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Glyoxylate aminotransferase mediated biosynthesis pathway of 5-aminolevulinic acid

A technology of glyoxylate transaminase and glycine transaminase, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems of loss of carbon source, long synthesis pathway, high toxicity of reaction solvent, etc., and achieve the effect of increasing yield

Active Publication Date: 2018-09-14
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] 5-ALA was mainly chemically synthesized in the early days, and the raw materials could be hippuric acid, succinic acid, tetrahydrofurfurylamine and levulinic acid, etc., but most of the chemical methods have high cost of raw materials, harsh reaction conditions, high toxicity of reaction solvents and high yields. Disadvantages such as low rate
In terms of raw material supply, glycine, as one of the raw materials for 5-ALA synthesis, has a long synthetic pathway, which is difficult to regulate and strengthen, and a molecule of 5,10-methylenetetrahydrofolate will be generated in the glycine synthetic pathway. source, but also the burden of metabolic regulation
At present, most studies still use exogenous glycine as the raw material supply, and a few regulations on the supply of glycine have not played a significant role

Method used

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  • Glyoxylate aminotransferase mediated biosynthesis pathway of 5-aminolevulinic acid
  • Glyoxylate aminotransferase mediated biosynthesis pathway of 5-aminolevulinic acid
  • Glyoxylate aminotransferase mediated biosynthesis pathway of 5-aminolevulinic acid

Examples

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Effect test

Embodiment 1

[0038] Example 1: In vitro verification of new pathways for 5-ALA biosynthesis

[0039] ①The glyoxylate transaminases from different sources were connected to the expression vector pet21a to construct recombinant expression vectors, respectively: alanine-glyoxylate transaminase pet21a-AGXT derived from Homo sapiens; Saccharomyces cerevisiae Alanine-glyoxylate transaminase pet21a-ScTA derived from Hyphomicrobium methylovorum; Alanine-glyoxylate transaminase pet21a-HmTA derived from Hyphomicrobium methylovorum;

[0040] ② Construction of isocitrate lyase gene expression vector pet21a-aceA;

[0041] ③ Construct the expression vector pet22b-sucCD of succinyl-CoA synthetase;

[0042] ④ Construct the expression vector pet22b-hemA of 5-ALA synthetase.

[0043] Transform the above expression vectors into the E. coli expression host E.coli BL21star (DE3), ferment at 37°C until the OD reaches 0.5-0.6, add 0.1mM IPTG to induce gene expression, continue to culture at 30°C for 10 hours, an...

Embodiment 2

[0046] Example 2: Construction of plasmids containing 5-ALA synthetase gene, glyoxylate transaminase gene and isocitrate lyase gene

[0047] (1) Gene synthesis and amplification: the 5-ALA synthetase gene hemA (shown in SEQ ID NO.1) and the glyoxylate transaminase gene AGXT (shown in SEQ ID NO.2) derived from Rhodobacter sphaeroides were combined (shown) were sent to Nanjing GenScript Biotech Co., Ltd. for synthesis to obtain plasmids pUC-hemA and pUC-AGXT. The isocitrate lyase gene aceA was amplified from Escherichia coli MG1655 with primers aceA-IF and aceA-IR for future use.

[0048] (2) Construction of plasmid pETDuet-1-hemA: the vector fragment (backbone) was amplified from plasmid pETDuet-1 with primers ACYCDuet-VecF and DuetDOWN1, and the insert fragment was amplified from plasmid pUC-hemA with primers DuetUP2 and T7Terminator (In-hemA), after the two fragments were purified and recovered, they were connected by means of Gibson assembly (Gibson assembly). The ligation...

Embodiment 3

[0053] Embodiment 3: Containing the construction of the recombinant strain of 5-ALA biosynthesis new pathway

[0054] Insert 100 μL of E. coli BL21star (DE3) into 20 mL of LB medium and store 100 μL of glycerol strains. Cultivate at 37 ° C and 220 r / min for 5 hours until the OD value reaches 0.5. Centrifuge 2 mL of the bacterial liquid, discard the supernatant, and The cell pellet was washed by re-spinning with 0.1M calcium chloride, the supernatant was discarded after centrifugation, and the washing was repeated. The cell pellet was re-spinned with 0.1M calcium chloride to obtain E.coliBL21star (DE3) cell competent cells.

[0055] Plasmid pETDuet-1-hemA and plasmid pRSFduet-1-AGXT-aceA were extracted from ToP10 (pETDuet-1-hemA) and ToP10 (pRSFduet-AGXT-aceA) obtained in Example 2, respectively.

[0056] Add 2 μL pETDuet-1-hemA plasmid to E.coli BL21star (DE3) competent cells, let it stand on ice for 30 minutes, place it in a water bath at 42°C for 90 seconds, then place it on...

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Abstract

The invention belongs to the technical fields of gene engineering and microbial fermentation, and in particular relates to a glyoxylate aminotransferase mediated biosynthesis pathway of 5-aminolevulinic acid. According to biosynthesis pathway, a 5-ALA synthase gene hemA is expressed by introducing heterologous glyoxylate aminotransferase, the expression of an isocitrate lyase gene aceA is simultaneously enhanced, and a production pathway of non-natural novel 5-aminolevulinic acid is established, so that efficient conversion from glucose to the 5-ALA is realized, the problem of exogenous addition of glycine in the current C4 production pathway is solved, and the molecular carbon lost during generation of glycine in the original C4 pathway can be avoided. After the biosynthesis pathway is adopted, the yield of the synthesized 5-ALA can be increased by 38%.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and specifically relates to a method for constructing a non-natural novel 5-aminolevulinic acid production pathway by introducing heterologous glycine transaminase. Background technique: [0002] 5-aminolevulinic acid (5-ALA) is an essential substance for organisms to synthesize chlorophyll, heme, and vitamin B12. 5-ALA has important applications in agriculture, medicine and organic synthesis. Studies have shown that 5-ALA regulates the synthesis of chlorophyll, improves photosynthesis efficiency and promotes the physiological functions of plant tissue differentiation. It can be used as a plant growth regulator in agriculture. In addition, it can also be used as a herbicide and insecticide, and it is biodegradable. It does not cause pests to develop resistance, and is a new type of green pesticide. However, due to the high market price and high applicatio...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/00C12R1/19C12R1/01C12R1/38C12R1/15
CPCC12N9/00C12N9/1096C12N9/88C12P13/005C12Y206/01C12Y401/03001
Inventor 曾安平任杰周立邦
Owner BEIJING UNIV OF CHEM TECH
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