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Genetic engineering strain capable of efficiently synthesizing melatonin and construction method and application of genetic engineering strain

A technology of genetically engineered bacteria and construction method, which is applied in the field of high-efficiency synthesis of melatonin, can solve the problems of low melatonin production and long time consumption

Inactive Publication Date: 2020-07-10
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the low yield and time-consuming problem of biosynthesis of melatonin at present, the present invention provides a genetically engineered bacterium for efficiently synthesizing melatonin. The genetically engineered bacterium overexpresses the tryptophan synthase gene TRP5 and knocks out the indole Amine 2,3-dioxygenase gene BNA2, the starting strain is Saccharomyces cerevisiae

Method used

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  • Genetic engineering strain capable of efficiently synthesizing melatonin and construction method and application of genetic engineering strain
  • Genetic engineering strain capable of efficiently synthesizing melatonin and construction method and application of genetic engineering strain
  • Genetic engineering strain capable of efficiently synthesizing melatonin and construction method and application of genetic engineering strain

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Experimental program
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Embodiment 1

[0044] Example 1. Construction of genetically engineered bacteria for the synthesis of melatonin.

[0045] 1. The TRP5 gene was obtained by PCR amplification, and then the TRP5 gene was ligated into the plasmid pYES2-ADH to construct the recombinant plasmid pYES2-ADH-TRP carrying the TRP5 gene. The specific method is as follows:

[0046] 1) Amplification of tryptophan synthase gene TRP5:

[0047] Query the sequence of the tryptophan synthase gene TRP5 from the NCBI database (GenBank ID: 852858), and entrust Qingdao Qingke Zixi Biotechnology Co., Ltd. to design the primers TRP5-F and TRP5-R sequences shown in Table 1. Yeast BY4741 genomic DNA was used as a template, and the tryptophan synthase gene TRP5 was obtained by PCR amplification. The 5'end and the 3'end respectively carry KpnI and XhoI restriction sites for ligation with the vector pYES2-ADH.

[0048] The amplification system and amplification procedures of the tryptophan synthase gene TRP5 are shown in Table 2 and Table 3, res...

Embodiment 2

[0100] Example 2. Fermentation experiment of recombinant Saccharomyces cerevisiae.

[0101] The recombinant Saccharomyces cerevisiae BY4741-ΔBNA2 constructed in Example 1 was subjected to a melatonin fermentation experiment. The strain contained high-copy TRP5 gene, and its BNA2 gene was successfully knocked out. The wild-type Saccharomyces cerevisiae BY4741 was used as a control strain to compare the degeneration of the respective strains. Melanin production.

[0102] 1. Strain activation and fermentation:

[0103] Take the strain out of the refrigerator at -80℃ and inoculate it into fresh liquid SC-Ura selective medium (recipe: 0.67g amino acid-free yeast nitrogen base, 2g glucose, 0.2g uracil amino acid mixture, dissolved in 100mL deionized In water, autoclave at 115°C for 15min), shake culture at 28°C, and activate once overnight. The seed liquid was inoculated into the fermentation medium (recipe: 0.67g amino acid-free yeast nitrogen base, 10g glucose, 0.2g uracil amino acid m...

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Abstract

The invention relates to a genetic engineering strain capable of efficiently synthesizing melatonin and a construction method and application of the genetic engineering strain. In view of the problemsof biosynthesis of the melatonin at present that the yield is low and consumed time is long, the invention provides the genetic engineering strain capable of efficiently synthesizing the melatonin. The genetic engineering strain over-expresses a tryptophane synthase gene TRP5, an iodolamine-2,3-dioxygenase gene BNA2 is knocked out, and saccharomyces cerevisiae serves as a starting strain. The melatonin is synthesized by using the genetic engineering strain constructed by the method, the efficiency of synthesis is increased obviously, the maximum yield reaches 25.7mg / L and is increased by 12 times or more compared with that of original strains. The genetic engineering strain can be applied to industrial production of the melatonin.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a genetically engineered bacterium for efficiently synthesizing melatonin and a construction method and application thereof. Background technique [0002] Melatonin is also known as pineal hormone, and its chemical name is N-acetyl-5-methoxytryptamine. It is an amine hormone produced by the pineal gland of mammals after being stimulated by darkness. It can improve Sleep quality of animal body. As the animal ages, the secretion of melatonin gradually decreases, which affects the sleep quality of the animal. More and more studies now show that in addition to treating insomnia, melatonin also has a variety of physiological functions such as anti-oxidation, anti-aging, immune regulation, and anti-cancer. It was initially believed that melatonin only exists in animals. Since the 1990s, scientists have successively discovered melatonin in many Chinese medicines and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/90C12P17/10C12R1/865
CPCC12N9/88C12N9/0069C12N15/81C12N15/905C12P17/10C12Y402/0102C12Y113/11052
Inventor 赵宏伟李静媛李高宁
Owner QINGDAO UNIV OF SCI & TECH
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