High-yield 5-aminolevulinic acid strain and its preparation method and application

A technology for producing aminolevulinic acid and bacterial strains, which is applied in the field of genetic engineering and microbial fermentation, and can solve the problems of ALA production by recombinant engineering bacteria and poor overall effect.

Active Publication Date: 2018-01-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above studies have partially attempted the intracellular supply of substrates for ALA synthesis, the overall effect is not good. Therefore, up to now, the method of direct exogenous addition is generally used for the substrate supply of ALA synthesis, especially now ALA synthesis The most commonly used C4 synthesis pathway in the human body, there is no report on the fermentation and production of ALA by recombinant engineering bacteria utilizing the C4 pathway in a medium with cheap glucose as the main carbon source

Method used

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  • High-yield 5-aminolevulinic acid strain and its preparation method and application
  • High-yield 5-aminolevulinic acid strain and its preparation method and application
  • High-yield 5-aminolevulinic acid strain and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Construction of a succinyl-CoA synthetase deletion mutant and a succinate dehydrogenase deletion mutant

[0091] Escherichia coli gene knockout adopts the classic Red recombination method, referring to relevant literature, and the specific operation is as follows: For the knockout of the succinyl-CoA synthetase structural gene sucCD gene, first, according to the genome sequence and auxiliary vector of Escherichia coli MG1655 published by NCBI The primers sucCD-1 and sucCD-2 were designed for the sequence of pKD13. See the primer sequence list for specific sequences, and the Kan resistance gene fragment with upstream and downstream homology arms of the sucCD gene was amplified using pKD13 as a template. The PCR amplification parameters were 94°C for 2min; 94°C for 20s, 64°C for 20s, 72°C for 1min, 30 cycles; 72°C for 5min. After the PCR product gel was recovered, the MG1655 / pKD46 strain was electrotransformed. For the preparation and transformation process of ...

Embodiment 2

[0093] Example 2. Construction of co-expression plasmids for phosphoenolpyruvate carboxylase ppc and ALA synthetase

[0094] Primers ppc-F and ppc-R were designed according to the genome sequence of Escherichia coli MG1655 published by NCBI, and the ppc gene fragment with its own promoter was amplified by PCR using the Escherichia coli MG1655 genome as a template, and the PCR amplification parameter was 94°C for 2 minutes; 94°C for 20s, 60°C for 20s, 72°C for 1.5min, cycle 30 times; 72°C for 5min. After the ppc gene fragment was recovered, it was treated with HindIII, and at the same time, the plasmid pZGA24 carrying ALA synthetase (see reference for the construction of pZGA24: Guo Xiaofei et al., using 5-aminolevulinic acid dehydratase-deficient recombinant Escherichia coli to synthesize 5-aminolevulinic acid , Journal of Tianjin University of Science and Technology, 2012, 27(4): 1-6) was also treated with this enzyme, the vector and fragments were recovered and ligated with ...

Embodiment 3

[0095] Example 3. Construction of co-expression plasmids for pyruvate carboxylase pyc and ALA synthetase

[0096] Primers pyc-1 and pyc-2 were designed according to the sequence of the promoter BBa_J23105 in BioBrick and the genome sequence of Rhizobium CFN42 published by NCBi, and the pyc gene fragment with the constitutive promoter sequence was amplified by PCR using the Rhizobium CFN42 genome as a template , PCR amplification parameters were 94°C for 2min; 94°C for 20s, 60°C for 20s, 72°C for 2min, 30 cycles; 72°C for 5min. After the target fragment is recovered, it is treated with phosphorylase, and at the same time, the pWSK29 vector is treated with restriction endonuclease PvuII and then dephosphorylase, and the obtained vector fragment is connected with the phosphorylated pyc gene fragment with T4 ligase, transformed into DH5α competent cells were spread on LB plates containing Amp, positive clones were picked to extract plasmids and verified by enzyme digestion, and th...

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Abstract

The invention discloses a method for constructing an ALA production strain, that is, enhancing the activity of related enzymes promoting the synthesis of oxaloacetate in the 5-aminolevulinic acid production strain or introducing exogenous related enzymes promoting the synthesis of oxaloacetate , such as phosphoenolpyruvate carboxylase or pyruvate carboxylase, and / or weaken the succinyl-CoA downstream metabolic pathway-related enzymes in the strain, such as succinyl-CoA synthetase or succinate dehydrogenase active. The invention also discloses an ALA high-yield strain constructed by the method and a method for preparing ALA by using the strain. The bacterial strain of the invention can produce ALA efficiently, at low cost and with low pollution without adding exogenous succinic acid.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and microbial fermentation. Specifically, the present invention relates to a high-yield strain of 5-aminolevulinic acid and its preparation method and application. Background technique [0002] 5-Aminolevulinic acid (5-aminolevulinic acid, ALA) is the precursor of tetrapyrrole compounds such as heme, chlorophyll, and vitamin B12, which are important components of cytochrome, hemoglobin, and chloroplast protein. , play an important role in life activities. Due to its degradable, non-toxic and residue-free characteristics, ALA has broad application prospects in the fields of medicine, agriculture, animal husbandry, and daily chemicals, and is an important high value-added bio-based chemical. As a new generation of photodynamic drugs, ALA can be used for cancer treatment, tumor diagnosis and skin disease treatment; as a plant growth regulator, ALA can greatly promote the growth of flow...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/77C12N15/74C12N1/21C12P13/00C12R1/19C12R1/15C12R1/01
CPCC12N9/0006C12N9/001C12N9/88C12N9/93C12P13/005C12Y401/01031C12Y101/01038C12Y203/01037C12Y401/01049C12P13/001
Inventor 郑平陈久洲蒲伟孙际宾马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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