Construction method and application of a recombinant bacterium producing lycopene

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

Active Publication Date: 2019-12-31
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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  • Construction method and application of a recombinant bacterium producing lycopene
  • Construction method and application of a recombinant bacterium producing lycopene
  • Construction method and application of a recombinant bacterium producing lycopene

Examples

Experimental program
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preparation example Construction

[0070] The preparation method of salt-free LB is as follows:

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The concentrations of chloramphenicol, ampicillin, and kanamycin in the examples were 34 μg / L, 50 μg / L, and 50 μg / L, respectively.

[0075] The preparation method of fermentation medium is as follows:

[0076] 1. 10% glycerin mother solution: Weigh 100g glycerol, add deionized water to 1L, sterilize at 121°C for 20min.

[0077] 2. Trace element mother liquor:. Comp...

Embodiment 1

[0094] Example 1, the relationship between the MEP pathway ispG and ispH gene expression intensity and β-carotene output

[0095] 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 CAR005D 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.

[0096] 1. Construction of mRS library to regulate dxr, ispD, ispE, ispG and ispH genes...

Embodiment 2

[0152] Example 2. The combination of ispG and ispH regulates the production of lycopene

[0153] In the above example 1, it can be seen that in the experiment of increasing the production of β-carotene by the combination regulation of ispG and ispH, the combination with the highest production of β-carotene is G4H14, that is, the corresponding promoters in mRSL-4::ispG and mRSL-14::ispH Combination, can effectively increase the expression of ispG and ispH, whether it has the same effect on all terpenoids.

[0154] In this example, the combination of mRSL-4::ispG and mRSL-14::ispH is used to regulate lycopene.

[0155] 1. Regulation of ispH gene in LYC010

[0156] Recombinant Escherichia coli LYC013 is the mRSL-14::ispH (sequence 32) in which the ispH gene in the genome of the starting strain LYC010 is pre-inserted for initiating the expression of the ispH gene.

[0157] The above replacement regulates the ispH gene through a two-step method to construct the LYC013 strain, and...

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Abstract

The invention discloses a building method for producing lycopene recombinant bacteria and application. The lycopene recombinant bacteria are optional one of first lycopene recombinant bacteria, second lycopene recombinant bacteria and third lycopene recombinant bacteria, wherein first lycopene recombinant bacteria are obtained by increasing the expression and / or activity of (E)-4-hydroxyl-3-methyl-2-butenyl-geranylgeranyl pyrophosphate synthase genes ispG and the (E)-4-hydroxyl-3-methyl-2-butenyl-geranylgeranyl pyrophosphate reductase genes ispH in lycopene escherichia coli; the second lycopene recombinant bacteria are obtained by regulating the expression and / or activity of the crt operon of the first lycopene recombinant bacteria; the third lycopene recombinant bacteria are obtained by regulating the expression and / or activity of the glpD genes of the second lycopene recombinant bacteria. Experiments show that the terpene compound synthesizing ability of recombinant escherichia coli can be increased effectively by expressing the ispG and ispH genes in the recombinant escherichia coli with certain terpene compound synthesizing ability in a coordinated manner, and terpene compounds include beta-carotene, lycopene and other terpene compounds.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of a recombinant bacterium producing lycopene. 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] Lycopene is a typical terpenoid; it is the main component of tomato red pigment and is an excellent antioxidant. Lycopene can prevent the damage caused by metabolite "free radicals" to human tissues and organs, and can be used as natural health food or medicine. Health food containing lycopene can prevent senile vision degradation, anti-aging and prevent cardiovascular disease. Lycopene also has a certain i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P5/02C12R1/19
CPCC12N9/0006C12N9/0093C12N15/70C12P5/007C12Y101/01008C12Y117/01002C12Y117/04
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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