Method for increasing heme synthesized from escherichia coli

A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of metabolic engineering, can solve the problem of not considering the relationship between gene expression intensity of heme synthesis pathway and synthetic heme

Active Publication Date: 2017-02-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current research on the heme synthesis pathway mainly focuses on the analysis of the influence of a single gene or several genes on heme production, without considering the relationship between the expression intensity of the heme synthesis pathway genes and the synthesis of heme

Method used

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  • Method for increasing heme synthesized from escherichia coli
  • Method for increasing heme synthesized from escherichia coli
  • Method for increasing heme synthesized from escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Amplification of gene fragments related to heme synthesis pathway

[0060] Using the E.coli DH5α genome as a template, hemB (989bp), hemC (956bp), hemD (755bp), hemE (1079bp), hemF (914bp), hemG (560bp ), hemH (977bp ) genes were amplified respectively, and then fused PCR fused the hemC and hemD fragments into hemC-hemD, and the hemG and hemH fragments into hemG-hemH. Primers are shown in Table 1.

[0061] Table 1 Primers

[0062]

Embodiment 2

[0063] Example 2 Construction of Escherichia coli Synthetic Heme Related Recombinant Engineering Strains

[0064]The fragment hemC-hemD was connected with the expression vectors pRSFDuet-2, pCDFDuet-2, pACYCDuet-2 respectively by using restriction sites PstⅠ and HindⅢ to obtain recombinant vectors pRSFDuet-2-hemC-hemD, pCDFDuet-2-hemC-hemD, pACYCDuet -2-hemC-hemD. The fragment hemB was connected with the vectors pRSFDuet-2-hemC-hemD, pCDFDuet-2-hemC-hemD, pACYCDuet-2-hemC-hemD respectively by BamHI and SacⅠ, and the recombinant vectors pRSFDuet-2-hemB-hemC-hemD, pCDFDuet- 2-hemB-hemC-hemD, pACYCDuet-2-hemB-hemC-hemD. The amplified hemE fragments were ligated into pRSFDuet-2-hemB-hemC-hemD, pCDFDuet-2-hemB-hemC-hemD, and pACYCDuet-2-hemB-hemC-hemD through restriction sites NdeI and XhoI, respectively, to obtain Recombinant plasmids pRSFDuet-2-hemB-hemC-hemD-hemE, pCDFDuet-2-hemB-hemC-hemD-hemE, pACYCDuet-2-hemB-hemC-hemD-hemE.

[0065] The fragment hemF amplified from the Es...

Embodiment 3

[0072] Embodiment 3 Recombinant bacteria shaking flask fermentation verification

[0073] With E.coli DH5α: pUC19-hemA-hemL as the control,

[0074] Recombinant strains:

[0075] (a) E.coli DH5α: pUC19-hemA-hemL+pRSFDuet-2-hemB-hemC-hemD-hemE+pCDFDuet-2-hemF-hemG-hemH;

[0076] (b) E.coli DH5α: pUC19-hemA-hemL+pRSFDuet-2-hemB-hemC-hemD-hemE+pACYCDuet-2-hemF-hemG-hemH;

[0077] (c) E.coli DH5α: pUC19-hemA-hemL+pCDFDuet-2-hemB-hemC-hemD-hemE+pRSFDuet-2-hemF-hemG-hemH;

[0078] (d) E.coli DH5α: pUC19-hemA-hemL+pCDFDuet-2-hemB-hemC-hemD-hemE+pACYCDuet-2-hemF-hemG-hemH;

[0079] (e) E.coli DH5α: pUC19-hemA-hemL+pACYCDuet-2-hemB-hemC-hemD-hemE+pRSFDuet-2-hemF-hemG-hemH;

[0080] (f) E. coli DH5α: pUC19-hemA-hemL+pACYCDuet-2-hemB-hemC-hemD-hemE+pCDFDuet-2-hemF-hemG-hemH.

[0081] Ferment the recombinant Escherichia coli (a), (b), (c), (d), (e), (f) and the control strain in 250mL Erlenmeyer flasks respectively, the inoculum size is 1%, and the initial glucose concentration is 20...

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Abstract

The invention discloses a method for increasing heme synthesized from escherichia coli and belongs to the technical field of metabolic engineering. Synthetic route genes of the heme from the escherichia coli are divided into two modules, the two modules are combined at random by the aid of plasmids with different copy numbers, and a heme synthetic route is enhanced on the basis of overexpressing glutamyl-tRNA reductase (a hemA code) and glutamyl aldehyde aminotransferase (hemL code). According to the method, a constructed recombinant escherichia coli engineering bacterium: E.coli DH 5a:Puc19-hemA-hemL+pACYCDuet-2-hemB-hemC-hemD-hemE+pRSFDuet-2-hemF-hemG-hemH is subjected to fermentation in a shaking flask so as to produce heme cells reaching 0.56uM/OD (micrometer/optical density). The method has the advantages that a metabolic engineering strategy is adopted to transform microbiological strains to synthesize the target product, the heme, and certain foundations are established for efficient heme production by microbiological methods.

Description

technical field [0001] The invention relates to a method for improving hemoglobin synthesis by Escherichia coli, belonging to the technical field of metabolic engineering. Background technique [0002] Heme is an iron-containing porphyrin derivative, composed of protoporphyrin and a ferrous iron atom. Its core is ferrous ion, which has the ability to carry oxygen and is an important component of the electron transport chain protein in aerobic respiration and anaerobic respiration. In addition, it is also the prosthetic group of many sensory regulatory proteins and enzymes, and participates in many important physiological and biochemical reaction processes in biological cells, such as electron transfer, gas transport, signal transduction in cells, superoxide reduction, etc. And is an excellent source of iron for many organisms. At present, heme is not only often used as a coloring agent in the food industry, such as intestinal products, but also widely used in clinical medi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P17/18C12N15/66C12R1/19
Inventor 康振陈坚堵国成翁焕娇
Owner JIANGNAN UNIV
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