CN113788838A - Red natural pigment compound and preparation method and application thereof - Google Patents

Abstract

The application discloses a red natural pigment and a preparation method and application thereof, wherein the molecular weight of the red natural pigment compound is 513, and the molecular formula is C₂₈H₁₉NO₉The compound is obtained by expressing synthetase PskI with a nucleotide sequence shown in SEQ ID No.1 obtained by whole gene synthesis in saccharomyces cerevisiae engineering bacteria, and has the advantages of good compatibility and low costThe compound has good water and oil repellency, good color rendering property, level dyeing property and color fastness, can be widely applied to the fields of synthetic fibers, plastics, rubber, leather, coatings, printing ink, paint, cosmetics, food additives and the like, is synthesized by utilizing engineering bacteria, has high yield, is not limited by raw material sources, is non-toxic and harmless, is easy to degrade and cannot cause pollution to the ecological environment.

Description

Red natural pigment compound and preparation method and application thereof

Technical Field

The application relates to the field of bioengineering, in particular to a novel red natural pigment and a preparation method and application thereof.

Background

In recent years, with the progress of technology and the improvement of the living standard of people, the application of the dye is more and more extensive, and particularly in the fields of food and cosmetics, the demand of non-toxic and environment-friendly dye is more and more large, which puts higher requirements on the development of the dye industry.

The current dyes can be mainly divided into two main categories according to their sources: natural dyes and synthetic dyes. The natural dye is earlier in application, most of the natural dyes are nontoxic and environment-friendly, but the natural dye has the limitations of limited content in the nature, complex extraction process, high production cost and the like. And most of natural dyes contain more hydroxyl groups, so that the water solubility is higher and the dyeing firmness is not high. Compared with natural dyes, the synthetic dyes have the advantages of bright color, no limitation of raw material sources, low production cost and the like, but have the defects of complex structure, high biotoxicity, difficult degradation or high degradation cost in nature and great damage to the ecological environment. The above-mentioned disadvantages of natural and synthetic dyes have severely restricted the rapid development of the dye industry and the further expansion of its application fields.

Disclosure of Invention

Aiming at the problems, the invention adopts biological materials to synthesize a novel non-toxic, environment-friendly, hydrophobic and oleophobic red natural dye compound (I), the dye belongs to naphthoquinone compounds, and the structural formula of the dye is shown as the following formula (I):

the compound (I) has a molecular weight of 513 and a molecular formula of C₂₈H₁₉NO₉The mass spectrum detection result is shown in figure 2; the characteristic ultraviolet absorption wavelength is 220nm, 307nm, 418nnm nm and 510nm, and the ultraviolet absorption spectrum is shown in figure 1.

The nuclear magnetic data of the compound (I) are: 1H-NMR (500MHz, DMSO-d6) delta 2.29(3H, s, H-11),2.27(3H, s, H-11 '), 3.95(2H, s, H-9),4.08(3H, s,6-OCH3),4.09(3H, s,6 ' -OCH3),6.32(1H, s,2, H-9 '), 6.94(1H, s, H-5),7.54(1H, s, H-7 '), 12.51(1H, s,2 ' -NH),12.62(1H, s, 2-OH); DEPTQ-13513C-NMR (125MHz, DMSO-d6) delta 30.0(C-11),13.0(C-11 '), 204.9(C-10),138.5 (C-10'), 38.5(C-9),106.1(C-9 '), 114.2(C-1),133.3 (C-1'), 157.5(C-2),126.0(C-2 '), 180.1(C-3),172.9 (C-3'), 106.3(C-3a),125.6(C-3b),110.5(C-3a '), 136.4(C-3 b'), 168.8(C-4),160.9(C-4 '), 103.5(C-5),113.1 (C-5'), 165.5(C-6),57.8(6-OCH3),160.2(C-6 '), 57.2 (6' -OCH3), 112.7-7), 106.2(C-7 '), 178.7(C-8),178.9 (C-8'),. The chemical structural formula is numbered as shown in figure 3.

Secondly, the application also provides a preparation method of the red natural dye (compound I), which comprises the following specific steps:

1) amplifying the PskI gene shown in SEQ ID NO.1 by polymerase chain reaction using primer F: 5'-CGggatcc ATGGCCGTTCCAACGAG-3' (SEQ ID NO.3) and primer R: 5'-CGgaattcCTAAACCCCAATGCTCTCC-3' (SEQ ID NO.4), ligating the PskI gene obtained by amplification into a pYES2 plasmid vector by restriction endonucleases BamHI and EcoRI, to obtain a recombinant plasmid pYES 2-PskI; then, the recombinant plasmid pYES2-PskI is transformed into saccharomyces cerevisiae engineering bacteria INVSC1 to obtain transformed engineering bacteria, and the transformed engineering bacteria are named as engineering bacteria S by the applicant;

2) selecting a single colony from a solid culture medium storing the engineering bacteria S, culturing the single colony in an SC culture medium in a shaking table at the temperature of 28 ℃ and the rpm of 200 for 24 hours to obtain a seed culture solution of the engineering bacteria S; adding a seed culture solution of the engineering bacteria S into the SC culture medium, and carrying out fermentation culture in a shaking table at the temperature of 28 ℃ and the rpm of 200 for 24 hours;

detecting the absorbance of the fermentation liquid by using a spectrophotometer under the wavelength of 600nm, adding 1% of yeast extract and 2% of peptone into the fermentation liquid when the absorbance of the fermentation liquid under the wavelength of 600nm reaches 0.8, placing the fermentation liquid in a shaker at 28 ℃, and fermenting and culturing for 72 hours at 200rpm to obtain a culture;

3) and (3) separating and purifying the culture: after the culture is finished, centrifuging the culture in a centrifuge at 850 Xg for 5 minutes to remove cells of the engineering bacteria S; extracting the fermentation liquor after removing the cells by using chloroform; repeating the process for 2 times to obtain extract containing compound I;

4) drying the extract obtained in step 3) to constant weight (preferably under vacuum at 38 deg.C) to obtain extract; subjecting the extract to silica gel column chromatography (preferably 200-300 mesh) to collect 100% chloroform-eluted fractions, and drying by rotary evaporation;

5) cleaning: oscillating and suspending the dried product by using 10mL of methanol, centrifuging for 10 minutes at 21000 Xg, discarding the supernatant, taking the precipitate, and repeating the process for 2 times to remove organic impurities to obtain a crude extract; then, ethyl acetate and n-hexane are respectively used for replacing methanol, the crude extract is cleaned by the same method, and the obtained product is dried, so that the pure red pigment compound I is obtained.

The dye I obtained by the method belongs to organic pigments, and can be widely applied to the fields of synthetic fibers, plastics, rubber, leather, coatings, printing ink, paint, cosmetics, food additives and the like.

The invention is with purified

The DNA of the genome PskI gene of the australis strain is used as a reference template, the transformed brand new synthetase PskI is obtained by whole-gene synthesis, the nucleotide sequence of the PskI gene is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No.1As shown in SEQ ID No.2, the improved synthetase PskI particularly and remarkably improves the yield of the compound I.

The application utilizes the reformed synthetase PskI gene to express in saccharomyces cerevisiae engineering bacteria INVSC1 for the first time to synthesize the novel hydrophobic and oleophobic natural red dye I. The method for synthesizing the red natural dye I by utilizing the engineering bacteria and the extraction process have the advantages of high dye yield, no limitation of raw material sources, easy control of the operation process and low production cost.

The red natural dye I is a new hydrophobic and oleophobic compound synthesized for the first time, has good color rendering property, level dyeing property and color fastness, is prepared from metabolites of saccharomyces cerevisiae thalli, is nontoxic and harmless, is easy to degrade, and cannot pollute the ecological environment.

Drawings

FIG. 1 shows the UV absorption spectrum of red pigment compound I.

FIG. 2 is a mass spectrometric detection of the red pigment compound I [ M-H ] +.

FIG. 3 is the chemical structural formula number of the haematochrome compound I.

FIG. 4 is a graph showing the results of detection of Compound I in example 3.

Detailed Description

The reagents and media components involved in the examples were:

SC medium (in mass percent): 0.062% of uracil-deficient defective amino acid mixture (DO Supplement-Ura), 6.4% of carbon source-free amino acid-free yeast nitrogen source (SC mineral Medium), 2% of glucose; pH 5.5.

Ethanol, methanol, acetone, ethyl acetate, chloroform, n-hexane, chloroform, glycerol, oils and DMSO were purchased from the company of biotechnology limited, prosperous, beijing dingguo.

The engineered Saccharomyces cerevisiae INVSC1 was purchased from Invitrogen.

The starting materials and the apparatuses referred to in the following examples were obtained from commercial sources unless otherwise specified.

EXAMPLE 1 preparation of Compound (I)

The specific procedure for the preparation of the red natural dye (compound I) of this example is as follows:

1) amplifying the PskI gene shown in SEQ ID NO.1 by using a front primer with the sequence shown in SEQ ID NO.3 and a rear primer with the sequence shown in SEQ ID NO.4 through a polymerase chain reaction, and connecting the amplified PskI gene into a pYES2 plasmid vector through restriction endonucleases BamHI and EcoRI to obtain a recombinant plasmid pYES 2-PskI; then, the recombinant plasmid pYES2-PskI is transformed into saccharomyces cerevisiae engineering bacteria INVSC1 to obtain transformed engineering bacteria, and the transformed engineering bacteria are named as engineering bacteria S by the applicant;

2) selecting a single colony from a solid culture medium storing the engineering bacteria S, putting the single colony in 2ml of SC culture medium, and culturing for 24 hours in a shaking table at the temperature of 28 ℃ and the rpm of 200 to obtain a seed culture solution of the engineering bacteria S; 1ml of seed culture solution of the engineering bacteria S is absorbed and added into the SC culture medium, and fermentation culture is carried out in a shaking table at the temperature of 28 ℃ and the rpm of 200 for 24 hours;

detecting the absorbance of the fermentation liquid by using a spectrophotometer under the wavelength of 600nm, adding 1% of yeast extract and 2% of peptone into the fermentation liquid when the absorbance of the fermentation liquid under the wavelength of 600nm reaches 0.8, placing the fermentation liquid in a shaker at 28 ℃, and fermenting and culturing for 72 hours at 200rpm to obtain a culture;

3) and (3) separating and purifying the culture: after the culture is finished, taking 200mL of culture, and centrifuging the culture in a centrifuge at 850 Xg for 5 minutes to remove cells of the engineering bacteria S; extracting the fermentation liquor after removing the cells by using 200mL of chloroform; this process was repeated 2 times, and the extract obtained comprised compound (I);

4) vacuum drying the extract obtained in the step 3) at 38 ℃ to constant weight to obtain an extract; subjecting the extract to silica gel column (200 mesh) chromatography to collect 100% chloroform-eluted fractions, and drying by rotary evaporation; then oscillating and suspending the dried product by using 10mL of methanol, centrifuging for 10 minutes at 21000 Xg, discarding the supernatant, and repeating the step for 2 times to remove organic impurities to obtain a crude extract;

then, the crude extract was washed in the same manner with ethyl acetate and n-hexane instead of methanol, respectively, and dried to obtain 0.276g of red solid powder.

The powder has the structural formula shown in the specification

As shown, the applicant named compound (I). The ultraviolet absorption spectrum is shown in figure 1, and the part indicated by the arrow in figure 1 is the compound (I). Mass spectrometric detection of map [ M-H]+ is shown in FIG. 2, and the chemical structural formula is shown in FIG. 3.

In this example, the total yield in 1L of fermentation broth was calculated based on the amount of red compound obtained from 200mL of culture, and the yield of compound I in the culture of the obtained engineering bacteria S was calculated to be the highest, which was 1.38g/L, indicating that the compound has a higher yeast biosynthesis rate.

EXAMPLE 2 solubility test of Compound (I)

At 25 ℃, 100mL of the solution was weighed: ethanol, methanol, acetone, ethyl acetate, chloroform, n-hexane, chloroform, glycerol, fats and DMSO were put into a beaker. 20g of the red pigment solid powder (i.e., compound I) obtained in example 1 was weighed and added to each beaker containing the solutions ethanol, methanol, acetone, ethyl acetate, chloroform, n-hexane, chloroform, glycerin, fats and DMSO. Gradually adding the solute of the red pigment solid powder, continuously stirring, and fully dissolving. Ensuring the supersaturation of the solution, filtering and taking out undissolved solid haematochrome, and drying the haematochrome for 24 hours at the temperature of 55 ℃ to ensure the complete volatilization of the solvent. The total mass of undissolved solid was weighed and the mass of solid dissolved in 100mL of solvent, i.e., the solubility of the solid at that temperature, was determined by the formula (20 g-mass of undissolved pigment ═ mass of dissolved pigment). The results of the solubility test for compound I are shown in table 1:

TABLE 1 solubility chart (25 ℃, unit: g/100mL solvent)

The detection results in table 1 show that the compound I is not soluble in other solvents except for good solubility in DMSO, and the hydrophobic and oleophobic characteristics of the compound I show that the compound I is not easy to fade due to washing, oil contamination and the like, and has good color fastness.

Example 3 analysis of Compound I production in Saccharomyces cerevisiae engineering bacterium INVsc1

1. The engineered strain of Saccharomyces cerevisiae INVSC1 containing the blank plasmid pYES2 was cultured for 72 hours in the same manner as in the preparation of example 1, and the culture was collected and prepared for use.

2. Taking the culture of the engineering bacteria S in the preparation method and the culture, performing liquid phase detection at a wavelength of 510nm, wherein the mobile phase is acetonitrile-water (0.1% formic acid), the flow rate is 1ml/min, and the elution method is gradient elution for 45min, wherein the acetonitrile accounts for 40min and the acetonitrile accounts for 5min, and the acetonitrile accounts for 15-80% and 80-90%.

The detection result is shown in FIG. 4, wherein the culture of the engineered saccharomyces cerevisiae INVSC1 containing the blank plasmid pYES2 is shown as A, which indicates that the synthesis of the compound I is not detected; the culture of the engineering bacterium S is shown as B, which indicates that the compound I is detected; PskI was shown to be a key enzyme for the synthesis of compound I.

Example 4 extraction, analysis and purification of Compound I

The engineered strain of Saccharomyces cerevisiae INVSC1 containing recombinant plasmid pYES2-PskI was cultured for 24 hours in the same manner as in example 1, and as a result, 0.34g of Compound I was isolated from 1L of the culture of the recombinant engineered strain S.

EXAMPLE 5 extraction, analysis and purification of Compound I

The engineered strain of Saccharomyces cerevisiae INVSC1 containing recombinant plasmid pYES2-PskI was cultured for 48 hours in the same manner as in example 1, and as a result, 0.78g of Compound I was isolated from 1L of the culture of the recombinant engineered strain S.

EXAMPLE 6 extraction, analysis and purification of Compound I

The engineered strain Saccharomyces cerevisiae INVSC1 containing recombinant plasmid pYES2-PskI was cultured for 72 hours in the same manner as in example 1, and as a result, 1.38g of Compound I was isolated from 1L of the culture of the recombinant engineered strain S.

EXAMPLE 7 extraction, analysis and purification of Compound I

The engineered strain of Saccharomyces cerevisiae INVSC1 containing recombinant plasmid pYES2-PskI was cultured for 96 hours in the same manner as in example 1, and as a result, 1.31g of Compound I was isolated from 1L of the culture of the recombinant engineered strain S.

EXAMPLE 8 measurement of physicochemical Properties of pigment

The compound (I) obtained in example 1 was used for physical and chemical property examination in a conventional manner: detecting the chromatic aberration parameters (DE, DC and DH) according to GB/T6688-; the particle size detection method is carried out according to GB/T19077-2016, and a detection instrument is Malvern 3000; the determination of the pH is carried out according to GB/T2390-2013, and the detection instrument is Mettler FE 28; the detection method of the intensity index is carried out according to GB/T6688-; the detection method of the conductivity index is carried out according to GB11007-89, and detection equipment is Mettler FE 30K; the viscosity index detection method is carried out according to GB/T10247-2008, the main equipment is a viscometer, the surface tension detection method is carried out according to GB/T22237-2008, and the main equipment is an Anmiao AS-120N tensiometer. The results are shown in table 2 below:

TABLE 2

As can be seen from table 2, the red powdered compound (I) obtained in example 1 has a small primary particle size, which is advantageous for applications in the downstream dyeing industry; meanwhile, the compound (I) has good detection results in color difference and aging rate, so that the biological pigment has good color rendering property, level dyeing property and color fastness, can replace the existing pigment, can be applied to the fields of synthetic fibers, plastics, rubber, leather, coatings, printing ink, paint, cosmetics, food additives and the like, and is environment-friendly.

The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Nanjing Hegu Biotechnology Ltd

<120> red natural pigment compound, preparation method and application thereof

<141> 2021-09-18

<150> 2020109868049

<151> 2020-09-18

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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acatctgccc tgaagcggga agtggccagg ctgggcaggg accagcagca ggaatgccct 180

ccctttgctt ctcttatcga cttggttcca agatggcggt cgagtacgct caatcctgcg 240

ctatgccagg cattgacttg tatttgccag ctcgcggcgt tcatgcggga acatggcgcc 300

caaggacgac cttaccctac accacagaac tcttgtctca ctggcctctg cacaggttcg 360

ctgtctgctg tcgcgtctag ttgcgcatcg agttcttccg acctgttgca cctaggtgtg 420

gagacggtca aggttgcgtt ccgcgttggt gcttgcgctt gggacttcgg cacgcgcctg 480

tccaacagtc gcgacgagat cggtcgctat caatcatgga ccgctgcact gtccggtgtc 540

agccatgatg atgttgctgc tactctcaag gagttttcga gcgagaggtc actggccaag 600

accacctctc cctacatctc cgcgaggatc gcccccaccg cggtctcggt ctccgccgca 660

cccagaatca tcacctctct gctctccgaa acacctcttg ggaagaagct tgtttccagg 720

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gtagtcgtaa atgattgtct gcgcaacacc atccgatggg atctcactcc agcagccctt 960

gctgccttca tcaaggggac ctctccagat gcttcatttt ccatcaattc gatagctact 1020

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gtcgccaccg caattctcaa acgtctggac gatgcaattg cggacaagga ccctgtcaag 1920

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ggtcagggtg cgcagttcac tggcatgggt gcagactatt atgctaggct accggacttc 2820

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ggcttgtcag aaatcaccct tgacaacgac aacatggagg ccactggctt tgtcaccttc 4500

aagaaggtct tgaacgaagg caaattccac accaaccctg ccttcatcga tgctctctcg 4560

caactcggcg gattcgtcat gaatgccaac gagggagtcg accttgacaa agaactcttc 4620

gtcaaccatg gatgggagaa cttccaactc ttcgagccta tctcgcccaa catgaactac 4680

cactctcacg ttaaaatggc tgaaggtgat gacaagctct ggtctggtga cgtcttcatc 4740

tttgacgatg ccaacaagct cactgctgtc tttaccaaga ttcagcttca gggtgttccc 4800

aagcgcttga tggagatcat cgtcaatggt gccaagaaga gattgggcag cctgcttggt 4860

cccgctcccg ctggctctgc tccttcaaag gccgcacctg cgcccaagaa ggctgctgct 4920

cctgtgtctg cacctcttcc ggctgctacc cacactgctg cgaagactgt caccaagacc 4980

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attggtgttg attcacttct gtcactcatg gtcagtagca ggatccgcga cgacattgga 5160

atcgatcttg actctgctgt cttcatggag gtcactacca ttggcaattt caaggctcac 5220

ctccgtggtc tgagtggtca cgtggagact gttactgaga ctgtggtcga agaagttgag 5280

gtacctgcgg ctgcttccgc cgctgctcct gtcgcggctc ccgccccaac tccattgcct 5340

accgcagctc ctgttgccga ggtcaacagc gaagcatgga acagtgtcct tagcattatc 5400

agtgaggaga gtggtcttgg cgtctctgat ctgactcccg agacttgctt caacgatatt 5460

ggcgttgatt ctctcctttc tctcgtcatc tgcagccgtt tccgcgacga gctcgagatc 5520

gacattcccg accgtgcgct cttcatggat tacactaccg tcgaggctct caagaaccac 5580

atcgtcggtg tcggctcacc ttcagagacc gatgttagca ccccaggcag tgtcaactct 5640

cctgcacttt cggcttcttc atcggtgtcc gacatgcggg acctgatgac ccccttcact 5700

cccgccactg agtggtcgga tgatgacaat gtcaaaggtt ttttcgacag taagccacgg 5760

ctcaagaagt ctgtctcctt cgaagttgtc gagcaagacc aggcttcctc agtcaagcca 5820

gcatggtcca tcgtccttca aggcagcccg aagcggtgtc ccaacaagtt tttcctcttt 5880

cccgacggat gcggtgccgc gacctcttat gtcaagatcc caaaggtcgc cgatagcaca 5940

gccgtcatcg gcttcaactc accctacatg aaaaatccat gggacatgaa caaccacacc 6000

ctctccgaag ttctcgcctc ctacatcgcc ggcgtccgca aacaccaaga gcacggccct 6060

taccacctcg gaggctggtc cgcagggggc atcctcgcct gcgccgtcgc ccaagaactc 6120

ctgcactcag gcgaagaaat cgccacactg accttgatcg actcccccgc cccagccgac 6180

ggcctcgacc gcctccccaa gcgcttcttc gaccactgca cctcggtcgg catcttcggc 6240

tccgaagtca ttaacgacgg caacggcgct cccaccaagg tccccgactg gctgatgccc 6300

cattttgaag ccaccatcga gctgctgcac gactataaag ctcccgtgtg gcctcaggac 6360

cgcaaggtgc cggacgtcaa cattatctgg gcgggcgact gcgcttttga cggggagaag 6420

tacgctctct tgcctggcgc gacggctgag gacgaggata ctgaggggat gaagtttttg 6480

acagagaaga ggacggattt ggggccaggg aggtggcagg agctgtttcc tgggaaggag 6540

ctgaagtgtg tggtgtcgga tggggaacat cattttagta tgatgagggg taagggagcg 6600

gagggtttga gagactttat caaggagagc attggggttt ag 6642

<210> 2

<211> 2213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Val Pro Thr Arg Val Tyr Val Phe Gly Asp Gln Thr Phe Asp

1 5 10 15

Ile Ala Asp Leu Leu Thr Lys Leu Val His Thr Tyr Asp Asp Pro Val

20 25 30

Leu Ser Asp Phe Phe Glu Arg Ala Thr Ser Ala Leu Lys Arg Glu Val

35 40 45

Ala Arg Leu Gly Arg Asp Gln Gln Gln Glu Cys Pro Pro Phe Ala Ser

50 55 60

Leu Ile Asp Leu Val Pro Arg Trp Arg Ser Ser Thr Leu Asn Pro Ala

65 70 75 80

Leu Cys Gln Ala Leu Thr Cys Ile Cys Gln Leu Ala Ala Phe Met Arg

85 90 95

Glu His Gly Ala Gln Gly Arg Pro Tyr Pro Thr Pro Gln Asn Ser Cys

100 105 110

Leu Thr Gly Leu Cys Thr Gly Ser Leu Ser Ala Val Ala Ser Ser Cys

115 120 125

Ala Ser Ser Ser Ser Asp Leu Leu His Leu Gly Val Glu Thr Val Lys

130 135 140

Val Ala Phe Arg Val Gly Ala Cys Ala Trp Asp Phe Gly Thr Arg Leu

145 150 155 160

Ser Asn Ser Arg Asp Glu Ile Gly Arg Tyr Gln Ser Trp Thr Ala Ala

165 170 175

Leu Ser Gly Val Ser His Asp Asp Val Ala Ala Thr Leu Lys Glu Phe

180 185 190

Ser Ser Glu Arg Ser Leu Ala Lys Thr Thr Ser Pro Tyr Ile Ser Ala

195 200 205

Arg Ile Ala Pro Thr Ala Val Ser Val Ser Ala Ala Pro Arg Ile Ile

210 215 220

Thr Ser Leu Leu Ser Glu Thr Pro Leu Gly Lys Lys Leu Val Ser Arg

225 230 235 240

Leu Pro Ile Thr Ala Pro Tyr His Ala Gly His Leu Tyr Thr Ala Glu

245 250 255

Asn Val Asp Ala Ile Leu Ala Asp Leu Lys Ala Thr Glu Gly His Thr

260 265 270

Thr Trp Pro Ser Leu Ser Leu Pro Ile Ile Ser Ser Val Thr Gly Glu

275 280 285

Phe Ile Pro Thr Ala Ser Phe Pro Asp Ala Leu Glu Val Val Val Asn

290 295 300

Asp Cys Leu Arg Asn Thr Ile Arg Trp Asp Leu Thr Pro Ala Ala Leu

305 310 315 320

Ala Ala Phe Ile Lys Gly Thr Ser Pro Asp Ala Ser Phe Ser Ile Asn

325 330 335

Ser Ile Ala Thr Ser Ala Glu Ser Leu Arg Pro Ala Ile Gln Lys Ile

340 345 350

Leu Gly Ser Thr Asn Gly Ile Thr Thr Ser Ile Thr Pro Thr Ser Ile

355 360 365

Asp Gln Gln Ser Val Gly Val Thr Gly Pro Ala Ala Lys Ser Lys Leu

370 375 380

Ala Ile Val Gly Met Ser Gly Arg Phe Pro Lys Ala Arg Ser Met Asn

385 390 395 400

Gly Phe Trp Asp Val Leu Asn Asn Gly Val Asp Cys His Glu Ile Val

405 410 415

Pro Ala Ala Arg Trp Pro His Glu Thr His Val Asn Asn Val Ala Asn

420 425 430

Pro Ala Lys Asn Val Ser Gly Thr Gly Phe Gly Cys Trp Leu His Asp

435 440 445

Ala Ala Glu Phe Asp Ala Arg Tyr Phe Asn Met Ser Pro Arg Glu Ala

450 455 460

Pro Gln Val Asp Pro Ala Gln Arg Met Ala Leu Leu Thr Ala Thr Glu

465 470 475 480

Ala Leu Glu Gln Ala Gly Ile Val Pro Asn Arg Thr Ala Ser Thr Glu

485 490 495

Lys Thr Arg Val Gly Val Tyr Phe Gly Ser Thr Ser Asn Asp Trp Met

500 505 510

Glu Thr Asn Ser Ala Gln Asp Ile Asp Thr Tyr Phe Ile Pro Gly Gly

515 520 525

Asn Arg Ala Phe Ile Pro Gly Arg Ile Asn Tyr His Phe Lys Phe Ser

530 535 540

Gly Pro Ser Tyr Thr Ile Asp Thr Ala Cys Ser Ser Ser Leu Ala Ala

545 550 555 560

Leu His Leu Ala Cys Asn Ala Leu Trp Lys Gly Glu Ile Asp Thr Ala

565 570 575

Ile Val Gly Gly Thr Asn Val Leu Thr Asn Pro Asp Met Thr Ala Gly

580 585 590

Leu Asp Arg Gly His Phe Leu Ser Arg Thr Gly Asn Cys Lys Thr Phe

595 600 605

Asp Glu Gly Ala Asp Gly Tyr Cys Arg Gly Glu Ala Val Ala Thr Ala

610 615 620

Ile Leu Lys Arg Leu Asp Asp Ala Ile Ala Asp Lys Asp Pro Val Lys

625 630 635 640

Ala Cys Ile Leu Ala Ile Ser Thr Asn His Asn Ala Glu Ala Glu Ser

645 650 655

Ile Thr Arg Pro His Val Gly Ala Gln Arg Glu Leu Phe Asp Lys Val

660 665 670

Leu Gly Gln Ala Gly Ile Asn Ala Ala Asn Ile Ser Tyr Cys Glu Leu

675 680 685

His Gly Thr Gly Thr Gln Ala Gly Asp Ala Gly Glu Thr Ala Ser Val

690 695 700

Ile Asn Thr Leu Ala Pro Leu Asp Gly Pro Tyr Ala Arg Thr Glu Ser

705 710 715 720

Gln Pro Leu Tyr Ile Gly Ala Ala Lys Ala Asn Ile Gly His Gly Glu

725 730 735

Ala Ala Ala Gly Val Thr Ser Leu Ala Lys Val Leu Leu Met Leu Glu

740 745 750

Asn Asn Thr Ile Pro Pro His Cys Gly Ile Lys Thr Lys Ile Asn Ser

755 760 765

Lys Ile Pro Glu Leu Ser Thr Arg Asn Thr Tyr Ile Ala Asn Lys Ser

770 775 780

Thr Pro Trp Val Arg Pro Lys Gly Gly Val Arg Gln Ile Met Leu Asn

785 790 795 800

Asn Phe Ser Ala Ala Gly Gly Asn Thr Ala Met Ile Leu Glu Asp Ala

805 810 815

Pro Glu Ile Ala Tyr Pro Ser Asp Ala Asp Val Arg Ser Ser His Val

820 825 830

Val Ala Val Ser Ala Lys Thr Pro Val Ser Leu Glu Asn Asn Leu Lys

835 840 845

Asn Leu Ile Asp Trp Ile Asp Asn Thr Glu Thr Asp Asp Leu Thr Leu

850 855 860

Pro Cys Leu Ser Tyr Thr Thr Thr Ala Arg Arg Ile His His Pro His

865 870 875 880

Arg Val Val Ala Val Gly Ser Asn Leu Ala Glu Ile Arg Met Ser Leu

885 890 895

Gln Leu Ser Leu Asp Thr Lys Asp Gly Ala Asn Arg Ser Lys Thr Ala

900 905 910

Pro Ser Tyr Val Phe Ser Phe Thr Gly Gln Gly Ala Gln Phe Thr Gly

915 920 925

Met Gly Ala Asp Tyr Tyr Ala Arg Leu Pro Asp Phe Arg Ala Asp Ile

930 935 940

Asp Gln Tyr Asp Gln Ile Ile Arg Gln Leu Glu Leu Pro Ser Ile Lys

945 950 955 960

Ala Leu Phe Arg Glu Pro Glu Thr Ala Phe Lys Asp Ala Thr Pro Thr

965 970 975

Met Leu Gln Leu Ala Ala Val Cys Leu Gln Met Ala Leu Ser Arg Phe

980 985 990

Trp Thr Ser Leu Gly Val Gln Pro Lys Ala Val Val Gly His Ser Leu

995 1000 1005

Gly Glu Tyr Ser Ala Leu Tyr Ala Ala Gly Val Leu Ser Gln Ala Asp

1010 1015 1020

Val Leu Ser Leu Val Gly Arg Arg Ala Glu Leu Leu Glu Thr His Cys

1025 1030 1035 1040

Glu Glu Gly Ser His Ala Met Leu Ala Ile Arg Ser Ser Ala Leu Gly

1045 1050 1055

Leu Thr Ser Leu Leu Gly Glu Pro Gly Lys Asp Tyr Glu Val Ser Cys

1060 1065 1070

Val Asn Gly Lys Glu Ser Ile Val Leu Gly Gly Thr Lys Glu Gln Met

1075 1080 1085

Asn Asn Ile Arg Pro Lys Leu Gly Arg Lys Leu Ser His Thr Phe Leu

1090 1095 1100

Glu Val Pro Tyr Ala Tyr His Thr Ala Gln Val Glu Pro Ile Leu Thr

1105 1110 1115 1120

Ser Leu Ser Thr Ile Ala Arg Gly Ile Lys Phe Asn Asp Pro Val Ile

1125 1130 1135

Pro Val Ile Ser Pro Ala Val Gly Lys Val Ile His Lys Ala Glu Glu

1140 1145 1150

Phe Pro Gln Asp Phe Ile Ser Arg His Cys Arg Gly Thr Val Asn Met

1155 1160 1165

His Asp Ala Leu Thr Val Ala Lys Gln Glu Gly Leu Leu Asp Glu Arg

1170 1175 1180

Met Met Gly Ile Glu Ile Gly Pro Ala Pro Val Val Cys Lys Met Val

1185 1190 1195 1200

Lys Glu Val Ala Gly Pro Ala Phe Gln Thr Phe Ala Ser Met Arg Gln

1205 1210 1215

Gly Asp Asp Ser Trp Lys His Leu Thr Leu Ala Leu Ser Lys Phe Gln

1220 1225 1230

Ala Ala Gly Ala Thr Ile Asp Trp Asn Ala Tyr Asn Lys Asp Phe Glu

1235 1240 1245

Ser Ala Gln Gln Val Leu Gln Ile Pro Ala Tyr Ser Trp Glu Tyr Lys

1250 1255 1260

Glu Tyr Trp Lys Gln Tyr Val Asn Asp Trp Ser Leu Arg Lys Gly Asp

1265 1270 1275 1280

Ala Pro Leu Arg Ile Val Thr Gly Gly Leu Asp Ser Ser Ser Ile Gln

1285 1290 1295

Lys Val Ile Lys Asp Glu Leu Ala Asn Asp Gly Gly Glu Leu Ile Val

1300 1305 1310

Glu Ser Asp Leu Val Arg Pro Asp Leu His Pro Met Val Gln Gly His

1315 1320 1325

His Val Tyr Gly Val Pro Leu Cys Thr Pro Ser Val Tyr Ala Asp Ile

1330 1335 1340

Ser Leu Thr Ile Gly Glu His Val Lys Asn Ile Leu Gly Gly Gly Gln

1345 1350 1355 1360

Asn Val Gly Val Glu Val Gly Lys Met Thr Ile Gln Ser Ala Leu Val

1365 1370 1375

Ala Asn Glu Glu Gly Arg Pro Gln Ile Leu Arg Ala Ile Gly Lys Ile

1380 1385 1390

Asn His Lys Glu Arg Thr Val His Ile Thr Phe His Ser Val Asn Lys

1395 1400 1405

Asp Gly Lys Ala Thr Glu Gln His Ser Asn Cys Met Val Arg Leu Tyr

1410 1415 1420

Asp Val Asp Lys Val Lys Lys Ala Ala Leu Ala Lys Val Pro Glu Val

1425 1430 1435 1440

Gln Gly Arg Met Lys Ser Leu His Asp Gln Thr Arg Ser Thr Gly Ser

1445 1450 1455

Thr Phe Arg Phe Ser Lys Ala Met Ile Tyr Lys Met Ile Gly Lys Leu

1460 1465 1470

Ala Asp Phe Asp Pro Asn Tyr Arg Gly Leu Ser Glu Ile Thr Leu Asp

1475 1480 1485

Asn Asp Asn Met Glu Ala Thr Gly Phe Val Thr Phe Lys Lys Val Leu

1490 1495 1500

Asn Glu Gly Lys Phe His Thr Asn Pro Ala Phe Ile Asp Ala Leu Ser

1505 1510 1515 1520

Gln Leu Gly Gly Phe Val Met Asn Ala Asn Glu Gly Val Asp Leu Asp

1525 1530 1535

Lys Glu Leu Phe Val Asn His Gly Trp Glu Asn Phe Gln Leu Phe Glu

1540 1545 1550

Pro Ile Ser Pro Asn Met Asn Tyr His Ser His Val Lys Met Ala Glu

1555 1560 1565

Gly Asp Asp Lys Leu Trp Ser Gly Asp Val Phe Ile Phe Asp Asp Ala

1570 1575 1580

Asn Lys Leu Thr Ala Val Phe Thr Lys Ile Gln Leu Gln Gly Val Pro

1585 1590 1595 1600

Lys Arg Leu Met Glu Ile Ile Val Asn Gly Ala Lys Lys Arg Leu Gly

1605 1610 1615

Ser Leu Leu Gly Pro Ala Pro Ala Gly Ser Ala Pro Ser Lys Ala Ala

1620 1625 1630

Pro Ala Pro Lys Lys Ala Ala Ala Pro Val Ser Ala Pro Leu Pro Ala

1635 1640 1645

Ala Thr His Thr Ala Ala Lys Thr Val Thr Lys Thr Val Ala Val Ala

1650 1655 1660

Pro Lys Ala Ser Gly Glu Ser Pro Ala Val Val Thr Ala Met Asn Ile

1665 1670 1675 1680

Ile Ser Glu Glu Ser Gly Ile Ala Leu Pro Glu Leu Thr Asp Asp Ser

1685 1690 1695

Arg Phe Asp Asp Ile Gly Val Asp Ser Leu Leu Ser Leu Met Val Ser

1700 1705 1710

Ser Arg Ile Arg Asp Asp Ile Gly Ile Asp Leu Asp Ser Ala Val Phe

1715 1720 1725

Met Glu Val Thr Thr Ile Gly Asn Phe Lys Ala His Leu Arg Gly Leu

1730 1735 1740

Ser Gly His Val Glu Thr Val Thr Glu Thr Val Val Glu Glu Val Glu

1745 1750 1755 1760

Val Pro Ala Ala Ala Ser Ala Ala Ala Pro Val Ala Ala Pro Ala Pro

1765 1770 1775

Thr Pro Leu Pro Thr Ala Ala Pro Val Ala Glu Val Asn Ser Glu Ala

1780 1785 1790

Trp Asn Ser Val Leu Ser Ile Ile Ser Glu Glu Ser Gly Leu Gly Val

1795 1800 1805

Ser Asp Leu Thr Pro Glu Thr Cys Phe Asn Asp Ile Gly Val Asp Ser

1810 1815 1820

Leu Leu Ser Leu Val Ile Cys Ser Arg Phe Arg Asp Glu Leu Glu Ile

1825 1830 1835 1840

Asp Ile Pro Asp Arg Ala Leu Phe Met Asp Tyr Thr Thr Val Glu Ala

1845 1850 1855

Leu Lys Asn His Ile Val Gly Val Gly Ser Pro Ser Glu Thr Asp Val

1860 1865 1870

Ser Thr Pro Gly Ser Val Asn Ser Pro Ala Leu Ser Ala Ser Ser Ser

1875 1880 1885

Val Ser Asp Met Arg Asp Leu Met Thr Pro Phe Thr Pro Ala Thr Glu

1890 1895 1900

Trp Ser Asp Asp Asp Asn Val Lys Gly Phe Phe Asp Ser Lys Pro Arg

1905 1910 1915 1920

Leu Lys Lys Ser Val Ser Phe Glu Val Val Glu Gln Asp Gln Ala Ser

1925 1930 1935

Ser Val Lys Pro Ala Trp Ser Ile Val Leu Gln Gly Ser Pro Lys Arg

1940 1945 1950

Cys Pro Asn Lys Phe Phe Leu Phe Pro Asp Gly Cys Gly Ala Ala Thr

1955 1960 1965

Ser Tyr Val Lys Ile Pro Lys Val Ala Asp Ser Thr Ala Val Ile Gly

1970 1975 1980

Phe Asn Ser Pro Tyr Met Lys Asn Pro Trp Asp Met Asn Asn His Thr

1985 1990 1995 2000

Leu Ser Glu Val Leu Ala Ser Tyr Ile Ala Gly Val Arg Lys His Gln

2005 2010 2015

Glu His Gly Pro Tyr His Leu Gly Gly Trp Ser Ala Gly Gly Ile Leu

2020 2025 2030

Ala Cys Ala Val Ala Gln Glu Leu Leu His Ser Gly Glu Glu Ile Ala

2035 2040 2045

Thr Leu Thr Leu Ile Asp Ser Pro Ala Pro Ala Asp Gly Leu Asp Arg

2050 2055 2060

Leu Pro Lys Arg Phe Phe Asp His Cys Thr Ser Val Gly Ile Phe Gly

2065 2070 2075 2080

Ser Glu Val Ile Asn Asp Gly Asn Gly Ala Pro Thr Lys Val Pro Asp

2085 2090 2095

Trp Leu Met Pro His Phe Glu Ala Thr Ile Glu Leu Leu His Asp Tyr

2100 2105 2110

Lys Ala Pro Val Trp Pro Gln Asp Arg Lys Val Pro Asp Val Asn Ile

2115 2120 2125

Ile Trp Ala Gly Asp Cys Ala Phe Asp Gly Glu Lys Tyr Ala Leu Leu

2130 2135 2140

Pro Gly Ala Thr Ala Glu Asp Glu Asp Thr Glu Gly Met Lys Phe Leu

2145 2150 2155 2160

Thr Glu Lys Arg Thr Asp Leu Gly Pro Gly Arg Trp Gln Glu Leu Phe

2165 2170 2175

Pro Gly Lys Glu Leu Lys Cys Val Val Ser Asp Gly Glu His His Phe

2180 2185 2190

Ser Met Met Arg Gly Lys Gly Ala Glu Gly Leu Arg Asp Phe Ile Lys

2195 2200 2205

Glu Ser Ile Gly Val

2210

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgggatccat ggccgttcca acgag 25

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cggaattcct aaaccccaat gctctcc 27

Claims (6)

1. A red natural pigment compound has a structural formula of

2. The method for preparing a red natural pigment compound according to claim 1, comprising the steps of:

1) amplifying a PskI gene with a nucleotide sequence shown as SEQ ID NO.1, and connecting the amplified PskI gene to a plasmid vector to obtain a recombinant plasmid;

then, the recombinant plasmid is transformed into saccharomyces cerevisiae engineering bacteria to obtain transformed engineering bacteria;

2) selecting a single bacterial colony of the transformed engineering bacteria obtained in the step 1) to perform fermentation culture in an SC (selective culture) medium, adding yeast extract with the final concentration of 1% and peptone with the final concentration of 2% when the absorbance of a fermentation solution reaches 0.8 under the wavelength of 600nm, and then continuing to culture for 72 hours to obtain a culture;

3) centrifuging the culture, and extracting supernatant with chloroform to obtain extractive solution;

4) drying the extract obtained in the step 3) to obtain an extract; subjecting the extract to silica gel column chromatography, collecting chloroform-eluted components, and rotary drying to obtain dried product;

5) oscillating and suspending the dried product by using methanol, centrifuging, removing supernatant, suspending the precipitate by using methanol again, and centrifuging to obtain a crude extract; then treating the crude extract with ethyl acetate and n-hexane respectively instead of methanol in the same way, and finally drying the obtained product to obtain the red natural pigment compound.

3. The method for producing a red natural pigment compound according to claim 2, wherein the amplification in step 1) is performed using primers having nucleotide sequences represented by SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. Use of the red natural pigment compound according to claim 1 for the preparation of dyes.

5. The nucleotide sequence of the PskI gene is shown as SEQ ID NO. 1.

6. Use of the PskI gene according to claim 5 for the preparation of red natural pigment compounds,the structural formula of the red natural pigment compound is shown in the specification

Images