A recombinant bacterium producing lycopene and its application

Abstract

The invention discloses a recombinant bacteria strain for producing lycopene and application of the recombinant bacteria strain. The recombinant bacteria strain 1 disclosed by the invention is constructed by improving enzyme activity of yak-based bisphosphate synthetase in recombinant Escherichia coli; the method for improving the enzyme activity of yak-based bisphosphate synthetase in recombinant Escherichia coli comprises the step of replacing an original regulation component of yak-based bisphosphate synthetase genes crtE in the recombinant Escherichia coli by an artificial regulation component 1. The strain disclosed by the invention is high in lycopene yield and has extremely high production and application values.

Description

technical field

[0001] The invention relates to a recombinant bacterium producing lycopene and its application. Background technique

[0002] Lycopene is the main component of tomato red pigment, which belongs to "carotenoid" substances and is an excellent antioxidant. The quenching effect of lycopene on singlet oxygen is twice that of β-carotene and 100 times that of vitamin E. Lycopene also has physiological functions such as preventing disease and cancer, enhancing the body's immunity and anti-aging, and has important uses in the fields of food, cosmetics and medicine. Lycopene can prevent the damage caused by metabolite "free radicals" to human tissues and organs. Because of its anti-free radical effect, it can be used as natural health food or medicine. At present, dozens of lycopene-containing products have appeared on the European market. health food and medicines. Health food containing lycopene can prevent senile vision degradation, anti-aging and prevent cardiov...

Examples

preparation example Construction

[0068] The preparation method of salt-free LB is as follows:

[0069] 1. 50% sucrose solution: Weigh 500g of sucrose, dissolve in 1L of ultrapure water, sterilize at 115°C for 20min.

[0070] 2. 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.

[0071] 3.6% 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.

[0072] The concentrations of chloramphenicol, ampicillin, and kanamycin in the examples were 34 μg / L, 50 μg / L, and 50 μg / L, respectively.

Embodiment 1

[0073] Example 1, Fermentation of Lycopene Recombinant Escherichia coli and Determination of Lycopene

[0074] 1. Drawing of lycopene standard curve

[0075] Accurately weigh 50mg of standard lycopene and add 1ml of dichloromethane to dissolve it, transfer the solution to a 250ml brown volumetric flask, dilute to 250ml with petroleum ether, and prepare a 200μg / ml stock solution (stored in a -80°C refrigerator middle). Use acetone for doubling dilution (2×, 4×, 8×, 16×, 32×), filter through a 0.45 μm microporous membrane into an HPLC sample bottle, and perform HPLC detection (using C18 column (4.6×250mm, 5μm); column temperature: 30℃; mobile phase: methanol:acetonitrile:dichloromethane=21:21:8 (volume ratio); flow rate: 1ml / min; injection volume: 20μl; Sample time: 20min; DAD light detection; detection wavelength is 480nm, with the peak area of ​​the standard product as the ordinate, and the lycopene concentration (mg / L) as the abscissa, the standard curve of lycopene is obt...

Embodiment 2

[0079] Example 2, Production of lycopene by knocking out the crtX and crtY genes of recombinant Escherichia coli CAR001

[0080] 1. Knockout the crtX and crtY genes of recombinant Escherichia coli CAR001 to construct recombinant bacteria LYC001 The recombinant bacteria LYC001 is to replace the zeaxanthin glycosyltransferase gene crtX and lycopene in the β-carotene synthesis gene cluster with recombinant Escherichia coli CAR001 The prime β-cyclase gene crtY was obtained. The zeaxanthin glycosyltransferase gene crtX is shown in SEQ ID No.1, and the lycopene β-cyclase gene crtY is shown in SEQ ID No.2. Two-step homologous recombination method was used to knock out the crtX and crtY genes, and at the same time inserted between the geranyl geranyl diphosphate synthase gene crtE and the phytoene dehydrogenase gene crtI suitable for gene expression in Escherichia coli The artificial RBS sequence (shown in SEQIDNo.3). A fragment for two-step homologous recombination is amplified by ...

technical field

[0001] The invention relates to a recombinant bacterium producing lycopene and its application. Background technique

[0002] Lycopene is the main component of tomato red pigment, which belongs to "carotenoid" substances and is an excellent antioxidant. The quenching effect of lycopene on singlet oxygen is twice that of β-carotene and 100 times that of vitamin E. Lycopene also has physiological functions such as preventing disease and cancer, enhancing the body's immunity and anti-aging, and has important uses in the fields of food, cosmetics and medicine. Lycopene can prevent the damage caused by metabolite "free radicals" to human tissues and organs. Because of its anti-free radical effect, it can be used as natural health food or medicine. At present, dozens of lycopene-containing products have appeared on the European market. health food and medicines. Health food containing lycopene can prevent senile vision degradation, anti-aging and prevent cardiov...

Examples

preparation example Construction

[0068] The preparation method of salt-free LB is as follows:

[0069] 1. 50% sucrose solution: Weigh 500g of sucrose, dissolve in 1L of ultrapure water, sterilize at 115°C for 20min.

[0070] 2. 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.

[0071] 3.6% 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.

[0072] The concentrations of chloramphenicol, ampicillin, and kanamycin in the examples were 34 μg / L, 50 μg / L, and 50 μg / L, respectively.

Embodiment 1

[0073] Example 1, Fermentation of Lycopene Recombinant Escherichia coli and Determination of Lycopene

[0074] 1. Drawing of lycopene standard curve

[0075] Accurately weigh 50mg of standard lycopene and add 1ml of dichloromethane to dissolve it, transfer the solution to a 250ml brown volumetric flask, dilute to 250ml with petroleum ether, and prepare a 200μg / ml stock solution (stored in a -80°C refrigerator middle). Use acetone for doubling dilution (2×, 4×, 8×, 16×, 32×), filter through a 0.45 μm microporous membrane into an HPLC sample bottle, and perform HPLC detection (using C18 column (4.6×250mm, 5μm); column temperature: 30℃; mobile phase: methanol:acetonitrile:dichloromethane=21:21:8 (volume ratio); flow rate: 1ml / min; injection volume: 20μl; Sample time: 20min; DAD light detection; detection wavelength is 480nm, with the peak area of ​​the standard product as the ordinate, and the lycopene concentration (mg / L) as the abscissa, the standard curve of lycopene is obt...

Embodiment 2

[0079] Example 2, Production of lycopene by knocking out the crtX and crtY genes of recombinant Escherichia coli CAR001

[0080] 1. Knockout the crtX and crtY genes of recombinant Escherichia coli CAR001 to construct recombinant bacteria LYC001 The recombinant bacteria LYC001 is to replace the zeaxanthin glycosyltransferase gene crtX and lycopene in the β-carotene synthesis gene cluster with recombinant Escherichia coli CAR001 The prime β-cyclase gene crtY was obtained. The zeaxanthin glycosyltransferase gene crtX is shown in SEQ ID No.1, and the lycopene β-cyclase gene crtY is shown in SEQ ID No.2. Two-step homologous recombination method was used to knock out the crtX and crtY genes, and at the same time inserted between the geranyl geranyl diphosphate synthase gene crtE and the phytoene dehydrogenase gene crtI suitable for gene expression in Escherichia coli The artificial RBS sequence (shown in SEQIDNo.3). A fragment for two-step homologous recombination is amplified by ...

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