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Genetically engineered bacterium for producing N-acetyl-5-methoxytryptamine as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and methoxytryptamine, applied in the field of genetic engineering, can solve the problems of high extraction cost of natural N-acetyl-5-methoxytryptamine, low toxic substances, and low content, so as to improve synthetic yield, The effect of increasing production

Pending Publication Date: 2021-10-22
河北维达康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the N-acetyl-5-methoxytryptamine in living organisms is safe and reliable, the extraction cost of natural N-acetyl-5-methoxytryptamine is too high due to its extremely small content
Although the chemical synthesis method can produce N-acetyl-5-methoxytryptamine in large quantities, it has disadvantages that cannot be ignored: toxic substances are used in the production process, complex and cumbersome synthesis steps, too many by-products, and low purity

Method used

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  • Genetically engineered bacterium for producing N-acetyl-5-methoxytryptamine as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing N-acetyl-5-methoxytryptamine as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing N-acetyl-5-methoxytryptamine as well as construction method and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0051] Through genetic engineering technology, the caffeic acid O-methyltransferase COMT gene is expressed on the plasmid, and the N-acetyl-5-methoxytryptamine biosynthetic pathway is rebuilt in Escherichia coli. With N-acetyl-5-serotonin as a substrate, N-acetyl-5-serotonin can be converted into N-acetyl-5-methoxytryptamine after whole-cell catalysis. Specific steps are as follows:

[0052] 1. Construction of pRSFDuet1-COMT recombinant plasmid

[0053] Use the first primer to amplify the caffeic acid O-methyltransferase COMT gene from the three artificially synthesized COMT genes, and connect the fragment to the multiple cloning site I of the plasmid vector pRSFDuet-1 by enzyme-cut ligation The recombinant plasmid pRSFDuet1-COMT was obtained.

[0054] 2. Preparation of chemically competent E. coli

[0055] Escherichia coli (Escherichia coli) BL21 (DE3) was inoculated onto LB plate medium without antibiotics and cultured overnight at 37°C. A well-growing single colony was ...

Embodiment 2

[0073] Through genetic engineering technology, the caffeic acid O-methyltransferase COMT gene and the methionine adenylyltransferase gene MAT are co-expressed on the plasmid to reconstitute N-acetyl-5-methionine in Escherichia coli Oxytryptamine biosynthetic pathway. With N-acetyl-5-serotonin as a substrate, N-acetyl-5-serotonin can be converted into N-acetyl-5-methoxytryptamine after whole-cell catalysis. Specific steps are as follows:

[0074] 1. Construction of pRSFDuet1-COMT-MAT ​​recombinant plasmid

[0075] The second primer was used to amplify the methionine adenylyltransferase gene MAT from six artificially synthesized MAT genes, and the fragments were respectively connected to the multiple cloning site II of the plasmid vector pRSFDuet1-COMT by enzyme-cut ligation The recombinant plasmid pRSFDuet1-COMT-MAT ​​was obtained.

[0076] 2. Preparation of chemically competent E. coli

[0077] Escherichia coli (Escherichia coli) BL21 (DE3) was inoculated onto LB plate med...

Embodiment 3

[0095] Through genetic engineering technology, the caffeic acid O-methyltransferase COMT gene and the methionine adenylyltransferase gene MAT are integrated and expressed in the genome to reconstruct N-acetyl-5-methoxy The biosynthetic pathway of tryptamine uses N-acetyl-5-serotonin as a substrate, and can convert N-acetyl-5-serotonin into N-acetyl-5-methoxytryptamine after whole-cell catalysis. Specific steps are as follows:

[0096] 1. Obtain the first step homologous recombination fragment I of knocking out TrpR

[0097] Using the pXZ-SC plasmid (Tan et al, 2013AEM) as a template, PCR amplification was performed using primers TrpR-F and TrpR-R, and a PCR product of about 3kb was obtained, which was the first step homologous recombination fragment DNA, including cat, sacB gene and 50bp upstream and downstream homology arms.

[0098] 2. Use the DNA fragment of the knockout TrpR gene for the first step of homologous recombination

[0099] The pTKred (Cox et al, 2010 Nuclei ...

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Abstract

The invention relates to a genetically engineered bacterium for producing N-acetyl-5-methoxytryptamine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium for producing the N-acetyl-5-methoxytryptamine comprises a caffeic acid O-methyltransferase gene and a methionine adenosine transferase gene or / and a methionine synthetase gene, and can be used for producing a large amount of caffeic acid O-methyltransferase with biological activity and methionine adenosine transferase or / and methionine synthetase in cells. According to the method, through overexpression of the caffeic acid-O methyltransferase (COMT) gene and the methionine adenosine transferase gene or / and the methionine synthetase gene, an N-acetyl-5-methoxytryptamine biosynthesis pathway is reconstructed, and the synthesis yield of N-acetyl-5-methoxytryptamine is obviously increased.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a genetic engineering bacterium for producing N-acetyl-5-methoxytryptamine, a construction method and application. Background technique [0002] N-acetyl-5-methoxytryptamine (melatonin) in vertebrates is a hormone produced in the brain by the pineal gland that regulates behavioral functions and circadian rhythms in animals. In addition to vertebrates, N-acetyl-5-methoxytryptamine has been found to be present in all known organisms, including bacteria, insects, fungi, animals and plants. N-acetyl-5-methoxytryptamine plays a variety of roles, from antioxidant activity to various biological functions of animals and plants, such as antioxidant effects, innate immunity, stem cell differentiation and DNA repair, etc., in plant growth, It also has diverse physiological roles in development and defense against a variety of abiotic and biotic stresses. [0003] The effects of N-acetyl-5...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/54C12N9/10C12P17/10C12R1/19
CPCC12N9/1007C12N9/1085C12N15/70C12N15/902C12P17/10C12Y201/01068C12Y205/01006
Inventor 冯斌王秋月展全乐王珂旭
Owner 河北维达康生物科技有限公司
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