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Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid

A technology of Corynebacterium glutamicum and aminolevulinic acid, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., to achieve the effect of promoting transportation and increasing yield

Inactive Publication Date: 2017-02-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to the search, there is no report of overexpressing the 5-aminolevulinic acid transporter gene of Corynebacterium glutamicum to increase the production of 5-ALA

Method used

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  • Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid
  • Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid
  • Corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of knockout plasmid pD-sacB and construction of overexpression 5-aminolevulinic acid synthase gene plasmid pXA

[0026] Construction of pD-sacB plasmid:

[0027] Firstly, the linear fragment of pK18mobsacB cut by HindIII was used as a template, and the sacB gene was amplified with the following primers sacB-1 / sacB-2. The sacB gene fragment was ligated with the plasmid pEC-XK99E after the MunI / EcoRV double digestion and the EcoRI / SmalI double digestion to obtain the plasmid pEC-XK99E-sacB. Use the following primers trcsacB-1 / trcsacB-2 to amplify the trcsacB fragment containing the trc promoter using the pEC-XK99E-sacB plasmid as a template.

[0028] Use the following primers pD-1 / pD-2, and use the pK18mobsacB plasmid as a template to amplify the pD fragment containing kanamycin resistance and E. The pD fragments were ligated to obtain plasmid pD-sacB.

[0029] Construction of pXA plasmid:

[0030] The 5-aminolevulinic acid synthase gene hemA o...

Embodiment 2

[0031] Example 2: Knockout of the lactate dehydrogenase encoding gene ldhA and knockout of the acetate production pathway genes pta-ackA, pqo and cat

[0032] Knockout of the gene ldhA encoding lactate dehydrogenase:

[0033] Using the genome of Corynebacterium glutamicum (C. glutamicum) ATCC 13032 as a template, using ldh-1 / ldh-2 as primers to amplify the upstream fragment of gene ldhA, and ldh-3 / ldh-4 as primers to amplify the downstream of gene ldhA fragment. After the two fragments were recovered by gel cutting, the fusion product of the two fragments was amplified using the equimolar proportion of the fragments as a template and ldh-1 / ldh-4 as primers. The fused fragment was digested with EcoRI / HindIII and ligated with pD-sacB after the same double digestion to obtain plasmid pD-ldhA.

[0034] Transfer the pD-ldhA plasmid into C.glutamicum ATCC 13032, and use kanamycin to screen the positive clones with successful recombination. The selected transformants were inoculate...

Embodiment 3

[0048] Example 3: Insertion of sod promoter and phosphoenolpyruvate carboxykinase encoding gene pck in front of ppc gene. Insertion of sod promoter in front of ppc gene

[0049] Using C. glutamicum ATCC13032 genome as a template and ppc-1 / ppc-2 as primers to amplify the upstream fragment of gene ppc. sod-1 / sod-2 are used to amplify the promoter of the sod gene. ppc-3 / ppc-4 is used to amplify the downstream fragment of the ppc gene. After the three fragments are cut and recovered, the equimolar proportion of the fragment is used as a template, and ppc-1 / ppc-4 is used as a primer to amplify the obtained Fusion product of three fragments. The fused fragment was digested with XbaI / HindIII and ligated with the plasmid vector pD-sacB after the same double digestion. The plasmid pD-ppc was obtained.

[0050] The constructed plasmid pD-ppc was transformed into the strain CG4 by electroporation, the positive clones with successful recombination were screened with kanamycin, and the ...

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Abstract

The invention discloses a corynebacterium glutamicum recombinant strain for producing 5-aminolevulinic acid. An establishing method comprises steps as follows: (1) a lactic dehydrogenase encoding gene ldhA and acetic acid producing genes pta-ackA, pqo and cat are knocked out from corynebacterium glutamicum ATCC 1303, and a strain CG4 is obtained; an sod promoter is inserted in front of a phosphoenolpyruvate carboxylase encoding gene in the CG4, and a strain CG5 is obtained; a phosphoenolpyruvate carboxykinase encoding gene pck is knocked out from the CG5, and a strain CG6 is obtained; (2) plasmids of an overexpressed 5-aminolevulinic acid synthase gene are transferred into the CG6, and a recombinant strain L is obtained; (3) 5-aminolevulinic acid transport protein plasmids are transferred into the L. The recombinant strain can promote 5-aminolevulinic acid to be transported outside corynebacterium glutamicum cells, and the yield of 5-aminolevulinic acid is 112.3% higher than that of a contrast strain.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, in particular to a recombinant strain of Corynebacterium glutamicum producing 5-aminolevulinic acid and its construction and application. Background technique [0002] 5-Aminolevulinic acid (5-ALA), molecular weight 131.13, melting point 118°C. It is a non-protein amino acid. Because 5-aminolevulinic acid has the characteristics of less side effects and good permeability, it has been widely used in the diagnosis of skin cancer, bladder cancer, digestive tract cancer, lung cancer and photodynamic therapy (PDT). [0003] 5-Aminolevulinic acid is the precursor of pyrrole compounds in organisms and has a wide range of applications. In agriculture, since 5-aminolevulinic acid is easily degraded in the environment, it is harmless to mammals and can selectively kill pests, so it can be widely used as a photodynamic insecticide. In addition, 5-aminolevulinic acid has also recei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/00C12R1/15
CPCC12N9/0006C07K14/34C12N9/1029C12N9/88C12P13/005C12Y203/01037C12Y401/01032
Inventor 王智文邹亚兰陈涛赵学明
Owner TIANJIN UNIV
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