Corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain

A technology of Corynebacterium glutamicum and aminolevulinic acid, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of low yield and yield of 5-aminolevulinic acid, complicated pathways, etc.

Inactive Publication Date: 2016-10-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are only a few reports about the production of 5-aminolevulinic acid by Corynebacterium glutamicum using the C5 pathway, but the pathway is relatively complicated, and the yield and yield of 5-aminolevulinic acid are very low
[0005] According to the search, there is no report about Corynebacterium glutamicum using C4 pathway to produce 5-aminolevulinic acid

Method used

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  • Corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain
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  • Corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Construction of knockout plasmid pD-sacB

[0025] 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.

[0026] 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 digested by AatII was ligated with the pD fragment digested by the same restriction enzyme to obtain plasmid pD-sacB.

Embodiment 2

[0027] Example 2: Knockout of the lactate dehydrogenase encoding gene ldhA and knockout of the acetate production pathway genes pta-ackA, pqo and cat

[0028] Knockout of the gene ldhA encoding lactate dehydrogenase:

[0029] Using Corynebacterium glutamicum (C.glutamicum) ATCC 13032 genome as a template, using ldh-1 (SEQ ID NO.7) / ldh-2 (SEQ ID NO.8) as primers to amplify the upstream fragment of gene ldhA, ldh- 3(SEQ ID NO.9) / ldh-4(SEQ ID NO.10) 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.

[0030] Transform the pD-ldhA plasmid into Corynebacterium glutamicum (C.glutamicum) ATCC 13032, use kanamycin to screen the positive clon...

Embodiment 3

[0044] Example 3: Insertion of a strong sod promoter in front of the ppc gene and knockout of the pck gene, the gene encoding phosphoenolpyruvate carboxykinase

[0045] A strong sod promoter was inserted in front of the ppc gene

[0046] C. glutamicum ATCC13032 genome was used as a template, and ppc-1 (SEQ ID NO. 23) / ppc-2 (SEQ ID NO. 24) was used as primers to amplify the upstream fragment of gene ppc. sod-1 (SEQ ID NO.25) / sod-2 (SEQ ID NO.26) was used to amplify the promoter of sod gene. ppc-3 (SEQ ID NO.27) / ppc-4 (SEQ ID NO.28) is used to amplify the downstream fragments of the ppc gene. After the three fragments are gel-cut and recovered, the equimolar ratio fragments are used as templates. 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.

[0047] The constructed plasmid was...

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Abstract

The invention discloses a corynebacterium glutamicum strain for production of 5-aminolevulinic acid and construction and application of corynebacterium glutamicum strain. A construction method includes: (1) deleting a lactic dehydrogenase coding gene 1dhA and acetic acid generation genes pta-ackA, pqo and cat in corynebacterium glutamicum to obtain a strain named CB4; inserting a strong sod promoter in front of a phosphoenolpyruvate carboxylase coding gene ppc in the strain CB4 to obtain a strain CB5; deleting a gene pck in the strain CB5 to obtain a strain CB6; (2) transferring plasmid pXA and plasmid pEP2<tuf>-rhtA into the strain CB6. The strain constructed according to the method is capable of generating 2.78g/L 5-aminolevulinic acid in a culture medium with 10g/L glucose serving as a carbon source, which lays a foundation for subsequent continuous feeding of a fermentation tank to increase yield of the 5-aminolevulinic acid.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and in particular relates to a Corynebacterium glutamicum strain for 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. 5-aminolevulinic acid has the characteristics of less side effects and good permeability, and has been widely used in the diagnosis of skin cancer, bladder cancer, digestive tract cancer, lung cancer and photodynamic therapy (PDT). At the same time, 5-aminolevulinic acid also has very important applications in agriculture such as plant growth regulators, herbicides and green pesticides. [0003] In nature, there are two main pathways for the biosynthesis of 5-aminolevulinic acid: the C4 pathway and the C5 pathway. The C4 pathway is the condensation of succinyl CoA and glycine under the act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N1/21C12P13/00C12R1/15
CPCC12N15/77C12N9/0006C12N9/0008C12N9/88C12P13/00C12Y101/01027C12Y101/01028C12Y401/01032
Inventor 王智文冯丽丽陈涛赵学明
Owner TIANJIN UNIV
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