CN107794263B - A gene for improving melanin yield and its application - Google Patents

Abstract

The invention discloses a gene for improving the yield of melanin. The gene is to mutate Tyr at the 280th position of the amino acid sequence of the encoded protein of gene Cjhppd into Phe, named as Cjhppd Y280F , and its nucleotide sequence is as SEQ ID NO : shown in 1; the application of this gene in the production of melanin is disclosed simultaneously. The gene Cjhppd Y280F provided by the invention can significantly increase the output of pyomelanin, and its use in producing pyomelanin has the advantages of simple method, low production cost, short production cycle, high yield, and controllability, and the produced pyomelanin It is a natural melanin that does not require animal or plant raw materials. Compared with artificially synthesized melanin, it has the advantages of safety and non-toxicity. The melanin produced by the invention can be used as a raw material for the preparation of health food, daily cosmetics, sunscreen lotion, sunscreen, black hair agent, bacterial cell protection and enzyme preparation of biological bacterial fertilizer and the like.

Description

A gene for improving melanin yield and its application

technical field

The invention relates to the technical field of genetic engineering, in particular to a gene for improving the yield of melanin and its application.

Background technique

Melanin is a kind of high-molecular-weight black pigment with unknown molecular structure. It is widely found in animals, plants and microorganisms. Its structure is complex and diverse, which can improve the ability of organisms to survive and compete. Melanin is a biological macromolecule, insoluble in water, insoluble in most organic solvents and acids. The structure of melanin is very complex, so far, researchers in the industry still lack a comprehensive and clear understanding of it. According to research, the basic structure of melanin is some covalently cross-linked indole rings, and the structure of melanin produced by different organisms is also different. Generally speaking, melanin is divided into four types, namely eumelanin, phaeomelanin, allomelanin and pyomelanin.

With the deepening of research, people have found that natural melanin has very powerful functions, such as anti-oxidation, anti-aging, anti-ultraviolet radiation, etc. Therefore, people can use natural melanin to prepare various functional daily cosmetics, such as sunscreen cream, sunscreen Creams, hair dyes, etc., can also be used as additives in wine, beverages, baby health products and public food to develop various health foods. In addition, people have found that natural melanin is also related to certain diseases, aging, skin color, etc. For example, in recent years, it has been found that some soluble melanin has a significant inhibitory effect on HIV in vitro. Therefore, natural melanin may become a new type of skin in the future. anti-AIDS drugs. In addition, natural melanin can also be used as a biosemiconductor material.

Compared with synthetic pigments, natural pigments have the advantages of safety, non-toxicity, and high nutritional value, which prompts people to obtain more natural pigments. At present, natural pigments are mainly extracted from animals, plants and microorganisms. Among them, the pigments extracted from animals and plants are expensive and their applications are limited. However, the use of microorganisms to produce natural pigments can not only overcome many shortcomings of natural pigments produced from animals and plants, but also facilitate industrialization. At present, it has been found that a variety of microorganisms can be used to produce pigments, such as producing carotenoids in Escherichia coli, Candida utilis, Saccharomyces cerevisiae and Zymomonas mobilis; The gene for lycopene synthesis was transferred into Candida proteogenes to obtain lycopene and phytoene; for example, Ruan Lifang cloned the melanin-producing mel gene in Pseudomonas maltophilia into the shuttle vector pHT3101, and the constructed The recombinant plasmid pHTAM was transferred into the Bacillus thuringiensis recipient strain BMB171 to obtain the recombinant strain RSA, and the mel gene was successfully expressed; for example, a gene HPDs cloned from Streptomyces avermitilis was expressed in Escherichia coli and was found to produce a A pigment produced by the oxidative polymerization of homogentisic acid; for example, Steven et al. cloned the hppd gene of the marine bacterium Shewanella colwellian D into Escherichia coli for fermentation to produce pyomelanin; Keon isolated the hppd gene from Mycosphaerella graminicola and cloned it into Escherichia coli In the process, pyomelanin was obtained in the fermentation broth; as Yao Hongbao disclosed in his master’s thesis, the hppd gene of Coptidis rhizome was expressed prokaryotically in Escherichia coli, and the expression product hppd of the recombinant bacteria catalyzed the oxidation of p-hydroxyphenylpyruvate to form homogentisic acid , when homogentisic acid is exposed to oxygen, it undergoes oxidative polymerization to form a brown pigment-pyomelanin. Although existing studies have produced melanin by transfecting specific genetically engineered bacteria, we found that the production of melanin in these studies was relatively low.

Contents of the invention

The purpose of the present invention is to provide a gene for increasing the yield of melanin and its application, which provides a high-efficiency production approach for the production of melanin.

The purpose of the present invention is achieved by the following technical solutions: a gene that improves the yield of melanin, the gene is the 280th Tyr mutation of the amino acid sequence of the encoded protein of the gene Cjhppd into Phe, named after Cjhppd Y280F , the gene The nucleotide sequence of Cjhppd Y280F is shown in SEQ ID NO: 1.

Wherein the nucleotide sequence of the gene Cjhppd is shown in SEQ ID NO: 3; the amino acid sequence of the encoded protein of the gene Cjhppd is shown in SEQ ID NO: 4.

A protein encoded by a gene whose nucleotide sequence is shown in SEQ ID NO: 1 to increase the yield of melanin, and whose amino acid sequence is shown in SEQ ID NO: 2.

A plasmid containing a gene whose nucleotide sequence is shown in SEQ ID NO: 1 to improve the yield of melanin, the plasmid is pET21d -Cjhppd Y280F ; the gene in the plasmid is under the regulation of the T7 expression framework.

An engineering strain containing a gene for increasing melanin yield as shown in SEQ ID NO: 1, which is a recombinant strain obtained by transferring the plasmid pET21d -Cjhppd Y280F into Escherichia coli BL21 (DE3) competent cells.

The present invention also discloses the application of the gene for improving the yield of melanin shown in SEQ ID NO: 1 in the production of melanin. The specific application method is as follows: the gene Cjhppd Y280F is connected to the plasmid vector and introduced into the Escherichia coli strain, Obtain recombinant bacteria capable of producing melanin, activate culture, induce expression to produce melanin, separate and extract to obtain melanin.

The plasmid vector is pET21d, and the Escherichia coli strain is BL21(DE3).

The induced expression means that after the recombinant bacteria are activated, they are inoculated in a 250 mL Erlenmeyer flask containing 50 mL of LB liquid fermentation medium (with 100 μg/mL ampicillin added) at a ratio of 1:50 by volume, at 220 r /min rotation speed, shaking culture at 37°C; when the bacterial concentration of the culture solution reaches A ₆₀₀ ≈0.6, add IPTG to a final concentration of 1 mmol/L, adjust the culture temperature to 30°C, induce the expression of the recombinant hppd enzyme protein, and continue to cultivate the recombinant The bacteria produced pigment, and the fermentation was stopped after 48 h.

In the present invention, the 280th position in the amino acid sequence of the encoded protein of the gene Cjhppd is mutated from the original tyrosine (Tyr, Y) to phenylalanine (Phe, F) by the method of molecular biology, and the mutated gene Cjhppd Y280F Connected to the plasmid and transformed into Escherichia coli, the expression vector and recombinant bacteria similar to the gene Cjhppd were obtained, and the melanin was produced by inducing expression. In particular, it was unexpectedly found that the mutated gene Cjhppd Y280F was more able to Significantly increase the production of melanin; and the melanin produced by the mutated gene Cjhppd Y280F belongs to the same pyomelanin as the melanin produced by the pre-mutated gene Cjhppd , and is also maintained under the conditions of light and temperature changes and the addition of oxidizing and reducing agents Stable characteristics; and the additives sucrose and glucose do not affect the stability of the pigment; it has ultraviolet protection effect on bacteria, and has anti-oxidation and possible enzyme protein activity protection.

Applying the gene Cjhppd Y280F provided by the present invention to the production of pyomelanin has the advantages of simple method, low production cost, short production cycle, high yield, and controllability, and the produced pyomelanin is natural melanin, which does not require animals or plants Compared with synthetic melanin, it has the advantages of non-toxicity and safety. The melanin produced by the invention can be used in the fields of health food industry, daily cosmetic industry, preparation of sunscreen cream, sunscreen and black hair care industry, bacterial cell protection and enzyme preparation of biological bacterial fertilizer and the like.

Description of drawings

Fig. 1 is the Fourier transform infrared spectrogram of the melanin produced by the recombinant strain obtained in Example 1 and Comparative Example 1.

Fig. 2 is the calibration curve of the mass concentration and OD value relation of the melanin that embodiment 4 makes.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials and reagents used in the examples can be obtained from commercial sources unless otherwise specified.

Part of the medium used in the following examples, the mother liquor formula are as follows:

LB liquid basic medium: NaCl 10 g/L, peptone 10 g/L, yeast powder 5 g/L, pH=7.0. For solid culture, 15 g/L of agar should be added to the LB liquid basic medium.

LB liquid fermentation medium: LB liquid basic medium + 1 g/L L-tyrosine.

Ampicillin stock solution (100 mg/mL): Dissolve 1 g of ampicillin sodium salt in a sufficient amount of sterilized water, finally dilute to 10 mL, and filter to sterilize with a 0.22 μm filter membrane.

Example 1

1. Mutation: The Fast Mutagenesis System of Beijing Quanshijin Biotechnology Co., Ltd. was used to design the mutation site with the recombinant plasmid pET21d -Cjhppd as the target plasmid, and the Tyr at position 280 of the amino acid sequence of the encoded protein of the gene Cjhppd was mutated into Phe, that is, the Y280 site is selected to be mutated into F, and the corresponding codon is mutated from TAT to TTT. The nucleotide sequence of the disclosed gene Cjhppd is shown in SEQ ID NO: 3, and its encoded amino acid sequence is shown in SEQ ID NO: 4; the nucleotide sequence of the mutated gene Cjhppd-Y280F is shown in SEQ ID NO: 1 Shown, its encoded amino acid sequence is shown in SEQ ID NO:2.

2. Design point mutation primers: the primer sequence (5'-3') is:

Cjhppd Y280F Fw: GCCAGATACAAACTTTTTTTGGAACATAA;

Cjhppd Y280F Rv: AAAGTTTGTATCTGGCTCTTTTCTCTTTG.

3. PCR system and conditions:

The PCR system is as follows:

Template (template) plasmid 0.2 μl

Forward Primer (forward primer) (10 μM) 1 μl

Reverse Primer (10 μM) 1 μl

TransStartFastPfuFly DNA Polymerase (TransStartFastPfuFly DNA Polymerase) 1μl

5×TransStart FastPfuFly Buffer (TransStartFastPfuFly buffer) 10 μl

dNTPs (deoxyribonucleoside triphosphates) (2.5 mM each) 5 μl

ddH ₂ O to final volume (add ultrapure water to final volume) 50 μl

The PCR conditions are as follows:

PCR products were detected by agarose gel electrophoresis at a mass ratio concentration of 1.5%;

Add 1 μl DMT enzyme to the PCR product, mix lightly, and incubate at 37°C for 1 h.

4. Conversion

(1) Add 5 μl DMT enzyme digestion product to 50 μl DMT competent cells (add the product when the competent cells are just thawed), flick and mix well, and ice-bath for 30 min;

(2) Precisely heat shock at 42°C for 45 sec, and immediately place on ice for 2 min;

(3) Add 500 μL of SOC equilibrated to room temperature, 250 rpm, and incubate at 37°C for 1 h;

(4) Centrifuge at 4000 rpm for 2 min, discard part of the supernatant, retain 100 μL, blow gently to suspend the bacteria, take all the bacteria solution to spread on the plate (the final concentration of Amp+ is 100 μg/mL), and incubate overnight at 37 °C;

5. Pick a single colony and shake it and send it to the sequencing company for sequencing (pick 3 clones), and add glycerol to preserve the strain.

6. Compare the sequencing results, take the strain with the correct nucleotide sequence for site mutation as shown in SEQ ID NO: 1, and the encoded amino acid sequence as shown in SEQ ID NO: 2, and shake the bacteria to extract the corresponding plasmid corresponding to the mutant strain .

7. Take point-mutated plasmids and original plasmids and transform them into Escherichia coli BL21 (DE3) competent cells.

(1) Add 1 μL of plasmid to 50 μL of Escherichia coli BL21 (DE3) competent cells (add the product when the competent cells are just thawed), flick and mix well, and ice-bath for 30 min;

(2) Heat shock at 42°C for 45 sec, and immediately place on ice for 2 min;

(3) Add 500 μL of SOC equilibrated to room temperature, 250 rpm, and incubate at 37°C for 1 h;

(4) Centrifuge at 4000 rpm for 2 minutes, discard part of the supernatant, retain 100 μl, blow gently to suspend the bacteria, take all the bacteria solution to smear (the final concentration of Amp+ is 100 μg/mL), and cultivate overnight at 37°C; pick After shaking the single colony, add glycerol to preserve the strain.

Perform point mutation on the target plasmid pET21d -Cjhppd through the above steps, and transform the point mutant plasmid pET21d -Cjhppd Y280F into clone competent cells, perform plasmid extraction on the single clone with correct sequencing, and transfer the plasmid pET21d -Cjhppd Y280F into expression Escherichia coli strain BL21 (DE3) competent cells, the recombinant bacteria.

Example 2

1. Activation culture of recombinant bacteria: The recombinant Escherichia coli strain BL21(DE3) transfected with plasmid pET21d -Cjhppd Y280F was activated and cultured, and under the ultra-clean workbench, the examples of preservation in -80°C refrigerator were picked with sterilized toothpicks respectively. 1. The obtained recombinant bacteria were inoculated in a 50mL Erlenmeyer flask containing 5mL LB liquid fermentation medium (additional 100μg/mL ampicillin), and cultured overnight for activation.

2. Induced expression and pigment production by recombinant bacteria

After the recombinant bacteria were activated, they were inoculated in a 250 mL Erlenmeyer flask filled with 50 mL of LB liquid fermentation medium (plus 100 μg/mL ampicillin) at a volume ratio of 1:50, and cultured with shaking at 220 r/min. When the bacterial concentration in the culture solution reached A ₆₀₀ ≈0.6, IPTG was added to a final concentration of 1 mmol/L to induce the expression of the recombinant hppd enzyme protein, and the recombinant bacteria were continued to be cultured to produce pigments. After 48 h, the fermentation was stopped, and the pigment production in the fermentation broth was measured.

3. Separation and extraction of pigment

The culture solution of the recombinant bacteria was filtered through one layer of filter paper and two layers of Miracloth to remove the bacteria, adjusted the pH of the filtrate to 2 with 6mol/L hydrochloric acid, let it stand for 4 hours, centrifuged at 8000r/min for 20min, and collected the precipitated crude pigment extract . The crude extract was dissolved in ultrapure water adjusted to pH 12 with 5 mol/L sodium hydroxide, mixed well, centrifuged at 8000r/min for 20min, the supernatant was adjusted to pH 2, and the pigment was precipitated at 8000r/min , centrifuged for 20min, collected the precipitate, and repeated the above steps twice; after that, washed the precipitate with absolute ethanol twice and acetone once, and centrifuged at 8000r/min for 20min until the supernatant was colorless; finally dried the precipitate at 60°C To constant weight, it is the pigment extract-pyomelanin.

Comparative example 1

With the plasmid pET21d- Cjhppd as the target plasmid, the nucleotide sequence of the gene Cjhppd is shown in SEQ ID NO: 3, and its encoded amino acid sequence is shown in SEQ ID NO: 4, which was transformed into the expression Escherichia coli BL21 (DE3) feeling In the state cells, the recombinant bacteria were obtained, and the same specific steps as in Example 2 were used to activate the strains, induce expression and produce pigments, measure the yield of pigments in the fermentation broth, separate and extract the pigments, and obtain pyomelanin.

Infrared Spectrum (IR) of the Pyomelanin Produced in Example 3

The pyomelanin that embodiment 2 and comparative example 1 obtains is scanned with KBr pressing tablet 600-4000cm-1, and its result is as shown in Figure 1, shows in the figure, the pyomelanin infrared spectrum that two kinds of bacterial strains produce are identical, illustrate as Same kind of melanin.

Example 4 Determination of Pigment Yield

1. Determination of pigment standard curve

The absorption spectrum and absorption peak of the pigment are measured by spectrophotometry, and the wavelength for measuring the pigment is selected.

Dissolve 50 mg of the pigment extracted in Example 3 with 1 mL of NaOH solution with a concentration of 1 mol/L, then add water to make up to 10 mL, and adjust the pH to 7.0 (all the pigment solutions used in this experiment are prepared according to this method), and prepare Make a 5 mg/mL pigment solution mother solution for later use. Dilute a certain number of times so that the mass concentration is 5mg/mL, 4mg/mL, 3mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, scan the ultraviolet-visible spectrum, and measure the absorbance at 400 nm. The mass concentration is the abscissa, the measured _OD400 is the ordinate, and the standard curve is made as shown in Figure 2.

2. Determination of pigments in fermentation broth

The fermentation broth in Example 2 and Comparative Example 1 was collected, and under the same conditions, the absorbance value at 400 nm was measured, and the absorbance value at 400 nm of the empty vector cell fermentation broth was subtracted under the same conditions to calculate the recombinant bacteria OD ₄₀₀ of the pigment in the fermentation broth. Then OD ₄₀₀ was substituted into the prepared standard curve to calculate the yield of the pigment in the fermentation broth of the recombinant bacteria, and the measurement results are shown in Table 1. The empty vector here refers to the bacterium containing the empty vector pET21d in the expression bacterium BL21 (DE3).

The pyomelanin production result (mg/mL) of table 1 embodiment 2 and comparative example 1

SEQUENCE LISTING

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Claims (6)

1. a kind of gene for improving melanin yield, which is characterized in that the gene is by geneCjhppdCoding albumen ammonia 280th Tyr of base acid sequence is mutated into Phe, is named asCjhppd Y280F, geneCjhppd Y280FNucleotides sequence Column are as shown in SEQ ID NO: 1.

2. a kind of albumen of the coded by said gene described in claim 1 for improving melanin yield, which is characterized in that its amino acid Sequence is as shown in SEQ ID NO: 2.

3. a kind of plasmid containing the gene as described in claim 1 for improving melanin yield.

4. a kind of engineered strain containing the gene as described in claim 1 for improving melanin yield.

5. a kind of application of gene described in claim 1 for improving melanin yield in production melanin.

6. application of the gene according to claim 5 for improving melanin yield in production melanin, which is characterized in that By geneCjhppd Y280FIt is connected on plasmid vector, imports in Escherichia coli, obtain the recombinant bacterium that can generate melanin Strain, activation culture, inducing expression, chromogenesis, separation and Extraction obtain melanin.