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Method for increasing content of intracellular heme in escherichia coli

A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of metabolic engineering and can solve problems such as increased cost and inconvenient operation

Active Publication Date: 2020-10-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although exogenous addition of ALA can increase hemoglobin content, it will cause problems such as inconvenient operation and increased cost

Method used

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  • Method for increasing content of intracellular heme in escherichia coli
  • Method for increasing content of intracellular heme in escherichia coli
  • Method for increasing content of intracellular heme in escherichia coli

Examples

Experimental program
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Embodiment 1

[0035] According to SEQ ID NO.1, each 500bp homology arm of its upstream and downstream was designed, through the method of Red homologous recombination (see Datsenko KA.One-step inactivation of chromosome genes in Escherichiacoli K-12 using PCR products.Proceedings of the specific steps National Academy of Sciences of the United States of America, 2000,97(12):6640-6645)) knock out the mscS gene in Escherichia coli BL21, after sequencing verification, obtain the genetically engineered bacterium that knocks out the mscS gene, and name it for WT-M.

[0036] Inoculate the genetically engineered bacteria WT-M into LB medium, activate overnight at 37°C, 200r / min, transfer 500μL of a certain concentration of bacterial solution to 50mL of LB medium the next day, and incubate at 37°C, 200r / min Under culture, measure the bacterial concentration in real time, and make a growth curve, such as figure 2 shown.

[0037] Inoculate strain WT into LB medium, activate overnight at 37°C, 200r...

Embodiment 2

[0041] According to SEQ ID NO.2, the upstream and downstream 50bp homology arms were designed, and the aroG gene was knocked out on the basis of WT-M through the Red homologous recombination method. After sequencing verification, the genetic engineering of knocking out the aroG gene was obtained. bacterium, and named it as WT-MG, and obtained the genetically engineered bacterium.

[0042] Inoculate the genetically engineered bacteria WT-MG into LB medium, activate overnight at 37°C, 200r / min, and take 500μL of the activated bacterial solution the next day and transfer it to 50mL of LB medium, at 37°C, 200r / min Under culture, measure the bacterial concentration in real time, and make a growth curve, such as figure 2 As shown, there was no significant difference in the growth of genetically engineered bacteria WT-MG and WT.

[0043] After culturing for 7 hours, the hemoglobin content was detected, and the hemoglobin content in WT-MG was 2.86 μmol / L.

Embodiment 3

[0045] The glutamyl-tRNA reductase hemA gene (nucleotide sequence shown in SEQ ID NO.3) was connected to the pET28a vector through HindⅢ and EcoR I to obtain the recombinant plasmid pET28a-hemA, and then the recombinant plasmid was transferred into JM109 , spread the bacterial solution on an LB plate, culture at 37°C until a single clone grows, pick a single clone and perform sequencing verification, and the correct verification is a positive transformant. Inoculate the positive transformants into LB liquid medium, cultivate for 8-12 hours, extract the recombinant plasmid pET28a-hemA from the bacterial liquid, introduce the recombinant plasmid into the bacterial strain WT-M obtained in Example 1, and obtain the engineering bacterium pEA-M, The hemoglobin concentration was detected by fluorescence method, and the results showed that the hemoglobin content of pEA-M reached 45.8 μmol L -1 .

[0046] The pET28a plasmid was transformed into BL21 with no load, and the engineered ba...

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Abstract

The invention discloses a method for increasing the intracellular heme content of escherichia coli, and belongs to the field of metabolic engineering. The method comprises the following steps: knocking out a gene mscS for encoding a small-conductivity mechanical sensitive ion channel protein, knocking out a gene aroG for encoding 3-deoxy-arabinoheptulose-7 phosphate synthase, or overexpressing a gene hemA for encoding glutamyl-tRNA reductase in escherichia coli. The constructed recombinant bacteria are cultured in an LB culture medium, the heme content can reach 47.6 [mu]mol.L<-1> and is remarkably improved compared with that of a control strain, and the recombinant bacteria have wide application value.

Description

technical field [0001] The invention relates to a method for increasing the intracellular hemoglobin content of Escherichia coli, specifically, the technology of combining gene knockout and overexpression, which belongs to the technical field of metabolic engineering. Background technique [0002] Heme is an important class of iron-containing porphyrins, involved in electron transfer, active oxygen decomposition, catalytic oxidation of substrates, control of gene expression, etc. In practical applications, heme can be used as a natural pigment for food additives chemically, as an iron supplement or anti-anemia drug for medical care, and for porphyria treatment in terms of disease treatment. Heme is an important cofactor of cellular respiration in most prokaryotic and eukaryotic organisms, and proteins with heme as a prosthetic group are crucial to various biological processes. In the biosynthesis of recombinant enzymes with heme as a cofactor, the deficiency of heme is an i...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/53C12N15/54C12N15/90C12N15/70C12P17/18C12R1/19
CPCC07K14/245C12N9/1085C12N9/0008C12N15/902C12N15/70C12P17/182C12Y205/01054C12Y102/0107C12N2800/80
Inventor 唐蕾潘梅刘爽欣
Owner JIANGNAN UNIV
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