WO2022007205A1 - 一种提高大肠杆菌胞内血红素含量的方法 - Google Patents

一种提高大肠杆菌胞内血红素含量的方法 Download PDF

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WO2022007205A1
WO2022007205A1 PCT/CN2020/118317 CN2020118317W WO2022007205A1 WO 2022007205 A1 WO2022007205 A1 WO 2022007205A1 CN 2020118317 W CN2020118317 W CN 2020118317W WO 2022007205 A1 WO2022007205 A1 WO 2022007205A1
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mscs
escherichia coli
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唐蕾
潘梅
刘爽欣
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Jiangnan University
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Definitions

  • the invention relates to a method for increasing the intracellular heme content of Escherichia coli, in particular, using a combination of gene knockout and overexpression, and belongs to the technical field of metabolic engineering.
  • Heme is an important class of iron-containing porphyrins, which are involved in electron transfer, decomposition of reactive oxygen species, catalytic oxidation of substrates, and control of gene expression.
  • heme can be used as a natural pigment for food addition in chemistry, as an iron supplement or an anti-anemia drug in medical care, and in porphyria treatment in terms of disease treatment.
  • Heme is an important cofactor for most prokaryotic and eukaryotic cellular respiration, and proteins with heme as a prosthetic group are essential for various biological processes.
  • heme-based recombinant enzymes In the biosynthesis of heme-based recombinant enzymes, the deficiency of heme is an important factor leading to low enzymatic activity, so an adequate supply of heme cofactor is crucial for soluble and functional hemoglobin production.
  • the recombinant protein with heme as the prosthetic group is expressed heterologously, the increase of the intracellular heme content is beneficial to increase the proportion of active protein.
  • Escherichia coli as an important genetically engineered host bacterium, mainly uses glutamic acid as a precursor to synthesize heme through the C5 pathway ( Figure 1). Glutamate is catalyzed by glutamyl-tRNA synthetase (encoded by gltX) and reductase (encoded by hemA) to synthesize 5-aminolevulinic acid (ALA), thereby synthesizing heme.
  • glutamyl-tRNA synthetase encoded by gltX
  • reductase encoded by hemA
  • ALA 5-aminolevulinic acid
  • exogenous addition of ALA can increase the heme content, it will lead to problems such as inconvenience of operation and increased cost. Therefore, if it is possible to increase the glutamate synthesis flux through the metabolic engineering of Escherichia coli, and then increase the content of ALA and heme, it is simple and economical, and has application value.
  • the present invention makes the intracellular carbon metabolism flow more to glutamate, and combines with overexpression of ALA synthesis key enzyme heMA, so as to increase the content of heme and reduce the amount of ALA or heme caused by exogenous addition of ALA or heme.
  • the cost increase and the inconvenience of operation are brought about.
  • the invention provides a recombinant Escherichia coli, which knocks out or silences the gene mscS encoding a small conductance class mechanosensitive ion channel protein, knocks out or silences the gene aroG encoding 3-deoxy-arabinoheptulose-7 phosphate synthase, Or overexpress the gene hemA encoding glutamyl-tRNA reductase.
  • the gene mscS and the gene aroG are knocked out or silenced in E. coli.
  • the gene mscS is knocked out or silenced in E. coli and the gene heMA is overexpressed.
  • the gene mscS and the gene aroG are knocked out or silenced in E. coli, and the gene heMA is overexpressed.
  • the NCBI accession number of the MscS protein is 2OAU_A
  • the GenBank number of the AroG protein is EFE3302334.1
  • the NCBI accession number of the glutamyl-tRNA reductase HemA is WP_021545525.
  • the nucleotide sequence of the mscS gene is shown in SEQ ID NO.1; the nucleotide sequence of the aroG gene is shown in SEQ ID NO.2; the hemA gene The nucleotide sequence is shown in SEQ ID NO.3.
  • Escherichia coli BL21 is used as the starting strain.
  • the present invention provides a method for constructing the recombinant bacteria Escherichia coli, which comprises knocking out the gene mscS or the gene aroG of the Escherichia coli genome by means of Red homologous recombination.
  • the homology arm of the gene shown in the nucleotide sequence of SEQ ID NO.1 is designed, and the homology arm is 400-600 bp upstream and downstream of the gene, and then the method of Red homologous recombination is used The gene mscS in the E. coli genome was knocked out.
  • the homology arm of the gene as shown in the nucleotide sequence of SEQ ID NO.2 is designed, and the homology arm is 40-60 bp upstream and downstream of the gene, and then the method of Red homologous recombination is used The gene aroG in the E. coli genome was knocked out.
  • the gene heMA is connected to the pET vector to construct an expression vector, and then the expression vector is transferred into a host cell.
  • the invention provides a method for improving heme production, which comprises knocking out the gene mscS encoding a small-conductance class mechanosensitive ion channel protein, and knocking out the gene encoding 3-deoxy-arabinoheptulose-7 phosphate synthase in Escherichia coli aroG, or overexpression of the gene hemA encoding glutamyl-tRNA reductase.
  • the gene mscS and the gene aroG are knocked out in E. coli.
  • the gene mscS is knocked out and the gene heMA is overexpressed in E. coli.
  • the gene mscS and the gene aroG are knocked out or silenced in E. coli, and the gene heMA is overexpressed.
  • the invention provides a method for producing heme, which uses the recombinant bacteria as a fermentation strain to produce heme.
  • the recombinant bacteria are added to the culture system in an amount of 1 mL/100 mL after overnight culture.
  • the culture system contains tryptone and yeast powder.
  • the culture is carried out at 30-37° C. for 6-8 hours.
  • the present invention also protects the recombinant Escherichia coli, or the application of the method in the production of heme.
  • the growth condition of the genetically engineered bacteria constructed in the present invention does not change significantly, but the intracellular heme content is greatly increased.
  • the obtained recombinant bacteria pEA-MG has After culturing in LB medium for 7 hours, the heme content was 47.6 ⁇ mol ⁇ L -1 , which was significantly higher than that of the starting strain and 23 times that of the starting strain.
  • Figure 1 is a diagram of the heme synthesis pathway of Escherichia coli.
  • Figure 2 is the growth curve diagram of each strain.
  • Figure 3 is a graph of heme content in each strain.
  • LB medium g/L: tryptone 10, yeast powder 5, NaCl 10, pH 7.0.
  • SEQ ID NO.1 design its upstream and downstream 500bp homology arms, through the method of Red homologous recombination (for specific steps, see Datsenko KA.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(12):6640-6645)) knocked out the mscS gene in Escherichia coli BL21. After sequencing and verification, a genetically engineered bacterium that knocked out the mscS gene was obtained. It was named WT-M.
  • the genetically engineered bacteria WT-M were inoculated into LB medium, activated overnight at 37°C, 200 r/min, and the next day, 500 ⁇ L of a certain concentration of bacterial liquid was transferred to 50 mL of LB medium, at 37°C, 200 r/min. Under culture, real-time determination of bacterial concentration, and make a growth curve, as shown in Figure 2.
  • the strain WT was inoculated into LB medium, activated overnight at 37°C, 200 r/min, and the next day, a small amount of 500 ⁇ L of the same bacterial solution was transferred to 50 mL of LB medium, and cultured at 37°C, 200 r/min, real-time. The bacterial concentration was measured, and production koji was produced, as shown in Figure 2.
  • the heme content was detected, and the heme content of WT and WT-M were 2.11 ⁇ mol/L and 2.35 ⁇ mol/L, respectively.
  • SEQ ID NO.2 design its upstream and downstream 50bp homology arms, through the method of Red homologous recombination, on the basis of WT-M, the aroG gene was knocked out, and the genetic engineering of the aroG gene was obtained by sequencing verification. bacteria, and named it WT-MG to obtain genetically engineered bacteria.
  • the genetically engineered bacteria WT-MG was inoculated into LB medium, activated overnight at 37°C, 200 r/min, and the next day, 500 ⁇ L of the activated bacterial solution was transferred to 50 mL of LB medium, at 37°C, 200 r/min. The bacterial concentration was measured in real time, and the growth curve was made. As shown in Figure 2, there was no significant difference in growth between the genetically engineered bacteria WT-MG and WT.
  • the glutamyl-tRNA reductase heMA gene (nucleotide sequence shown in SEQ ID NO. 3) was connected to the pET28a vector through Hind III and EcoR I to obtain the recombinant plasmid pET28a-hemA, and then the recombinant plasmid was transferred into JM109 In , the bacterial solution was spread on the LB plate, cultured at 37°C until a single clone was grown, and the single clone was picked and verified by sequencing, and the correct one was the positive transformant.
  • the positive transformants were inoculated into LB liquid medium, cultured for 8 to 12 hours, the recombinant plasmid pET28a-hemA was extracted from the bacterial liquid, and the recombinant plasmid was introduced into the strain WT-M obtained in Example 1 to obtain the engineered strain pEA-M, The heme concentration was detected by fluorescence method. The results showed that the heme content of pEA-M reached 45.8 ⁇ mol ⁇ L -1 .
  • the pET28a plasmid was empty-transformed into BL21 to obtain engineering bacteria pET.
  • the engineering bacteria pET was cultured using the above-mentioned culture method. After the cultivation, the heme concentration was detected by fluorescence method. The results showed that the heme content of pET reached 1.7 ⁇ mol ⁇ L. -1 .
  • Example 3 The specific embodiment is the same as in Example 3, except that the recombinant plasmid pET28a-hemA constructed in Example 3 was transformed into the strain WT-MG obtained in Example 2 to obtain engineering strain pEA-MG, and the heme concentration was detected by fluorescence method. , the results showed that the heme content of pEA-MG reached 47.6 ⁇ mol ⁇ L -1 , which was 23 times that of the starting strain WT.
  • Example 3 The specific embodiment is the same as Example 3, except that the recombinant plasmid pET28a-hemA constructed in Example 3 was transformed into BL21 to obtain engineering bacteria pEA, and the heme concentration was detected by fluorescence method. The results showed that the heme content of pEA reached 34.1 ⁇ mol ⁇ L -1 .

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Abstract

一种提高大肠杆菌胞内血红素含量的方法，在大肠杆菌中敲除编码小电导类机械敏感型离子通道蛋白的基因mscS、敲除编码3-脱氧-阿拉伯庚酮糖-7磷酸合成酶的基因aroG，或过表达了编码谷氨酰-tRNA还原酶的基因hemA。构建得到的重组菌在LB培养基中进行培养，血红素含量可达47.6μmol·L -1。

Description

一种提高大肠杆菌胞内血红素含量的方法 技术领域
本发明涉及一种提高大肠杆菌胞内血红素含量的方法，具体地，利用基因敲除和过表达相结合的技术，属于代谢工程技术领域。
背景技术
血红素是一类重要的含铁卟啉类化合物，参与电子转移、活性氧分解、底物的催化氧化、基因表达的控制等。在实际应用中，血红素在化学方面可作为天然色素用于食品添加，在医疗保健方面可用作补铁剂或抗贫血药，在治疗疾病方面可用于卟啉症治疗等。血红素是大多数原核和真核生物细胞呼吸的重要辅助因子，以血红素为辅基的蛋白对各种生物学过程至关重要。在以血红素为辅基的重组酶生物合成中，血红素的不足是导致酶活性不高的重要因素，因此血红素辅因子的充足供应对于可溶性和功能性血红蛋白生产至关重要。在异源表达以血红素为辅基的重组蛋白时，胞内血红素含量的增加有利于提高活性蛋白的比例。
大肠杆菌(E.coli)作为重要的基因工程宿主菌，主要以谷氨酸为前体物经C5途径合成血红素(图1)。谷氨酸在谷氨酰-tRNA合成酶(gltX编码)和还原酶(hemA编码)的催化下合成5-氨基乙酰丙酸(ALA)，进而合成血红素。虽然外源添加ALA可以提高血红素含量，但会导致操作不便、成本增加等问题。所以如果能够通过对大肠杆菌的代谢工程改造，加大谷氨酸合成通量，进而提高ALA和血红素的含量，简便经济，具有应用价值。
发明内容
本发明通过大肠杆菌的代谢工程改造，使胞内的碳代谢流更多地流向谷氨酸，结合过表达ALA合成关键酶hemA，以提高血红素的含量，减少因外源添加ALA或血红素带来的成本增加及操作不便等问题。
本发明提供了一种重组大肠杆菌，敲除或沉默编码小电导类机械敏感型离子通道蛋白的基因mscS、敲除或沉默编码3-脱氧-阿拉伯庚酮糖-7磷酸合成酶的基因aroG，或过表达编码谷氨酰-tRNA还原酶的基因hemA。
在本发明的一种实施方式中，在大肠杆菌中敲除或沉默基因mscS和基因aroG。
在本发明的一种实施方式中，在大肠杆菌中敲除或沉默基因mscS并过表达基因hemA。
在本发明的一种实施方式中，在大肠杆菌中敲除或沉默基因mscS和基因aroG，并过表达基因hemA。
在本发明的一种实施方式中，所述MscS蛋白NCBI登录号为2OAU_A，所述AroG蛋白 GenBank号为EFE3302334.1；所述谷氨酰-tRNA还原酶HemA在NCBI上的登录号为WP_021545525。
在本发明的一种实施方式中，所述mscS基因的核苷酸序列如SEQ ID NO.1所示；所述aroG基因的核苷酸序列如SEQ ID NO.2所示；所述hemA基因的核苷酸序列如SEQ ID NO.3所示。
在本发明的一种实施方式中，以大肠杆菌BL21为出发菌株。
本发明提供一种构建所述重组菌大肠杆菌的方法，所述方法是通过Red同源重组的方法将大肠杆菌基因组的基因mscS或基因aroG敲除。
在本发明的一种实施方式中，设计如SEQ ID NO.1核苷酸序列所示的基因的同源臂，同源臂为基因上下游各400～600bp，再通过Red同源重组的方法将大肠杆菌基因组的基因mscS敲除。
在本发明的一种实施方式中，设计如SEQ ID NO.2核苷酸序列所示的基因的同源臂，同源臂为基因上下游各40～60bp，再通过Red同源重组的方法将大肠杆菌基因组的基因aroG敲除。
在本发明的一种实施方式中，将基因hemA连接至pET载体上，构建得到表达载体，再将表达载体转入宿主细胞。
本发明提供一种提高血红素产量的方法，在大肠杆菌中敲除编码小电导类机械敏感型离子通道蛋白的基因mscS、敲除编码3-脱氧-阿拉伯庚酮糖-7磷酸合成酶的基因aroG，或过表达编码谷氨酰-tRNA还原酶的基因hemA。
在本发明的一种实施方式中，在大肠杆菌中敲除基因mscS和基因aroG。
在本发明的一种实施方式中，在大肠杆菌中敲除基因mscS并过表达基因hemA。
在本发明的一种实施方式中，在大肠杆菌中敲除或沉默基因mscS和基因aroG，并过表达基因hemA。
本发明提供一种生产血红素的方法，以所述重组菌为发酵菌株生产血红素。
在本发明的一种实施方式中，将所述重组菌过夜培养后以1mL/100mL的量加入培养体系。
在本发明的一种实施方式中，所述培养体系中含有胰蛋白胨和酵母粉。
在本发明的一种实施方式中，在30～37℃条件下培养6～8h。
本发明还保护所述重组大肠杆菌，或所述方法在生产血红素中的应用。
本发明的有益效果：
本发明构建的基因工程菌与出发菌株相比生长状况没有显著改变，但胞内血红素含量大 幅提升，在敲除了基因aroG和mscS，并过表达基因hemA后，得到的重组菌pEA-MG在LB培养基中培养7h后，血红素含量为47.6μmol·L -1，较出发菌株显著提升，是出发菌株的23倍。
附图说明
图1为大肠杆菌血红素合成途径图。
图2为各菌株生长曲线图。
图3为各菌株中血红素含量图。
具体实施方式
1、LB培养基(g/L)：胰蛋白胨10，酵母粉5，NaCl 10，pH7.0。
2、血红素浓度测定：
采用荧光法检测血红素浓度，具体为：取培养后的适量菌体，使得OD 600×菌液体积(mL)＝8(例如：若菌体的OD 600值为0.4，则取20mL菌液)，于4℃，12000r/min离心5min后得到菌体沉淀，水洗后加入1.5mL琥珀色离心管；向1.5mL离心管加入500μL 20mmol/L的草酸，于4℃暗室过夜静置16h；在各离心管中加入500μL 2mol/L的草酸，取一半样品(用于卟啉浓度检测)于常温下反应作为实验对照组，另一半样品(用于卟啉和血红素总浓度检测)于95℃中加热30min；自然冷却后12000r/min离心5min，取200μL于黑色96孔板，进行荧光值检测(激发波长为400nm，发射波长为620nm)，两组差值即为待测血红素浓度。
3、大肠杆菌感受态的制备：参见Takara感受态制备试剂盒说明书(Competent Cell Preparation Kit)。
实施例1
根据SEQ ID NO.1，设计其上下游各500bp同源臂，通过Red同源重组的方法(具体步骤参见Datsenko KA.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):6640-6645))敲除大肠杆菌BL21中的mscS基因，经测序验证，得到敲除mscS基因的基因工程菌，并将其命名为WT-M。
将基因工程菌WT-M接种至LB培养基中，在37℃，200r/min过夜活化，次日取一定浓度的菌液500μL转接于50mL的LB培养基中，在37℃，200r/min下培养，实时测定菌浓，并制作生长曲线，如图2所示。
将菌株WT接种至LB培养基中，在37℃，200r/min过夜活化，次日取少量相同的菌液 500μL转接于50mL的LB培养基中，在37℃，200r/min下培养，实时测定菌浓，并制作生产曲，如图2所示。
由图2可知，基因工程菌WT-M与WT在生长上无显著差异。
培养7h后，检测血红素含量，WT与WT-M的血红素含量分别为2.11μmol/L和2.35μmol/L。
实施例2
根据SEQ ID NO.2，设计其上下游各50bp同源臂，通过Red同源重组的方法，在WT-M的基础上，敲除aroG基因，经测序验证，得到敲除了aroG基因的基因工程菌，并将其命名为WT-MG，得到基因工程菌。
将基因工程菌WT-MG接种至LB培养基中，在37℃，200r/min过夜活化，次日取活化后的菌液500μL转接于50mL的LB培养基中，在37℃，200r/min下培养，实时测定菌浓，并制作生长曲线，如图2所示，基因工程菌WT-MG与WT在生长上无显著差异。
培养7h后，检测血红素含量，WT-MG血红素含量为2.86μmol/L。
实施例3
将谷氨酰-tRNA还原酶hemA基因(核苷酸序列如SEQ ID NO.3所示)通过Hind Ⅲ和EcoR I，连接至pET28a载体上得到重组质粒pET28a-hemA，再将重组质粒转入JM109中，将菌液涂布于LB平板上，在37℃下培养至长出单克隆，挑取单克隆并进行测序验证，验证正确的即为阳性转化子。将阳性转化子接种至LB液体培养基中，培养8～12h，从菌液中提取重组质粒pET28a-hemA，将重组质粒导入实施例1得到的菌株WT-M中，得到工程菌pEA-M，采用荧光法检测血红素浓度，结果表明pEA-M的血红素含量达到45.8μmol·L -1。
将pET28a质粒空载转化至BL21中，得到工程菌pET，利用上述培养方法，将工程菌pET进行培养，培养结束后采用荧光法检测血红素浓度，结果表明pET的血红素含量达到1.7μmol·L -1。
实施例4
具体实施方式同实施例3，区别在于，将实施例3构建得到的重组质粒pET28a-hemA转化至实施例2得到的菌株WT-MG中，得到工程菌pEA-MG，采用荧光法检测血红素浓度，结果表明pEA-MG的血红素含量达到47.6μmol·L -1，是出发菌株WT的23倍。
实施例5
具体实施方式同实施例3，区别在于，将实施例3构建得到的重组质粒pET28a-hemA转化至BL21中，得到工程菌pEA，采用荧光法检测血红素浓度，结果表明pEA的血红素含量 达到34.1μmol·L -1。
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 (17)

  1. 一种重组大肠杆菌，其特征在于，敲除或沉默了编码小电导类机械敏感型离子通道蛋白的基因mscS、敲除或沉默了编码3-脱氧-阿拉伯庚酮糖-7磷酸合成酶的基因aroG，并过表达编码谷氨酰-tRNA还原酶的基因hemA；所述基因mscS的核苷酸序列如SEQ ID NO.1所示；所述基因aroG的核苷酸序列如SEQ ID NO.2所示；所述基因hemA的核苷酸序列如SEQ ID NO.3所示。
  2. 一种重组大肠杆菌，其特征在于，敲除或沉默了编码小电导类机械敏感型离子通道蛋白的基因mscS、敲除或沉默了编码3-脱氧-阿拉伯庚酮糖-7磷酸合成酶的基因aroG，或过表达编码谷氨酰-tRNA还原酶的基因hemA。
  3. 根据权利要求2所述的重组菌，其特征在于，所述基因mscS的核苷酸序列如SEQ ID NO.1所示；所述基因aroG的核苷酸序列如SEQ ID NO.2所示；所述基因hemA的核苷酸序列如SEQ ID NO.3所示。
  4. 根据权利要求2所述的重组菌，其特征在于，在大肠杆菌中敲除或沉默基因mscS和基因aroG。
  5. 根据权利要求2所述的重组菌，其特征在于，在大肠杆菌中敲除或沉默基因mscS并过表达基因hemA。
  6. 根据权利要求2所述的重组菌，其特征在于，在大肠杆菌中敲除或沉默基因mscS和基因aroG，并过表达基因hemA。
  7. 根据权利要求2所述的重组菌，其特征在于，以大肠杆菌BL21为出发菌株。
  8. 构建权利要求1～7任一所述重组大肠杆菌的方法，其特征在于，通过Red同源重组的方法将大肠杆菌基因组的基因mscS或基因aroG敲除。
  9. 根据权利要求8所述的方法，其特征在于，
    设计如SEQ ID NO.1核苷酸序列所示的基因的同源臂，同源臂为基因上下游各400～600bp，再通过Red同源重组的方法将大肠杆菌基因组的基因mscS敲除；或
    设计如SEQ ID NO.2核苷酸序列所示的基因的同源臂，同源臂为基因上下游各40～60bp，再通过Red同源重组的方法将大肠杆菌基因组的基因aroG敲除。
  10. 根据权利要求8所述的方法，其特征在于，将基因hemA连接至pET载体上，构建得到表达载体，再将表达载体转入宿主细胞。
  11. 一种提高血红素产量的方法，其特征在于，在大肠杆菌中敲除编码小电导类机械敏感型离子通道蛋白的基因mscS、敲除编码3-脱氧-阿拉伯庚酮糖-7磷酸合成酶的基因aroG，或过表达编码谷氨酰-tRNA还原酶的基因hemA。
  12. 根据权利要求11所述的方法，其特征在于，在大肠杆菌中敲除或沉默基因mscS和 基因aroG。
  13. 根据权利要求11所述的重组菌，其特征在于，在大肠杆菌中敲除或沉默基因mscS并过表达基因hemA。
  14. 根据权利要求11所述的重组菌，其特征在于，在大肠杆菌中敲除或沉默基因mscS和基因aroG，并过表达基因hemA。
  15. 一种生产血红素的方法，其特征在于，将权利要求1～7任一所述重组菌加入培养体系中生产血红素。
  16. 根据权利要求15所述的方法，其特征在于，培养体系中含有胰蛋白胨和酵母粉；在30～37℃条件下培养6～8h。
  17. 权利要求1～7任一所述重组大肠杆菌，或权利要求8～16任一所述方法在生产血红素中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975837A (zh) * 2022-07-12 2023-04-18 浙江工业大学 大肠杆菌zjut-mhol及其在产血红素加氧酶-1中的应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109182232B (zh) * 2018-07-30 2022-04-19 浙江工业大学 重组大肠杆菌zjut-ho1及其在制备胆绿素中的应用
CN111593061A (zh) * 2020-07-06 2020-08-28 江南大学 一种提高染料脱色过氧化物酶活性的方法
CN116162583B (zh) * 2022-11-30 2025-09-23 江南大学 一种提高重组大肠杆菌血红素合成的方法
CN116410914A (zh) * 2023-05-18 2023-07-11 华中农业大学 一种发酵脱臭解淀粉芽胞杆菌工程菌及其在血红素合成中的应用
CN117603930B (zh) * 2024-01-23 2024-04-16 黑龙江新和成生物科技有限公司 一种表达突变型西罗血红素合酶的重组菌

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133056A1 (en) * 2010-04-23 2011-10-27 Instytut Biologii Doświadczalnej Im. M. Nenckiego Pan Mutant strains of escherichia coli, a method of testing potential antibacterial agents using said strains as well as a testing kit
CN106434509A (zh) * 2016-10-12 2017-02-22 江南大学 一种提高大肠杆菌合成血红素的方法
CN110591989A (zh) * 2018-08-03 2019-12-20 中国科学院天津工业生物技术研究所 一株高产l-色氨酸工程菌株及其应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1499739B1 (en) * 2002-04-22 2016-03-30 Novozymes, Inc. Methods for increasing homologous recombination of a nucleic acid sequence
CN104694612B (zh) * 2015-02-12 2017-12-29 新疆阜丰生物科技有限公司 一种工业化发酵高产l‑色氨酸的方法
US20160376564A1 (en) * 2015-06-26 2016-12-29 MetaMixis, Inc. Nucleic Acids and Polypeptides for Utilizing Plant Biomass
US20190282628A1 (en) * 2016-01-11 2019-09-19 Synlogic Inc. Recombinant bacteria engineered to treat diseases and disorders associated with amino acid metabolism and methods of use thereof
KR102168039B1 (ko) * 2017-12-12 2020-10-21 한국과학기술원 대사공학적으로 조작된 미생물을 이용한 헴의 세포외 생산방법
CN109943544A (zh) * 2019-04-02 2019-06-28 江南大学 一种提高重组染料脱色过氧化物酶酶活的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133056A1 (en) * 2010-04-23 2011-10-27 Instytut Biologii Doświadczalnej Im. M. Nenckiego Pan Mutant strains of escherichia coli, a method of testing potential antibacterial agents using said strains as well as a testing kit
CN106434509A (zh) * 2016-10-12 2017-02-22 江南大学 一种提高大肠杆菌合成血红素的方法
CN110591989A (zh) * 2018-08-03 2019-12-20 中国科学院天津工业生物技术研究所 一株高产l-色氨酸工程菌株及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FU WEIQI: "Studies on 5-Aminoleyulinate Production via Recombinants Optimization and Process Design", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 1 April 2009 (2009-04-01), pages 1 - 139, XP055886411 *
PAN MEI, LIU SHUANGXIN, TANG LEI: "Enhancing Heme Synthesis in Escherichia coli by Regulating Glutamic Acid Metabolism and Over-Expressing hemA", MICROBIOLOGY CHINA, vol. 48, no. 3, 23 July 2020 (2020-07-23), pages 701 - 709, XP055886430, DOI: 10.13344/j.microbiol.china.200434 *
TOBLAS DASSLER , THOMAS MAIER , CHIRISTOPH WINTERHALTER , AUGUST BOCK: "Identification of a major facilitator from escherichia coli involved in efflux of metabolites of the cystiene pathway", MOLECULAR MICROBIOLOGY, vol. 36, no. 5, 1 June 2000 (2000-06-01), GB , pages 1101 - 1112, XP000992212, ISSN: 0950-382X, DOI: 10.1046/j.1365-2958.2000.01924.x *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975837A (zh) * 2022-07-12 2023-04-18 浙江工业大学 大肠杆菌zjut-mhol及其在产血红素加氧酶-1中的应用

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