、谷氨酸(E)、甘氨酸(G)、色氨酸(W)或亮氨酸(L)的RosR突变体。
本发明另提供一种重组微生物，所述重组微生物表达上述RosR突变体。
优选，所述重组微生物的出发菌株为谷氨酸棒杆菌。
本发明另提供一种上述重组微生物的如下任一种应用：
(1)在发酵生产L-谷氨酰胺及其衍生物中的应用；
(2)在用于生产L-谷氨酰胺及其衍生物的微生物遗传育种中的应用；
(3)在提高发酵生产L-谷氨酰胺及其衍生物产量上的应用。
本发明还提供一种L-谷氨酰胺的生产方法，其包括以重组微生物进行发酵培养的步骤，所述重组微生物如上所述。
本发明的有益效果至少在于：
本发明的通过实验证实，突变菌株MHZ-0513-3-rosRV34A与出发菌株MHZ-0513-3相比，谷氨酰胺产量由28.9g/L提高到32.8g/L，产酸提高13.5％，副产物谷氨酸、丙氨酸减少，菌株MHZ-0513-3-ΔrosR摇瓶发酵谷氨酰胺产量从28.9g/L提高到30.2g/L，产酸提高4.5％，由此可见，RosR点突变V34A利于谷氨酰胺的积累，效果优于ΔrosR。当RosR第34位氨基酸由缬氨酸(V)突变为谷氨酸(E)、甘氨酸(G)、色氨酸(W)、亮氨酸(L)后，谷氨酰胺产量均有提升，副产物谷氨酸和丙氨酸减少，且优于ΔrosR，其中突变为色氨酸(W)后，谷氨酰胺产量提高到34.6g/L，产酸提高19.7％。显而易见，该位点突变成其他氨基酸后也利于谷氨酰胺及其衍生物的生产。
本发明提供了一种新的更有效地提升谷氨酰胺产量的生物发酵方法。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。下述实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买得到的常规产品。
本发明实施例中涉及的引物名称及序列如表1所示。
表1引物序列(SEQ ID No.1-9)
| 引物 | 序列 |
| UP-F | ctagTCTAGACAACAATGGAGTCGGTGTACT |
| UP-R | TCAGGTTGTGGTTTTCCGCGAGGGTCTCATCTAGTGT |
| DN-F | CGCGGAAAACCACAACCTGA |
| DN-R | cccAAGCTTTGGCTGATTGAGTTTTTCTTTCTTC |
| 鉴定-F | ACACTAGATGAGACCCTCAC |
| ID-F | AACTTGTCTACTGCGAAGAA |
| ID-R | ATTCCAACCGTAGGTCTCTGCA |
| P82 | CTCGTATGTTGTGTGGAATTGTG |
| P85 | CGCCCTGAGTGCTTGCGGCA |
实施例1质粒pK18-rosRV34A构建和重组菌株MHZ-0513-3-rosRV34A构建
菌株MHZ-0513-3-rosRV34A具体构建过程如下：
利用Phusion超保真聚合酶(New England BioLabs)，以谷氨酸棒杆菌MHZ-0513-3的基因组为模板，以UP-F/UP-R为引物，制备重组片段UP，以DN-F/DN-R为引物，制备重组片段DN，所得片段经琼脂糖凝胶回收试剂盒(天根)纯化，随后以片段UP、DN为模板，以UP-F/DN-R为引物制备重组片段，所得重组片段经琼脂糖凝胶回收试剂盒(天根)纯化，然后利用XbaI/HindIII进行消化，同时将pK18-mobsacB(武汉淼灵生物科技有限公司)利用XbaI/HindIII进行消化，并用T4 DNA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，XbaI/HindIII酶切鉴定得到片段插入pK18mobsacB的阳性克隆，进一步通过用P82/P85引物测序(Invitrogen公司)鉴定插入的片段正确。将所得到的质粒命名为pK18-rosRV34A。
包含上下游同源臂部分的野生型RosR的核苷酸序列如SEQ ID No.10所示。RosRV34A突变体的氨基酸序列如SEQ ID No.11所示。野生型RosR的氨基酸序列如SEQ IDNo.12所示。
将pK18-rosRV34A转入谷氨酸棒杆菌MHZ-0513-3中，在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为33℃，倒置培养。将筛得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为33℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。将所筛选出的菌株进一步进行表型验证，挑选KanS的重组子利用鉴定-F/DN-R验证点突变重组子，通过摸索退火温度，获得含点突变的重组子，将得到的阳性重组子用ID-F/ID-R扩增测序，验证获得的为目的突变菌株，并命名为MHZ-0513-3-rosRV34A。
根据文献(2022，Xiangfei Li，MarR-type transcription factor RosRregulates glutamate metabolism network and promotes accumulation of L-glutamate in CorynebacteriumglutamicumG01)报道，用同上方法构建质粒pK18-ΔrosR，菌株MHZ-0513-3-ΔrosR。
谷氨酸棒杆MHZ-0513-3已在CN106701649A中公开，其分类命名为谷氨酸棒杆菌Corynebacteriumglutamicum，于2016年11月30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.13405。
实施例2 MHZ-0513-3-rosRV34A突变菌株生产谷氨酰胺的性能
发酵验证谷氨酰胺产量的方法：将冻存于-80℃甘油管中的菌株接种于下述斜面培养基中进行活化，于33℃下培养24h后长出菌苔，从新鲜活化的斜面上挑取菌苔，接种于下述种子培养基中，于33℃，100rpm下振荡培养至对数生长中后期，培养时间为5h，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33℃，150rpm振荡培养48h。结果如表2所示(OD562培养液在562nm的浊度并表示细胞量，Gln(g/L)表示积累的L-谷氨酰胺的量)。
培养基配方如下：
斜面培养基：脑心浸液37g/L，琼脂1.8％，121℃0.1MPa灭菌20min；
种子培养基：葡萄糖50g/L，尿素5g/L，KH2PO4 2.0g/L，MgSO4·7H2O 1.0g/L，玉米浆30g/L，pH 7.0；
发酵培养基：葡萄糖90g/L，(NH4)2SO4 40g/L，KH2PO4 2.0g/L，MgSO4·7H2O 1.0g/L，玉米浆10g/L，CaCO3 50g/L，pH 7.0。
表2突变菌株谷氨酰胺含量检测
| 菌株 | OD<sub>562</sub> | Gln(g/L) | 产酸提高率％ | Glu(g/L) | Ala(g/L) |
| MHZ-0513-3 | 43.3 | 28.9 | -- | 2.2 | 2.5 |
| MHZ-0513-3-rosR<sup>V34A</sup> | 43.2 | 32.8 | 13.5 | 1.6 | 1.9 |
| MHZ-0513-3-ΔrosR | 43.5 | 30.2 | 4.5 | 1.9 | 2.2 |
如表2所示，在MHZ-0513-3中RosR第34位氨基酸由缬氨酸(V)突变为丙氨酸(A)，即GTG突变为GCG后，获得菌株MHZ-0513-3-rosRV34A，摇瓶发酵谷氨酰胺产量从28.9g/L提高到32.8g/L，产酸提高13.5％，副产物谷氨酸(Glu)和丙氨酸(Ala)减少，菌株MHZ-0513-3-ΔrosR摇瓶发酵谷氨酰胺产量从28.9g/L提高到30.2g/L，产酸提高4.5％，由此可见，rosRV34A突变更利于谷氨酰胺的积累和副产物的减少。
实施例3 RosR第34位氨基酸突变成其他氨基酸生产谷氨酰胺的性能
鉴于RosR第34位氨基酸由缬氨酸(V)突变为丙氨酸(A)后，谷氨酰胺产量有所提升，本实施例接着研究了RosR第34位氨基酸由缬氨酸(V)突变为谷氨酸(E)、甘氨酸(G)、色氨酸(W)、亮氨酸(L)后的发酵性能，各突变菌株构建方法参考实施例1，各突变菌株发酵方法参见实施例2。发酵结果如表3所示。
表3突变菌株谷氨酰胺含量检测
发酵结果表明，RosR第34位氨基酸由缬氨酸(V)突变为谷氨酸(E)、甘氨酸(G)、色氨酸(W)、亮氨酸(L)后，突变株谷氨酰胺产量均有提升，副产物谷氨酸、丙氨酸均减少，且效果都优于ΔrosR菌株，其中突变为色氨酸(W)后，谷氨酰胺产量提高到34.6g/L，产酸提高19.7％，且副产物谷氨酸和丙氨酸最少。显而易见，该位点突变成其他氨基酸也有利于谷氨酰胺及其衍生物的生产。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 一种RosR突变体及其重组微生物与应用
<130> KHP221115987.6
<160> 12
<170> SIPOSequenceListing 1.0
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<211> 31
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ctagtctaga caacaatgga gtcggtgtac t 31
<210> 2
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
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tcaggttgtg gttttccgcg agggtctcat ctagtgt 37
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
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cgcggaaaac cacaacctga 20
<210> 4
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
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cccaagcttt ggctgattga gtttttcttt cttc 34
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acactagatg agaccctcac 20
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<213> 人工序列(Artificial Sequence)
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aacttgtcta ctgcgaagaa 20
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<212> DNA
<213> 人工序列(Artificial Sequence)
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attccaaccg taggtctctg ca 22
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcgtatgtt gtgtggaatt gtg 23
<210> 9
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgccctgagt gcttgcggca 20
<210> 10
<211> 1006
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caacaatgga gtcggtgtac tcggtgaatt caccgcgaac cttggtaacc attgcgtggc 60
gagccacgaa cttgatttcg gtgtgtgcag ggtcgagggt ccaggtgcca gtcaacttgc 120
tcattgtgaa gtccttttcg gtgaaagtgt gtttcgtttt taacggtacc gacactcttg 180
catctgtgca gtttgtgtcg ctgccgttgt tcgtttctat cctaatcaag attgatgacg 240
tgtcaacaat atttctaaaa aacttgatgt ggaaactaaa atccgcaggt gggggcgtga 300
ataaaattgt ttgttgggcc ggagagtggg ctgctgtgta tcgagctttt aacagggggt 360
tctttgaata aatcttcagg agcaggctag ggtaggtgat atgacaacac cacgatggct 420
ctccactgaa gagcaacaac tctggcgcat gatcttgtct gcaacccgca aaatggaacg 480
cacactagat gagaccctcg tggaaaacca caacctgacc acttcagaat ttgcagtact 540
agttactctt tctgaggcaa caggtcagca aatgcgcctg cgagacatgt gccaagaact 600
agattgggac cgcagtagaa cctcccacca agtcacccgc atggacaaaa agggcttagt 660
ggccaaggtt aaatgcgcag gtgacgcacg aggtgtgaac gtagaaatca ccccggaagg 720
tgaacgacgc ctcaaggatg ccgtacctgc tcatgtagaa acagtccgcc aactagtttt 780
cgaccccatg gaagaacgcc acatggaagg acttcgttcc tacctcaccg cagtgttgaa 840
ctccaacacc tgcattgaga tcaacaacca acgcgcggca gagctgtaag ggtttacttg 900
gagcgttttc tcaggggttt ttaggggttg ggagagggga aatccccgat gtgctctagg 960
ttcttattgg cgatgattga agaagaaaga aaaactcaat cagcca 1006
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<211> 162
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Met Thr Thr Pro Arg Trp Leu Ser Thr Glu Glu Gln Gln Leu Trp Arg
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Met Ile Leu Ser Ala Thr Arg Lys Met Glu Arg Thr Leu Asp Glu Thr
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Leu Ala Glu Asn His Asn Leu Thr Thr Ser Glu Phe Ala Val Leu Val
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Thr Leu Ser Glu Ala Thr Gly Gln Gln Met Arg Leu Arg Asp Met Cys
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Gln Glu Leu Asp Trp Asp Arg Ser Arg Thr Ser His Gln Val Thr Arg
65 70 75 80
Met Asp Lys Lys Gly Leu Val Ala Lys Val Lys Cys Ala Gly Asp Ala
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Arg Gly Val Asn Val Glu Ile Thr Pro Glu Gly Glu Arg Arg Leu Lys
100 105 110
Asp Ala Val Pro Ala His Val Glu Thr Val Arg Gln Leu Val Phe Asp
115 120 125
Pro Met Glu Glu Arg His Met Glu Gly Leu Arg Ser Tyr Leu Thr Ala
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Val Leu Asn Ser Asn Thr Cys Ile Glu Ile Asn Asn Gln Arg Ala Ala
145 150 155 160
Glu Leu
<210> 12
<211> 162
<212> PRT
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Met Thr Thr Pro Arg Trp Leu Ser Thr Glu Glu Gln Gln Leu Trp Arg
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Met Ile Leu Ser Ala Thr Arg Lys Met Glu Arg Thr Leu Asp Glu Thr
20 25 30
Leu Val Glu Asn His Asn Leu Thr Thr Ser Glu Phe Ala Val Leu Val
35 40 45
Thr Leu Ser Glu Ala Thr Gly Gln Gln Met Arg Leu Arg Asp Met Cys
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Gln Glu Leu Asp Trp Asp Arg Ser Arg Thr Ser His Gln Val Thr Arg
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Met Asp Lys Lys Gly Leu Val Ala Lys Val Lys Cys Ala Gly Asp Ala
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Arg Gly Val Asn Val Glu Ile Thr Pro Glu Gly Glu Arg Arg Leu Lys
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Asp Ala Val Pro Ala His Val Glu Thr Val Arg Gln Leu Val Phe Asp
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Pro Met Glu Glu Arg His Met Glu Gly Leu Arg Ser Tyr Leu Thr Ala
130 135 140
Val Leu Asn Ser Asn Thr Cys Ile Glu Ile Asn Asn Gln Arg Ala Ala
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Glu Leu
Claims (8)
1.一种RosR突变体，其特征在于，以野生型RosR的氨基酸序列为参考序列，所述RosR突变体含有第34位缬氨酸被取代的突变；所述野生型RosR的氨基酸序列如SEQ ID NO.12所示。
2.根据权利要求1所述的RosR突变体，其特征在于，所述RosR突变体含有第34位缬氨酸被丙氨酸、谷氨酸、甘氨酸、色氨酸或亮氨酸取代的突变。
3.根据权利要求2所述的RosR突变体，其特征在于，所述RosR突变体含有第34位缬氨酸被色氨酸取代的突变。
4.一种DNA分子，其特征在于，以SEQ ID NO.10为参考序列，所述DNA分子含有第500-502位碱基由SEQ ID NO.10中的GTG突变为GCG、GAG、GGG、TGG或CTG的突变。
5.一种重组微生物，其特征在于，所述重组微生物表达权利要求1-3任一项所述的RosR突变体。
6.根据权利要求5所述的重组微生物，其特征在于，所述重组微生物的出发菌株为谷氨酸棒杆菌。
7.权利要求5或6所述的重组微生物的如下任一种应用：
(1)在发酵生产L-谷氨酰胺及其衍生物中的应用；
(2)在用于生产L-谷氨酰胺及其衍生物的微生物遗传育种中的应用；
(3)在提高发酵生产L-谷氨酰胺及其衍生物产量上的应用。
8.一种L-谷氨酰胺的生产方法，其特征在于，包括以重组微生物进行发酵培养的步骤，所述重组微生物如权利要求5或6所述。
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| CN113881611A (zh) * | 2021-02-02 | 2022-01-04 | 江南大学 | 一种提高谷氨酸棒杆菌合成l-谷氨酸产量的方法 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001000844A2 (en) * | 1999-06-25 | 2001-01-04 | Basf Aktiengesellschaft | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001000844A2 (en) * | 1999-06-25 | 2001-01-04 | Basf Aktiengesellschaft | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
Non-Patent Citations (3)
| Title |
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| MICHAEL BUSSMANN: "RosR (Cg1324), a hydrogen peroxide-sensitive MarR-type transcriptional regulator of Corynebacterium glutamicum", J BIOL CHEM, vol. 285, no. 38, pages 29305 - 18 * |
| TATJANA WALTER ET AL.: "Physiological Response of Corynebacterium glutamicum to Indole", MICROORGANISMS, vol. 8, no. 12, pages 1945 * |
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| CN113881611A (zh) * | 2021-02-02 | 2022-01-04 | 江南大学 | 一种提高谷氨酸棒杆菌合成l-谷氨酸产量的方法 |
| CN113881611B (zh) * | 2021-02-02 | 2022-12-13 | 江南大学 | 一种提高谷氨酸棒杆菌合成l-谷氨酸产量的方法 |
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