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Crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction method thereof+

A technology of aminolevulinic acid, Corynebacterium glutamicum, applied in the directions of microorganism-based methods, bacteria, microorganisms, etc.

Inactive Publication Date: 2017-05-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] According to the search, there is no report on the production of 5-aminolevulinic acid by using the penicillin-binding protein knockout strain

Method used

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  • Crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction method thereof+
  • Crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction method thereof+
  • Crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and construction method thereof+

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of knockout plasmid pD-sacB and construction of overexpression 5-aminolevulinic acid synthase gene plasmid pXA

[0031] Construction of pD-sacB plasmid

[0032] 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 (SEQ ID NO.1) / sacB-2 (SEQ ID NO.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. The following primers trcsacB-1 (SEQ ID NO.3) / trcsacB-2 (SEQ ID NO.4) were used to amplify the trcsacB fragment containing the trc promoter using the pEC-XK99E-sacB plasmid as a template.

[0033] Use the following primers pD-1 (SEQ ID NO.5) / pD-2 (SEQ ID NO.6), use the pK18mobsacB plasmid as a template to amplify the pD fragment containing kanamycin resistance and the Escherichia coli replicon, and finally The fragment trcsacB digest...

Embodiment 2

[0036] Example 2: Knockout of the lactate dehydrogenase encoding gene ldhA and knockout of the acetate production pathway genes pta-ackA, pqo and cat

[0037] Knockout of the gene ldhA encoding lactate dehydrogenase:

[0038] Using Corynebacterium glutamicum (C.glutamicum) ATCC 13032 genome as a template, using ldh-1 (SEQ ID NO.9) / ldh-2 (SEQ ID NO.10) as primers to amplify the upstream fragment of gene ldhA, ldh- 3 (SEQ ID NO.11) / ldh-4 (SEQ ID NO.12) are primers for amplifying the downstream fragment of gene ldhA. 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.

[0039]Transfer the pD-ldhA plasmid into C.glutamicum ATCC 13032, and use kanamycin to screen the positive clones with successful reco...

Embodiment 3

[0053] Example 3: Insertion of a strong sod promoter in front of the ppc gene and knockout of the gene pck encoding phosphoenolpyruvate carboxykinase

[0054] A strong sod promoter was inserted in front of the ppc gene

[0055] Using the C. glutamicum ATCC13032 genome as a template and using ppc-1 (SEQ ID NO. 25) / ppc-2 (SEQ ID NO. 26) as primers to amplify the upstream fragment of gene ppc. sod-1 (SEQ ID NO. 27) / sod-2 (SEQ ID NO. 28) was used to amplify the promoter of sod gene. ppc-3 (SEQ ID NO.29) / ppc-4 (SEQ ID NO.30) is used to amplify the downstream fragment of the ppc gene. After the three fragments are cut and recovered, the equimolar ratio of the fragments is used as a template. Using ppc-1 / ppc-4 as primers, the fusion product of the three fragments was amplified. 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.

[0056] The constructed plasmid was transfer...

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Abstract

The invention discloses a crynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid and a construction method thereof. The construction method comprises the steps of 1, knocking out lactic dehydrogenase encoding gene idhA, and acetic acid generation genes pta-ackA, pqo and cat in the corynebacterium glutamicum, inserting a strong sod promoter in front of a phosphoenolpyruvate carboxylase encoding gene ppc, knocking out a phosphoenolpyruvate carboxykinase pck, thus acquiring the strain CB6; 2, knocking out a penicillin-binding protein encoding gene pbpla in the strain CB6, thus acquiring the strain CB7; transferring the plasmid pXA of a built over-expressed 5-aminolevulinic acid synthase gene into the strain CB7, thus acquiring the engineering strain L2. According to the engineering strain provided by the invention, the 5-aminolevulinic acid is produced in a medium in which 10g / L glucose is added as a carbon source; compared with reference strains, the engineering strain is respectively improved by 13.53% and above.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and in particular relates to an engineering strain of Corynebacterium glutamicum producing 5-aminolevulinic acid and its construction and application. Background technique [0002] 5-Aminolevulinic acid has a molecular weight of 131.13 and a melting point of 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-aminolevulini...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12P13/00C12R1/15
CPCC12N9/0006C12N9/1029C12N9/88C12N15/77C12N2800/22C12P13/005C12Y101/01027C12Y203/01037C12Y401/01C12Y401/01031
Inventor 王智文冯丽丽陈涛赵学明
Owner TIANJIN UNIV
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