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Genetically engineered bacterium for producing 5-hydroxytryptophan, and construction method and application of genetically engineered bacterium

A technology of hydroxytryptophan and genetically engineered bacteria, applied in the field of genetically engineered bacteria and its construction, can solve the problems that the natural product extraction method is difficult to meet the market demand of 5-HTP, unstable, and the market price of 5-HTP remains high. , to achieve good application prospects, good growth traits, and the effect of convenient trait improvement.

Active Publication Date: 2021-10-26
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can obtain 5-HTP products with high purity, its raw materials need to be purchased from Africa, which makes the supply of raw materials often affected by seasons, supply regions, and even international relations, and is very unstable, which leads to 5-HTP Market prices remain high all year round
[0004] It can be seen that the natural product extraction method has become increasingly difficult to meet the growing market demand for 5-HTP, and various companies are actively looking for and developing other more efficient and convenient methods 5-HTP production method

Method used

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  • Genetically engineered bacterium for producing 5-hydroxytryptophan, and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing 5-hydroxytryptophan, and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing 5-hydroxytryptophan, and construction method and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0088] Construction of strain E.coli HTP10

[0089] 1. Methods of gene editing

[0090] The gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 mediated genome editing. Metabolic Engineering, 2015, 31: 13-21.), the method For the two plasmid maps used see Figure 10 . Among them, pREDCas9 carries the elimination system of gRNA expression plasmid pGRB, the Red recombination system of λ phage and the Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0091] Such as Figure 9 As shown, the specific steps of the method are as follows:

[0092] 1.1 pGRB plasmid construction

[0093] The pur...

Embodiment 2

[0149] Utilize the Escherichia coli genetically engineered bacterium described in Example 1 to produce 5-hydroxytryptophan by fermentation in shake flasks

[0150] 1. Medium

[0151] 1.1 Incline medium

[0152] Glucose 1-3g / L, peptone 5-10g / L, beef extract 5-10g / L, yeast extract powder 2-5g / L, sodium chloride 2-5g / L, agar powder 15-30g / L, dissolved in water And adjust the volume to the required volume, adjust the pH to 7.0-7.2 with sodium hydroxide, sterilize in a high-pressure steamer at 121°C for 20 minutes, and then pack into test tubes.

[0153] 1.2 Seed medium

[0154] Glucose 20-40g / L, yeast extract powder 2-5g / L, ammonium sulfate 1-5g / L, potassium dihydrogen phosphate 1-5g / L, anhydrous magnesium sulfate 0.5-2g / L, ferrous sulfate heptahydrate 10 -30mg / L, manganese sulfate monohydrate 10-30mg / L, V H 0.1-0.5mg / L, V B1 0.5-1mg / L, trace element mixture 1-2mL / L, phenol red: 2% of the constant volume, adjust the pH to 7.0-7.2 with sodium hydroxide, and sterilize in a hi...

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Abstract

The invention provides a genetically engineered bacterium for producing 5-hydroxytryptophan, and a construction method and application of the genetically engineered bacterium. Firstly, a tryptophan synthesis pathway in escherichia coli and a metabolic flux related to tryptophan in a whole amino acid metabolic network are analyzed and reconstructed, and supply of 5-HTP precursor tryptophan is enhanced; and then a human-derived type-2 tryptophan hydroxylase mutant and a tetrahydropterin synthesis and regeneration approach are introduced, a tryptophan intracellular hydroxylation reaction is broken through, the plasmid-free genetically engineered bacterium which has a clear genetic background and is capable of efficiently and stably producing 5-HTP through a microbial direct fermentation method is obtained, and the plasmid-free genetically engineered bacterium has a good application value.

Description

technical field [0001] The invention relates to the field of compound biotechnology production, in particular to a genetically engineered bacterium for producing 5-hydroxytryptophan and its construction method and application. Background technique [0002] 5-hydroxytryptophan (5-hydroxytryptophan, 5-HTP), the chemical name is 5-hydroxy-3-indolyl-α-alanine, is the precursor of neurotransmitter 5-hydroxytryptamine biosynthesis, can effectively Treats a variety of conditions such as depression, fibromyalgia, obesity, insomnia, and chronic headaches, among others. At present, more than 50 kinds of preparations have been developed for health medicines with 5-HTP as the main component, and they have been marketed in more than 30 countries around the world. With the gradual deepening of people's research on the function of 5-HTP, its market demand is still expanding, which makes 5-HTP one of the most commercially valuable small varieties of amino acids. [0003] At present, my co...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/53C12N15/54C12N15/55C12N15/31C12N15/70C12P13/22C12R1/19
CPCC12N9/88C12N9/1247C12N9/0071C12N9/0006C12N9/1085C12N9/78C12N9/0028C12N15/70C07K14/245C12P13/227C12Y401/99001C12Y207/07006C12Y114/16004C12Y401/03027C12Y205/01054C12Y101/01095C12Y305/04016C12Y402/03012C12Y101/01153C12Y105/01034C12Y402/01096
Inventor 徐庆阳金敏张震余子辰师丹阳
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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