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Recombination microorganism for producing beta-carotene, construction method and application

A technology of carotene and recombinant bacteria, applied in the biological field, can solve the problem of reducing expression intensity and producing β-carotene by recombinant Escherichia coli

Active Publication Date: 2017-02-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the 1-deoxy-xylulose-5-phosphate reductoisomerase gene (dxr), (E)-4-hydroxy-3-methyl-2-butenyl-pyrophosphate synthase gene ( ispG), (E)-4-hydroxy-3-methyl-2-butenyl-pyrophosphate reductase gene (ispH) expression strength reduced the ability of recombinant E. coli to produce β-carotene (Yuan et al .,2006)

Method used

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  • Recombination microorganism for producing beta-carotene, construction method and application
  • Recombination microorganism for producing beta-carotene, construction method and application
  • Recombination microorganism for producing beta-carotene, construction method and application

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preparation example Construction

[0067] The preparation method of salt-free LB is as follows:

[0068] 50% sucrose solution: Weigh 500g of sucrose, dissolve it in a small amount of ultrapure water, and dilute to 1L, sterilize at 115°C for 20 minutes.

[0069] 10% salt-free sucrose LB medium: Weigh 5g of yeast extract, 10g of peptone in 800ml of water, sterilize at 115°C for 20min. Add 200ml of 50% sucrose solution after sterilization.

[0070] 6% salt-free sucrose LB medium: Weigh 5g yeast extract, 10g peptone, 15g agar powder, dissolve in 880ml water, sterilize at 115°C for 20min. Add 120ml of 50% sucrose solution after sterilization.

[0071] The concentrations of chloramphenicol, ampicillin, and kanamycin in the examples were 34 μg / L, 50 μg / L, and 50 μg / L, respectively.

[0072] Primers used in the present invention in table 1

[0073]

[0074]

[0075]

[0076] Table 2 is the bacterial strain constructed in the present invention

[0077]

[0078]

[0079]

[0080]

Embodiment 1

[0081] Embodiment 1, explore the relationship between MEP pathway dxr, ispD, ispE, ispG and ispH gene expression intensity and β-carotene output

[0082] In order to study the MEP pathway in CAR005 (Zhao J, Li Q, Sun T, et al. Engineering central metabolic modules of Escherichia coli for improving beta-carotene production. Metabolicengineering 2013; 17:42-50. Both CAR001 and CAR005 are from this article) The relationship between the expression intensity of each gene and the production of β-carotene, the mRS library was established to regulate dxr, ispD, ispE, ispG and ispH genes, 15 strains were randomly selected, and the production of β-carotene was determined. According to the production of β-carotene, Then select several representative strains and use the real-time quantitative PCR method to measure the gene expression level, and study the relationship between the expression level and the yield of β-carotene.

[0083] 1. Construction of mRS library to regulate dxr, ispD, is...

Embodiment 2

[0125] Embodiment 2, the recombinant bacterium obtained by combination regulation of ispG and ispH

[0126] The ispG artificial regulatory elements of ispG-1, ispG-4, and ispG-11 in the ispG library regulation, and the ispH artificial regulatory elements of ispH-3, ispH-4, and ispH-14 in the ispH library were combined and regulated in CAR005 .

[0127] 1. Construction of recombinant Escherichia coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14

[0128]Recombinant Escherichia coli G1H3 is the mRSL regulatory element mRSL-1::ispG (SEQ ID NO: 21) that pre-inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the front of the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-3::ispH (SEQ ID NO: 29);

[0129] Recombinant Escherichia coli G1H4 is the mRSL regulatory element mRSL-1::ispG (SEQ ID NO: 21) that pre-inserts the ispG gene in the genome of the ...

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Abstract

The invention discloses a method for finding and removing MEP route rate-limiting step and an application thereof in production of terpene compound. The invention provides a method for constructing recombinant bacteria. The expression and / or activity of (E)-4-hydroxy-3-methyl-2-butenyl-pyrophosphoric acid synthetase gene ispG and (E)-4-hydroxy-3-methyl-2-butenyl-pyrophosphoric acid reductase gene ispH for producing beta-carotene escherichia coli are promoted, so as to obtain the recombinant bacteria. An experiment proves that the cytotoxicity is caused by the intracellular accumulation of MEP route intermediate HMBPP in the recombinant escherichia coli having a certain beta-carotene compounding capacity and the coordinative expression for ispG and ispH genes can effectively increase the capacity of the recombinant escherichia coli for compounding the terpene compound.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the discovery and removal of the rate-limiting steps of the MEP pathway and its application in the production of terpene compounds, in particular to a recombinant microorganism for producing beta-carotene, its construction method and its application. Background technique [0002] Terpenoids are a class of compounds that exist in nature and are composed of isoprene as a structural unit. Many terpenoids have good pharmacological activity and are the main active ingredients of traditional Chinese medicine and natural herbal medicine. Some terpenoids have been developed into effective drugs widely used in clinical practice. [0003] Beta-carotene and lycopene are two typical terpenoids. β-carotene has the functions of anti-oxidation, detoxification, anti-cancer, prevention of cardiovascular diseases, prevention and treatment of cataracts and liver protection. In addition, β-carotene, kn...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P23/00C12R1/19
CPCC12N9/0093C12N15/70C12P23/00C12Y117/01002C12Y117/04
Inventor 张学礼李清艳唐金磊毕昌昊
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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