WO2018018569A1 - 一种通过敲除黄素还原酶提高l-精氨酸产量的方法 - Google Patents

一种通过敲除黄素还原酶提高l-精氨酸产量的方法 Download PDF

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WO2018018569A1
WO2018018569A1 PCT/CN2016/092179 CN2016092179W WO2018018569A1 WO 2018018569 A1 WO2018018569 A1 WO 2018018569A1 CN 2016092179 W CN2016092179 W CN 2016092179W WO 2018018569 A1 WO2018018569 A1 WO 2018018569A1
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corynebacterium
arginine
nadph
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frd2
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饶志明
满在伟
徐美娟
杨套伟
张显
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/01038—FMN reductase (NADPH) (1.5.1.38)

Definitions

  • the invention relates to a method for improving the yield of L-arginine by knocking out flavin reductase, and belongs to the technical field of amino acid fermentation production of amino acids.
  • L-arginine is a semi-essential amino acid in humans and animals. It is a synthetic precursor of various biologically active substances and has a variety of unique physiological and pharmacological effects. With the continuous deep research and understanding of the biological functions of arginine, arginine is increasingly used in the pharmaceutical, food, and feed industries.
  • the production method of L-arginine is a hydrolysis method and a fermentation method.
  • Most of the existing manufacturers in China still mainly use the protein hydrolysis method to produce L-arginine, which has serious environmental pollution and low yield, and is not suitable for large-scale production.
  • the process of producing L-arginine by fermentation is relatively simple and environmentally friendly, and has great development potential.
  • domestic production of L-arginine by microbial fermentation has generally low acid production levels and high costs, and production levels and production cannot meet domestic demand. Therefore, it is very important to increase the fermentation acid production level and glucose utilization rate of L-arginine.
  • Corynebacterium crenatum SDNN403 (this strain was studied by the research group for many years, and a strain was obtained by the traditional mutagenesis method, the preservation number CGMCC NO: 0890, patent number: ZL 03112896.3), and the high-yield L-arginine C. crenatum.
  • the L-arginine anabolic pathway of this strain was systematically analyzed in the previous work. The feedback inhibition and feedback repression regulation in L-arginine anabolism were studied, and the feedback inhibition in the strain was relieved.
  • the metabolic modification of the key enzyme gene of the competitive bypass metabolism of arginine synthesis is carried out, and the expression of key enzyme gene clusters of the arginine synthesis pathway of the target product is enhanced, and the competitive branches such as proline and glutamine are weakened.
  • the metabolic flux of the pathway ultimately concentrates the catabolism of L-glutamate in the anabolic flux of L-arginine, thereby increasing the yield of arginine.
  • Active oxygen species including superoxide anion Hydroxyl radical OH ⁇ and hydrogen peroxide H 2 O 2 are by-products in the process of biological aerobic metabolism and are highly toxic to cells. After long-term evolution of cells, a system of resistance to reactive oxygen species has been formed. For example, superoxide dismutase can convert superoxide anion O 2- to H 2 O 2 , and catalase can decompose H 2 O 2 into H. 2 O and O 2 .
  • the decomposition of cells after the production of reactive oxygen species is a passive defense process. The generated reactive oxygen species cannot be immediately removed, and a small amount of active oxygen can still react with intracellular substances, thereby causing damage to the cells. If the synthesis of reactive oxygen species can be directly reduced, the damage of reactive oxygen species to cells can be fundamentally reduced.
  • H 2 O 2 is one of intracellular reactive oxygen species and has high oxidative activity and toxicity. It has been reported that flavin reductase can use NAD(P)H to reduce oxidized flavin FMN, FAD and riboflavin to reduced flavin FMNH 2 , FADH 2 and reduced riboflavin, which can be further catalyzed
  • NAD(P)H NAD(P)H
  • FAD and riboflavin to reduced flavin FMNH 2
  • FADH 2 reduced riboflavin
  • the present invention provides a method for increasing the yield of L-arginine.
  • the present invention identifies a putative NADPH-dependent FMN reductase, analyzes its final catalytic product, and ultimately promotes cell growth by knocking out the putative NADPH-dependent FMN reductase gene frd1 and/or frd2 in C. crenatum. L-arginine synthesis.
  • a first object of the present invention is a recombinant strain of Corynebacterium cloacae having an increased L-arginine production, which is a Corynebacterium cloacae lacking the NADPH-dependent FMN reductase gene frd1 and/or frd2.
  • amino acid sequence of the NADPH-dependent FMN reductase gene frd1 is shown in SEQ NO. 3, and the nucleotide sequence is shown in SEQ NO.
  • amino acid sequence of the NADPH-dependent FMN reductase gene frd2 is shown in SEQ NO. 4, and the nucleotide sequence is shown in SEQ NO.
  • the Corynebacterium cloacae recombinant strain is obtained by knocking out a NADPH-dependent FMN reductase gene in Corynebacterium crenatum CGMCC NO: 0890.
  • the recombinant strain of Corynebacterium cloacae is constructed by using the Corynebacterium crenatum CGMCC NO:0890 genome as a template to obtain a frd1 and/or frd2 gene deletion fragment, and then obtaining the obtained frd1 and / or frd2 gene deletion fragment is ligated to the pK18mobsacB linearization vector and transferred to E.
  • coli select positive transformants, construct plasmid pK18mobsacB- ⁇ frd1 and / or pK18mobsacB- ⁇ frd2; electroporate pK18mobsacB- ⁇ frd1 and / or pK18mobsacB- ⁇ frd2 plasmid Transformation of Corynebacterium crenatum CGMCC NO: 0890, first cultured on a solid medium plate containing kanamycin to obtain the first homologous recombinant transformant, and then the target transformant was subjected to stress twice in sucrose-containing medium. Recombinant screening, and identification of the second homologous recombinant transformants, identified the correct strains named 403 ⁇ frd1, 403 ⁇ frd2, 403 ⁇ frd12.
  • a second object of the present invention is to provide a method for synthesizing L-arginine, which comprises fermenting a recombinant strain of Corynebacterium cloacae according to any one of claims 1 to 5 as a production strain.
  • the fermentation is carried out at 28 to 32 °C.
  • the fermentation medium component for fermentation glucose 120 g/L, corn syrup 40 g/L, biotin 8 ⁇ 10 -5 g/L, histidine 5 ⁇ 10 -4 g / L, manganese sulfate 0.02 g / L, ammonium sulfate 20 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1.5 g / L, ferrous sulfate 0.02 g / L.
  • a third object of the present invention is to provide a method for promoting L-arginine synthesis by knocking out flavin reductase by knocking out a NADPH-dependent FMN reductase gene of Corynebacterium bluntii Bacteria, L-arginine was synthesized using recombinant bacteria as a production strain.
  • amino acid sequences of the NADPH-dependent FMN reductase genes frd1, frd2 are shown in SEQ NO. 3 and SEQ NO. 4, respectively.
  • Corynebacterium cloacae is Corynebacterium crenatum CGMCC NO: 0890.
  • a fourth object of the present invention is to provide L-arginine produced by the recombinant bacteria, and the use of the recombinant bacteria in the pharmaceutical, food, and feed industries.
  • the knockout of the frd1 or frd2 gene of the present invention can increase the L-arginine production of the strain by 16.46% and 3.16%, respectively.
  • the L-arginine yield of the recombinant bacteria 403 ⁇ frd1 and 403 ⁇ frd2 of the present invention after fermentation for 60 hours was 18.4 g/L and 16.3 g/L.
  • the strain used in the present invention is Corynebacterium crenatum SDNN403, which is a high-yield arginine mutant strain selected by the laboratory, and the preservation number CGMCC No. 0890 has been disclosed in the patent document No. ZL 03112896.3.
  • Known biological materials are known.
  • C. crenatum SDNN403 ie Corynebacterium crenatum CGMCC NO.0890
  • the putative NADPH-dependent FMN reductase encoding gene frd1 corresponding to C. glutamicum ATCC13032 gene cg3223
  • frd2 were amplified by PCR.
  • the primer sequences are as follows (the nucleotide sequences are shown in SEQ ID NO: 5 to SEQ ID NO: 8, respectively):
  • frd1 and frd2 gene fragments were ligated with the pET-28a linearization plasmid, and heat-transformed into E. coli BL21, and positive transformants were picked to obtain recombinant strains BL21/pET-28a-frd1 and BL21/pET-28a-frd2. .
  • the recombinant strains BL21/pET-28a-frd1 and BL21/pET-28a-frd2 were induced to overexpress the target proteins Frd181 and Frd188. After ultrasonic cell disruption, the target protein Frd181 and Frd188 were obtained by nickel column affinity chromatography.
  • the target proteins Frd181 and Frd188 were purified by nickel column affinity chromatography.
  • the protein purification was analyzed by SDS-PAGE. The results showed that the purification effect was better.
  • Reaction system 0.1 M pH 7.5 Tris-HCl, 75 ⁇ M NAD (P) H, 50 ⁇ M flavin (FMN, FAD and riboflavin) and an appropriate amount of enzyme solution.
  • NAD 340 (P) H a By measuring the change in OD monitoring consumption of NAD 340 (P) H a.
  • the phenol red-horseradish peroxidase method and biosensor analyzer were used to determine the formation of H 2 O 2 in the reaction system.
  • Frd181 and Frd188 are H 2 O 2 NAD(P)H-dependent flavin reductase.
  • the frd1 and frd2 gene deletion fragments were obtained by the overlap extension PCR method using the C. crenatum SDNN403 genome as a template.
  • the primer sequences are as follows (the nucleotide sequences are shown in SEQ ID NO: 9 to SEQ ID NO: 16, respectively):
  • frd1 and frd2 gene deletion fragments were ligated to the pK18mobsacB linearization vector, and transferred to E. coli JM109, and positive transformants were picked to construct plasmids pK18mobsacB- ⁇ frd1 and pK18mobsacB- ⁇ frd2.
  • the pK18mobsacB- ⁇ frd1 and pK18mobsacB- ⁇ frd2 plasmids were electroporated into C. crenatum SDNN403, which was applied to a solid medium plate containing LBG+Km after 1800V, 5ms electroporation, and cultured for 24 to 36 hours at 30 °C for the first time. Homologous recombination transformants grow. The target transformants were separately subjected to stress secondary recombination screening in sucrose-containing medium, and finally streaked on LBG plates and multiple transformants were picked, and the second homologous recombination strain was subjected to PCR. Respond to the identification of the wild type/gene deletion type. The correct strains were identified as 403 ⁇ frd1 and 403 ⁇ frd2, respectively. The frd2 gene was further knocked out in the 403 ⁇ frd1 strain to obtain strain 403 ⁇ frd12.
  • Fermentation medium components glucose 120g / L, corn syrup 40g / L, biotin 8 ⁇ 10 -5 g / L, histidine 5 ⁇ 10 -4 g / L, manganese sulfate 0.02g / L, ammonium sulfate 20g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1.5 g / L, ferrous sulfate 0.02 g / L.
  • the fermentation temperature was 30 ° C and the shaking speed was 220 r/min. Fermentation 60 h L-arginine yield 403 ⁇ frd1 was 18.4 g / L, 403 ⁇ frd 2 was 16.3 g / L, 403 ⁇ frd12 was 18.7 g / L.

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Abstract

提供了一种通过敲除黄素还原酶提高L-精氨酸产量的方法。以钝齿棒杆菌SDNN403基因组为模板，通过重叠延伸PCR获取黄素还原酶基因缺失片段，并连接pK18mobsacB，得到敲除质粒pK18mobsacB-Δfrd1和pK18mobsacB-Δfrd2，通过电击转化钝齿棒杆菌SDNN403，二次筛选得到重组菌株403Δfrd1和403Δfrd2。摇瓶发酵发现，通过敲除黄素还原酶基因，L-精氨酸产量明显提高。

Description

一种通过敲除黄素还原酶提高L-精氨酸产量的方法 技术领域
本发明涉及一种通过敲除黄素还原酶提高L-精氨酸产量的方法，属于微生物发酵生产氨基酸技术领域。
背景技术
L-精氨酸是人和动物体内的一种半必需氨基酸，是多种生物活性物质的合成前体，具有多种独特的生理和药理作用。随着对精氨酸的生物学功能不断地深入研究和了解，精氨酸在医药、食品、饲料工业上的用途越来越广泛。
L-精氨酸的生产方法有水解法和发酵法。我国现有的生产厂家目前大多仍主要采用蛋白质水解法来生产L-精氨酸，该方法对环境污染严重且收率不高，不适用于大规模生产。发酵法生产L-精氨酸工艺相对简单且对环境友好，具有很大的发展潜力。但是，国内以微生物发酵生产L-精氨酸，产酸水平普遍较低，成本较高，生产水平和产量不能满足国内需求。因此，提高L-精氨酸的发酵产酸水平和葡萄糖利用率非常重要。
Corynebacterium crenatum SDNN403(该菌株为本课题组多年研究，产用传统诱变方法获得一株菌株，保藏编号CGMCC NO:0890，专利号为：ZL 03112896.3)，为高产L-精氨酸C.crenatum。前期工作中对该菌株的L-精氨酸合成代谢途径进行了系统的分析。对L-精氨酸合成代谢中的反馈抑制与反馈阻遏调控作用进行了研究，解除了菌株中的反馈抑制。同时对精氨酸合成的竞争旁路代谢关键酶基因进行敲除等代谢改造，在增强目标产物精氨酸合成途径关键酶基因簇表达的同时，削弱脯氨酸、谷氨酰胺等竞争支路途径的代谢流，最终使L-谷氨酸分解代谢集中在L-精氨酸的合成代谢流从而提高了精氨酸的产量。
活性氧包括超氧阴离子
Figure PCTCN2016092179-appb-000001
氢氧自由基OH·和过氧化氢H2O2等，是生物有氧代谢过程中的副产物，对细胞具有很高的毒性。经过长期进化细胞已经形成一套抵抗活性氧毒害的作用体系，如超氧化物歧化酶可以将超氧阴离子O2-转化为H2O2，过氧化氢酶可以将H2O2分解成H2O和O2。细胞产生活性氧后再进行分解是一种被动防御的过程，产生的活性氧不可能被立即清除干净，少量活性氧仍然可以与胞内物质发生反应，从而对细胞造成损伤。如果可以直接减少活性氧的合成，则可以从根本上降低活性氧对细胞的危害。
H2O2作为细胞内活性氧的一种，具有很高的氧化活性和毒性。有报道指出黄素还原酶可以利用NAD(P)H将氧化型黄素FMN、FAD和核黄素还原为还原型黄素FMNH2、FADH2和还原型核黄素，之后可以进一步催化将还原型黄素的电子转移给胞内其他电子受体。如果电 子受体为O2，则可能生成H2O2。
对Corynebacterium glutamicum ATCC13032的基因组检索发现基因cg1150(基因组NCBI Accession Number:BX927151)和基因cg3223(基因组NCBI Accession Number:BX927156)编码假定的NADPH-依赖型FMN还原酶。但是，目前为止这两个基因的功能并不明确，还没有相关研究报道。
虽然目前已有报道对菌株的代谢途径进行改造以提高L-精氨酸产量，但有必要开发新的方法以进一步促进L-精氨酸的合成。
发明内容
为了解决上述问题，本发明提供了一种提高L-精氨酸产量的方法。本发明鉴定了假定的NADPH-依赖型FMN还原酶，分析了其最终催化产物，最终通过敲除C.crenatum中假定的NADPH-依赖型FMN还原酶基因frd1和/或frd2促进了菌体生长和L-精氨酸合成。
本发明的第一个目的一种L-精氨酸产量提高的钝齿棒杆菌重组菌，所述重组菌是缺失了NADPH-依赖型FMN还原酶基因frd1和/或frd2的钝齿棒杆菌。
在本发明的一种实施方式中，所述NADPH-依赖型FMN还原酶基因frd1的氨基酸序列如SEQ NO.3所示，核苷酸序列如SEQ NO.1所示。
在本发明的一种实施方式中，所述NADPH-依赖型FMN还原酶基因frd2的氨基酸序列如SEQ NO.4所示，核苷酸序列如SEQ NO.2所示。
在本发明的一种实施方式中，所述钝齿棒杆菌重组菌是将Corynebacterium crenatum CGMCC NO:0890中的NADPH-依赖型FMN还原酶基因敲除得到的。
在本发明的一种实施方式中，所述钝齿棒杆菌重组菌的构建方法是：以Corynebacterium crenatum CGMCC NO:0890基因组为模板，获得frd1和/或frd2基因缺失片段，然后将获得的frd1和/或frd2基因缺失片段与pK18mobsacB线性化载体连接，并转入E.coli，挑取阳性转化子，构建质粒pK18mobsacB-Δfrd1和/或pK18mobsacB-Δfrd2；将pK18mobsacB-Δfrd1和/或pK18mobsacB-Δfrd2质粒电击转化Corynebacterium crenatum CGMCC NO:0890，先在含有卡那霉素的固体培养基平板上进行培养得到第一次同源重组转化子，再分别将目标转化子在含蔗糖的培养基中进行胁迫二次重组筛选，并对第二次同源重组转化子进行鉴定，鉴定正确的菌株命名为403Δfrd1、403Δfrd2、403Δfrd12。
本发明的第二个目的是提供一种合成L-精氨酸的方法，所述方法是利用权利要求1-5任一所述的钝齿棒杆菌重组菌为生产菌株进行发酵培养。
在本发明的一种实施方式中，所述发酵是在28～32℃下进行。
在本发明的一种实施方式中，用于发酵的发酵培养基成分：葡萄糖120g/L，玉米浆40g/L，生物素8×10-5g/L，组氨酸5×10-4g/L，硫酸锰0.02g/L，硫酸铵20g/L，硫酸镁0.5g/L，磷酸二氢钾1.5g/L，硫酸亚铁0.02g/L。
本发明的第三个目的是提供一种通过敲除黄素还原酶促进L-精氨酸合成的方法，所述方法是将钝齿棒杆菌的NADPH-依赖型FMN还原酶基因敲除得到重组菌，以重组菌为生产菌株合成L-精氨酸。
在本发明的一种实施方式中，所述NADPH-依赖型FMN还原酶基因frd1、frd2的氨基酸序列分别如SEQ NO.3、SEQ NO.4所示。
所述钝齿棒杆菌为Corynebacterium crenatum CGMCC NO:0890。
本发明的第四个目的是提供所述重组菌生产得到的L-精氨酸，以及重组菌在医药、食品、饲料工业上的应用。
本发明的有益效果：
本发明敲除frd1或者frd2基因，能够使菌株的L-精氨酸产量分别提高16.46％、3.16％。本发明的重组菌403Δfrd1、403Δfrd2发酵60h后L-精氨酸产量为18.4g/L、16.3g/L。
具体实施方式
本发明所使用的菌株为Corynebacterium crenatum SDNN403，是本实验室筛选得到的高产精氨酸突变菌株，保藏编号CGMCC NO.0890，已经在专利号为：ZL 03112896.3的专利文献中公开过了，是已知的生物材料。
实施例1：假定的NADPH-依赖型FMN还原酶Frd181和Frd188的纯化和功能鉴定
发明人对假定的NADPH-依赖型FMN还原酶Frd181和Frd188进行了过表达、纯化和功能鉴定。具体如下：
(1)过表达和纯化
以C.crenatum SDNN403(即Corynebacterium crenatum CGMCC NO.0890)基因组为模板，利用PCR的方法引物扩增假定的NADPH-依赖型FMN还原酶的编码基因frd1(对应C.glutamicum ATCC13032基因cg3223)和frd2(对应C.glutamicum ATCC13032基因cg1150)，引物序列如下(核苷酸序列分别如SEQ ID NO:5～SEQ ID NO:8所示)：
28a-frd1F：
Figure PCTCN2016092179-appb-000002
28a-frd1R：
Figure PCTCN2016092179-appb-000003
28a-frd2F：
Figure PCTCN2016092179-appb-000004
28a-frd2R：
Figure PCTCN2016092179-appb-000005
将获得的frd1和frd2基因片段与pET-28a线性化质粒连接，并热激转化E.coli BL21，挑取阳性转化子，获得重组菌BL21/pET-28a-frd1和BL21/pET-28a-frd2。诱导重组菌BL21/pET-28a-frd1和BL21/pET-28a-frd2过表达目标蛋白Frd181和Frd188。超声波细胞破碎后镍柱亲和层析纯化获得目标蛋白Frd181和Frd188。
经过镍柱亲和层析纯化获得目标蛋白Frd181和Frd188，利用SDS-PAGE分析蛋白纯化情况，结果表明纯化效果较好。
(2)对蛋白Frd181和Frd188进行功能鉴定。
反应体系：0.1M pH7.5Tris-HCl，75μM NAD(P)H，50μM黄素(FMN、FAD和核黄素)和适量酶液。通过测定OD340的变化监测NAD(P)H的消耗情况。同时采用酚红-辣根过氧化物酶方法和生物传感分析仪测定反应体系中H2O2的生成情况。
对Frd181和Frd188进行功能鉴定的结果表明，Frd181在FMN和FAD存在条件下能够氧化NADPH，在FMN、FAD和核黄素存在条件下能够氧化NADH，并且在FAD和核黄素存在条件下具有很高的NADH氧化活性。Frd188在FMN和FAD存在条件下能够氧化NADPH和NADH。H2O2生成分析表明Frd181和Frd188催化反应体系中均有H2O2生成。以上结果表明Frd181和Frd188为产H2O2NAD(P)H-依赖型黄素还原酶。
实施例2：基因敲除菌株的构建
以C.crenatum SDNN403基因组为模板，重叠延伸PCR方法获得frd1和frd2基因缺失片段，引物序列如下(核苷酸序列分别如SEQ ID NO:9～SEQ ID NO:16所示)：
Δfrd1-1：
Figure PCTCN2016092179-appb-000006
Δfrd1-2：
Figure PCTCN2016092179-appb-000007
Δfrd1-3：
Figure PCTCN2016092179-appb-000008
Δfrd1-4：
Figure PCTCN2016092179-appb-000009
Δfrd2-1：
Figure PCTCN2016092179-appb-000010
Δfrd2-2：
Figure PCTCN2016092179-appb-000011
Δfrd2-3：
Figure PCTCN2016092179-appb-000012
Δfrd2-4：
Figure PCTCN2016092179-appb-000013
将获得的frd1和frd2基因缺失片段与pK18mobsacB线性化载体连接，并转入E.coli JM109，挑取阳性转化子，构建质粒pK18mobsacB-Δfrd1和pK18mobsacB-Δfrd2。
将pK18mobsacB-Δfrd1和pK18mobsacB-Δfrd2质粒电击转化C.crenatum SDNN403，经1800V，5ms电击后涂布于含有LBG+Km固体培养基平板上，30℃培养24～36h，第一次 同源重组转化子长出。再分别将目标转化子在含蔗糖的培养基中进行胁迫二次重组筛选，最终在LBG平板上进行划线分离并挑取多个转化子，通过PCR对发生第二次同源重组的菌株进行回复野生型/基因缺失型的鉴定。鉴定正确的菌株分别命名为403Δfrd1和403Δfrd2。在403Δfrd1菌株中继续敲除frd2基因，得到菌株403Δfrd12。
实施例3：frd1或者frd2基因敲除对L-精氨酸合成的影响
403Δfrd1、403Δfrd2、403Δfrd12摇瓶发酵。发酵培养基成分：葡萄糖120g/L，玉米浆40g/L，生物素8×10-5g/L，组氨酸5×10-4g/L，硫酸锰0.02g/L，硫酸铵20g/L，硫酸镁0.5g/L，磷酸二氢钾1.5g/L，硫酸亚铁0.02g/L。发酵温度为30℃，摇床转速220r/min。发酵60h L-精氨酸产量403Δfrd1为18.4g/L，403Δfrd2为16.3g/L，403Δfrd12为18.7g/L。
对照实施例：C.crenatum SDNN403摇瓶发酵，发酵培养基成分与发酵条件同实施例3。发酵60h L-精氨酸产量为15.8g/L。
以上数据说明，敲除frd1和/或者frd2基因，能够使菌株的L-精氨酸产量分别提高16.46％、3.16％。
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。
Figure PCTCN2016092179-appb-000014
Figure PCTCN2016092179-appb-000015
Figure PCTCN2016092179-appb-000016
Figure PCTCN2016092179-appb-000017
Figure PCTCN2016092179-appb-000018
Figure PCTCN2016092179-appb-000019
Figure PCTCN2016092179-appb-000020

Claims (10)

  1. 一种钝齿棒杆菌重组菌，其特征在于，所述重组菌是缺失了NADPH-依赖型FMN还原酶基因frd1和/或frd2的钝齿棒杆菌。
  2. 根据权利要求1所述的钝齿棒杆菌重组菌，其特征在于，所述NADPH-依赖型FMN还原酶基因frd1、frd2的氨基酸序列分别如SEQ NO.3、SEQ NO.4所示。
  3. 根据权利要求1所述的钝齿棒杆菌重组菌，其特征在于，所述钝齿棒杆菌重组菌是将Corynebacterium crenatum CGMCC NO:0890中的NADPH-依赖型FMN还原酶基因敲除得到的。
  4. 根据权利要求1所述的钝齿棒杆菌重组菌，其特征在于，所述NADPH-依赖型FMN还原酶基因frd1、frd2的的核苷酸序列分别如SEQ NO.1或者SEQ NO.2所示。
  5. 一种合成L-精氨酸的方法，其特征在于，所述方法是利用权利要求1-5任一所述的钝齿棒杆菌重组菌为生产菌株进行发酵培养。
  6. 根据权利要求5所述的方法，其特征在于，所述发酵是在28～32℃下进行；用于发酵的发酵培养基成分：葡萄糖120g/L，玉米浆40g/L，生物素8×10-5g/L，组氨酸5×10-4g/L，硫酸锰0.02g/L，硫酸铵20g/L，硫酸镁0.5g/L，磷酸二氢钾1.5g/L，硫酸亚铁0.02g/L。
  7. 一种通过敲除黄素还原酶促进L-精氨酸合成的方法，其特征在于，所述方法是将钝齿棒杆菌的NADPH-依赖型FMN还原酶基因frd1和/或frd2敲除得到重组菌，以重组菌为生产菌株合成L-精氨酸。
  8. 根据权利要求7所述的方法，其特征在于，所述NADPH-依赖型FMN还原酶基因frd1、frd2的的氨基酸序列分别如SEQ NO.3、SEQ NO.4所示。
  9. 根据权利要求7所述的方法，其特征在于，所述钝齿棒杆菌为Corynebacterium crenatum CGMCC NO:0890。
  10. 权利要求1-4任一所述的钝齿棒杆菌重组菌在医药、食品或者饲料工业上的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10997558B2 (en) 2017-02-20 2021-05-04 Vspatial, Inc. System and method for creating a collaborative virtual session

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102251946B1 (ko) * 2019-10-31 2021-05-17 대상 주식회사 yeeO 유전자 불활성에 의해 방향족 아미노산 생산능력이 향상된 균주
CN113735282B (zh) * 2021-09-18 2022-11-22 江南大学 一种老黄酶oye2蛋白及其在铬污染中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0007476B1 (de) * 1978-08-01 1982-03-24 Roche Diagnostics GmbH Verfahren und Reagens zur Bestimmung eines oxidierten Pyridin-coenzyms
WO2001044447A1 (en) * 1999-12-15 2001-06-21 Syngenta Participations Ag Compositions and methods for halogenation reactions
CN1441055A (zh) * 2003-02-25 2003-09-10 江南大学 一种生产l－精氨酸的菌株及其诱变方法与利用该菌株生产l－精氨酸的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0007476B1 (de) * 1978-08-01 1982-03-24 Roche Diagnostics GmbH Verfahren und Reagens zur Bestimmung eines oxidierten Pyridin-coenzyms
WO2001044447A1 (en) * 1999-12-15 2001-06-21 Syngenta Participations Ag Compositions and methods for halogenation reactions
CN1441055A (zh) * 2003-02-25 2003-09-10 江南大学 一种生产l－精氨酸的菌株及其诱变方法与利用该菌株生产l－精氨酸的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [O] 12 July 2017 (2017-07-12), XP055460264, Database accession no. WP-003857758.1 *
DATABASE GENBANK [O] 2 November 2016 (2016-11-02), XP055460268, Database accession no. WP-003856821.1 *
TAO, SHUAI: "Proteomic Research of L-arginine Hyper Producing Corynebacterium Crenatum in Different Oxygen-supplying Strategy", CHINA MASTER'S THESES FULL-TEXT DATABASE (ELECTRONIC JOURNAL) ENGINEERING TECHNOLOGY I , 2011, 31 August 2011 (2011-08-31), pages B018 - 46, ISSN: 1674-0246 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10997558B2 (en) 2017-02-20 2021-05-04 Vspatial, Inc. System and method for creating a collaborative virtual session
US11403595B2 (en) 2017-02-20 2022-08-02 vSpatial, Inc Devices and methods for creating a collaborative virtual session

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