CN114317296A - Preparation method of bethest forest - Google Patents

Abstract

The invention discloses a preparation method of bethest forest, belonging to the field of microbial fermentation and natural product purification. The invention provides a method for preparing a bethest Gaulin, which comprises knocking out polyketide synthase CTB1 gene on a cercospora JNU001 genome with a preservation number of CCTCC NO of M2017842 by gene knockout to obtain a mutant strain cercospora JNU001 delta CTB1, reducing metabolic byproducts of the cercospora and facilitating the preparation of the bethest Gaulin, preparing a crude product of the bethest Gaulin by using a microbial fermentation technology, preliminarily purifying the crude product by using a thin layer chromatography method, and preparing the high-purity bethest Gaulin by using a semi-preparative liquid chromatography method.

Description

Preparation method of bethest forest

Technical Field

The invention relates to a preparation method of bethest forest, belonging to the fields of microbial fermentation and natural product purification.

Background

Beigraine is a natural product with a complex polycyclic structure, and is a yellow toxin isolated from Cercospora betacola, which has antibacterial activity and phytotoxicity and can be absorbed by K⁺And removing H⁺Depolarizing the transmembrane potential of plant cells while being plasma membrane-dependent in vitro⁺-Mg²⁺The ATP enzyme of the channel has special inhibition effect and also has the effect of inhibiting the growth of cancer cells.

At present, the preparation difficulty of the bethest forest is increased due to the fact that wild cercospora species produce cercospora sp in a large quantity, and in order to find a simple and easy-to-operate preparation method of the bethest forest with high purity, starting from two aspects, firstly, the gene mutant strain is constructed to carry out microbial fermentation, and secondly, the high-purity bethest forest is prepared through a simple and easy-to-operate purification process.

Because the preparation of the bethenazone is limited at present, the biological activity and the pharmacological activity of the bethenazone are not developed, and therefore a simple and easy-to-operate preparation method of high-purity bethenazone needs to be found, and a foundation can be laid for the application and development of the bethenazone.

Disclosure of Invention

The method comprises the steps of obtaining mutant strains through gene knockout, reducing metabolites of cercospora, facilitating preparation of the bethanensis, preparing a crude product of the bethanensis by using a microbial fermentation technology, preliminarily purifying by using a thin layer chromatography, and preparing the high-purity bethanensis by using a semi-preparative liquid chromatography.

The invention provides a genetically engineered bacterium, which is characterized in that the genetically engineered bacterium is obtained by knocking out a polyketide synthase CTB1 gene on a cercospora JNU001 genome with the preservation number of CCTCC NO: M2017842 and is named cercospora JNU001 delta CTB 1.

The Cercospora JNU001 is described in the Chinese patent publication No. CN 109055237B.

In one embodiment of the invention, the nucleotide sequence of the gene encoding polyketide synthase CTB1 is shown in SEQ ID NO. 1.

In one embodiment of the invention, the genetically engineered bacterium cercospora JNU001 Δ CTB1 is cultured as follows: the above Cercospora JNU001 [ 001 ] CTB1 was cultured in a shaker at a constant temperature of 25 ℃ and 135rpm under continuous and sufficient light.

The invention also provides a preparation method of the bethest forest, which is prepared by fermenting the genetic engineering bacteria.

In one embodiment of the invention, the genetically engineered bacteria are inoculated into a fermentation medium for liquid fermentation culture to prepare a fermentation broth, and the fermentation broth is extracted to prepare the bedigold.

In one embodiment of the invention, the culture time of the liquid fermentation culture is 8-15 days, the culture temperature is 25-30 ℃, and the rotation speed is 100-150 rpm.

In one embodiment of the invention, the fermentation culture time is 8 days.

In one embodiment of the invention, the fermentation medium comprises: 15-25 g/L glucose, 1-3 g/L soybean peptone, 0.5-2 g/L sodium acetate, 1-10 mg/L L-phenylalanine, 50-200 mg/L sodium benzoate, 80-200 mg/L potassium dihydrogen phosphate, 0.5-2 mg/L biotin, 3-10 mg/L calcium nitrate, 0.5-2 mg/L pyridoxal phosphate, 0.5-2 mg/L calcium pantothenate, 0.5-2 mg/L thiamine hydrochloride, 2-10 mg/L manganese chloride, 2-10 mg/L ferric chloride, 0.5-2 mg/L copper nitrate, 0.5-5 mg/L magnesium sulfate, and 1-5 mg/L zinc sulfate.

In one embodiment of the present invention, the fermentation broth obtained is extracted by using a mixed solution of dichloromethane and methanol at a volume ratio of (4: 1) to (10: 1) at a rotation speed of: 100rpm to 300rpm, and the extraction time is 0.5 to 1 hour.

In one embodiment of the present invention, the method further comprises a purification step of primarily purifying the prepared bethilin by thin layer chromatography, and preparing high-purity bethilin using semi-preparative liquid chromatography.

In one embodiment of the present invention, the ratio of the developing solvent of the thin layer chromatography preparation plate in the thin layer chromatography is such that the volume ratio of dichloromethane, methanol and formic acid is (100:10:1) - (200:10: 1).

In one embodiment of the invention, the preparative column of the semi-preparative liquid chromatography is a C18 column with a specification of 4.6 mm × 250 mm.

In one embodiment of the present invention, the mobile phase of the semi-preparative liquid chromatography is acetonitrile and water, and 0.1% by volume of formic acid is added.

In one embodiment of the invention, the semi-preparative liquid chromatography is performed under gradient elution with 45% to 90% acetonitrile for 35min at a flow rate of 3 mL/min.

The invention also provides application of the gene engineering bacteria in preparing the bethilin or products containing the bethilin.

In one embodiment of the invention, the product is a pharmaceutical or chemical.

Advantageous effects

(1) According to the invention, from gene knockout, cercosporal synthesis key gene CTB1 is knocked out, the cercosporal delta CTB1 mutant strain is obtained, metabolic byproducts of cercosporal are greatly reduced, and the preparation of the Bedta Gaultheria is facilitated.

(2) The method for preparing high-purity betheuchin solves the problem that the application of the betheuchin is limited due to the fact that the source of the betheuchin is rare and no purification method exists.

(3) The method for preparing high-purity bethiavalicarb sinensis has the advantages of simple operation, few preparation steps and high preparation purity, so that the bethiavalicarb sinensis has wide application prospect in the aspect of inhibiting the growth of cancer cells.

Biological material preservation

A Cercospora JNU001(Cercospora sp.JNU001) is classified and named Cercospora JNU001(Cercospora sp.JNU001), and is preserved in the China center for type culture collection in 12 and 25 months in 2017, with the preservation number of CCTCC NO: m2017842, the preservation unit address is Wuhan, Wuhan university in China.

Drawings

FIG. 1: cercospora JNU001 Δ CTB1 in positive form.

FIG. 2: cercospora JNU001 Δ CTB1 in reverse form.

FIG. 3: preparing a betheuchrine mass spectrogram.

FIG. 4: preparing the bethijialin¹And (4) H spectrum.

FIG. 5: preparing the bethijialin¹³And (4) C spectrum.

FIG. 6: preparing a Bedbigold COSY spectrum.

FIG. 7: and preparing a BEDIGalin HMBC spectrum.

FIG. 8: and preparing the Beigold HSQC spectrum.

FIG. 9: preparing a betigalloline ROESY spectrum.

FIG. 10: and (5) detecting the image by high performance liquid chromatography of the betel.

FIG. 11: structure of betheuchrine.

FIG. 12: beth upslin standard curve.

Detailed Description

The invention is further illustrated with reference to specific examples.

The lywallases mentioned in the following examples were purchased from sigma, and the plasmids pGD-GFP mentioned in the following examples are disclosed in the "differentiation of the Ergosterol Biosynthetic Pathway Results in incorporated Membrane viability, gaming over production and characterization of Extracellular vectors in localized Fermentation" articles.

The media involved in the following examples are as follows:

resistance screening medium: 10g/L glucose, 20g potato, 15-20g agar and 50mg/L hygromycin B.

S-7 solid medium: 15-20g/L agar, 20g/L glucose, 2g/L soytone, 1g/L sodium acetate, 5 mg/LL-phenylalanine, 100mg/L sodium benzoate, 136mg/L potassium dihydrogen phosphate, 1mg/L biotin, 6.5mg/L calcium nitrate, 1mg/L pyridoxal phosphate, 1mg/L calcium pantothenate, 1mg/L thiamine hydrochloride, 5mg/L manganese chloride, 2mg/L ferric chloride, 1mg/L copper nitrate, 3.6mg/L magnesium sulfate, 2.5mg/L zinc sulfate.

S-7 liquid fermentation medium: 20g/L glucose, 2g/L soybean peptone, 1g/L sodium acetate, 5mg/L L-phenylalanine, 100mg/L sodium benzoate, 136mg/L potassium dihydrogen phosphate, 1mg/L biotin, 6.5mg/L calcium nitrate, 1mg/L pyridoxal phosphate, 1mg/L calcium pantothenate, 1mg/L thiamine hydrochloride, 5mg/L manganese chloride, 2mg/L ferric chloride, 1mg/L copper nitrate, 3.6mg/L magnesium sulfate, 2.5mg/L zinc sulfate.

PDB culture medium: 10g/L glucose and 200g potato.

A method for culturing Cercospora JNU001 in the following examples:

inoculating Cercospora delta CTB1 mutant strain on S-7 plate, growing at 25 deg.C for 5-10 days, sealing with sealing film, and storing in refrigerator at 4 deg.C. Preparing 1L S-7 liquid culture medium in 2L shake flask, sterilizing at 115 deg.C for 15min, cooling, perforating the plate with a perforator to obtain stipe of 5mm × 5mm, adding 3 pieces into 1L S-7 liquid culture medium, culturing at 25 deg.C and 135rpm in a continuous and recombinant shaking table under continuous illumination for 8-10 days.

The buffers referred to in the following examples are as follows:

and (3) permeation buffer solution: dissolving 1.42g of disodium hydrogen phosphate and 144g of magnesium sulfate in deionized water, and diluting to constant volume with 1000mL of distilled water for later use; 0.276g of sodium dihydrogen phosphate and 29g of magnesium sulfate are dissolved in deionized water, 200mL of distilled water is added to a constant volume for standby, and the two prepared solutions are mixed and adjusted to pH 5.8.

STC buffer solution: 218.6g of sorbitol, 0.47g of calcium chloride and 1000mL of distilled water are added to the constant volume, filtered and sterilized, and then stored in a refrigerator at 4 ℃.

PEG6000 buffer solution: 250g of PEG6000, 11.098g of calcium chloride, 44.73g of potassium chloride and 1000mL of distilled water for constant volume, filtering and sterilizing, and storing in a refrigerator at 4 ℃.

The detection methods referred to in the following examples are as follows:

content detection of the betigraine:

(1) and (5) making a standard curve.

A standard curve was prepared by using purified Betadine having the highest purity, and the sample was diluted to ten gradients of 0.02mg/mL, 0.04mg/mL, 0.06mg/mL, 0.08mg/mL, 0.1mg/mL, 0.12mg/mL, 0.14mg/mL, 0.16mg/mL, 0.18mg/mL, and 0.2mg/mL, and methanol was used as a solvent, and a Waters 2695 liquid chromatograph was used to detect the sample loading amount of 50. mu.L, and the standard curve was obtained by calculation based on the UV absorption at 254 nm.

(2) Betel content detection

Extracting 1L of fermentation liquor for 3 times, performing rotary evaporation drying on the extraction liquid after extraction, re-dissolving the obtained sample by using 20mL of methanol, detecting by using a Waters 2695 liquid chromatograph, calculating the peak area according to the ultraviolet absorption value under 254nm, substituting into a standard curve, and calculating to obtain the content of the bethanlin.

(3) Method for calculating purity of betigulin

Dissolving a proper amount of prepared betheylene in methanol, detecting the purity of the betheylene by using a Waters 2695 liquid chromatograph, and using a mobile phase A: acetonitrile (containing 0.1% formic acid), B: water (containing 0.1% of formic acid), gradient elution is carried out under the liquid phase condition, the mobile phase A5% -85%, the elution time is 50min, the detection wavelength is 200nm-600nm, peak area integration is carried out in an ultraviolet detection signal diagram by using liquid chromatography software, and when the integral of the peak area of the Betadine is more than 95%, the high-purity Betadine is prepared.

Example 1: construction of Cercospora JNU001 [ 001 ] CTB1

The method comprises the following specific steps:

(1) preparing protoplasts:

culturing Cercospora JNU001 in S-7 solid culture medium at 25 deg.C for 5-6 days, and culturing its mycelium in 100mL S-7 liquid culture medium for 5 days. Taking out mycelium, grinding with sterilized mortar, adding 100mL S-7 liquid culture medium, culturing for 18 hr, collecting appropriate amount of mycelium, centrifuging at 6000 Xg for 10min, washing with osmotic buffer twice, and discarding supernatant. 30mL of a permeation buffer containing 1g of lywallzyme was added, and the mixture was incubated at 30 ℃ and 120rpm for 2 to 3 hours to prepare a suspension. The suspension is filtered and collected by 6 layers of gauze, equal volume of STC buffer solution is gently added, 5000g of the suspension is centrifuged for 10min, and the supernatant is discarded. Adding 2 times of STC buffer solution, centrifuging for 5-8 min at 6000g, discarding supernatant, adding an appropriate amount of STC buffer solution for heavy suspension, and subpackaging.

(2) Construction of a knockout plasmid:

an upstream sequence (nucleotide sequence is shown as SEQ ID NO. 2) and a downstream sequence (nucleotide sequence is shown as SEQ ID NO. 3) outside the reading frame of the CTB1 gene are found from the whole genome of cercospora JNU001, 1500bp are selected for cloning, hygromycin resistance genes are inserted into the upstream sequence and the downstream sequence, and a complete plasmid is constructed after a vector fragment of plasmid pGD-GFP is connected.

(3) Cloning of the exogenous fragment: comprises an upstream sequence, a hygromycin B resistance gene and a downstream sequence, the sequence of a cloned fragment is about 20 mu g, and the cloned fragment is purified for later use.

(4) And (3) exogenous fragment transformation: and (3) adding 200 mu L of STC buffer solution into 200 mu L of protoplast to dissolve the prepared exogenous fragment, gently mixing, and incubating on ice for 1 h. Add 1.25mL PEG6000 buffer, gently rotate and mix, incubate for 30min at room temperature. 5mL of PDB medium containing 1.2mol/L sorbitol was added and the mixture was cultured for 12 hours. Spread on plates containing antibiotics.

(5) And (3) transformant screening: after the transformant grows out, extracting a genome, carrying out PCR verification, and screening a correct transformant to obtain the cercospora JNU001 delta CTB1 (shown in figures 1-2) with the CTB1 gene knocked out.

Example 2: cercospora JNU001 delta CTB1 fermentation culture

The method comprises the following specific steps:

(1) cercospora JNU 001. delta. CTB1 prepared in example 1 was inoculated onto S-7 solid medium, grown at 25 ℃ for 5 to 10 days, sealed with a sealing film, and stored in a refrigerator at 4 ℃.

(2) Preparing 1L S-7 liquid culture medium in 2L shake flask, sterilizing at 115 deg.C for 15min, and cooling; perforating the plate for culturing cercospora JNU001 delta CTB1 obtained in the step (1) by using a perforator to obtain stipes with the size of 5mm multiplied by 5mm, putting 3 pieces of the stipes into a prepared 1L S-7 liquid culture medium, placing the mixture into a shaking table with the temperature of 25 ℃, the rpm of 135rpm and continuous illumination and recombination, and culturing for 8-10 days to prepare a fermentation liquid containing mycelia.

(3) And (3) separating the mycelium from the fermentation liquor containing the mycelium collected in the step (2) by using a Buchner funnel, and adding a mixed solution of dichloromethane and methanol (wherein the volume ratio of dichloromethane to methanol is 5: 1) into the harvested fermentation liquor according to the volume fraction of 50%. Stirring and extracting for 1h by using a magnetic stirrer, repeating for 3 times, separating by using a separating funnel, collecting an organic phase, and rotatably evaporating a dichloromethane solvent at 37 ℃ to prepare a crude product of the Betadine solid.

Example 3: preparation of pure betel nut and high forest product

The method comprises the following specific steps:

(1) dissolving the crude solid product of the betheumarine obtained in the example 2 by using dichloromethane, wherein the concentration of the dissolved solid product is 5mg/mL, uniformly loading the dissolved solid product on a thin-layer chromatography preparation plate, separating by using a developing solvent with dichloromethane and methanol to formic acid volume ratio of (100:10:1) to (200:10:1), drying by using a blower after the first chromatography is finished, continuously separating by using a developing solvent with dichloromethane and methanol to formic acid volume ratio of (100:10:1) to (200:10:1), collecting target bands, and collecting target bands by using a developing solvent with dichloromethane to methanol volume ratio of 15: 1, and rotationally evaporating dichloromethane and methanol at 37 ℃ for later use to prepare a primarily purified Bedtiegolin sample.

(2) And (2) dissolving the sample prepared in the step (1) by using methanol, wherein the concentration of the dissolved sample is 1.5mg/mL, and performing suction filtration by using a 0.22-micron PTFE filter membrane for later use. The preparation of a Baiye forest is carried out by using an Agilent 1260-prep liquid chromatograph, the detector is an ultraviolet detector, the wavelength is 254nm, and the semi-preparative chromatographic column is an Agilent Eclipse XDB-C18, and the specification is 9.4mm multiplied by 250 mm. The mobile phases used were acetonitrile and water, to which 0.1% by volume of formic acid was added. The semi-preparative chromatographic column was mounted on 1260-prep liquid phase, equilibrated with 45% acetonitrile at a flow rate of 3mL/min, and loaded at 600. mu.L each time after equilibration. The liquid phase condition is gradient elution, 45 percent to 90 percent acetonitrile gradient elution is carried out for 35 minutes, and the peak is collected after the peak is produced. And mixing the collected samples, and rotationally evaporating acetonitrile and water at 37 ℃ to obtain the pure product of the betheucha forest.

Example 4: identification of high-purity bethilin and detection of content

The method comprises the following specific steps:

(1) an appropriate amount of the purified betheynia sinensis product obtained in example 3 was dissolved in methanol, and purity of the betheynia sinensis product was measured by a Waters 2695 liquid chromatograph at a detection wavelength of 200nm to 600nm, and when integration of peak area was performed in a detection signal diagram and the integration of the betheia sinensis peak area was 95% or more, the purified betheia sinensis product was obtained with purity as high as 95% or more (as shown in fig. 3).

(2) Taking the high-purity Bedeighine prepared in the step (1), adjusting the final concentration to 50 mu g/mL, and carrying out liquid phase mass spectrometry (shown in figure 4).

Dissolving another 5mg in 600 μ L of deuterated acetone, and performing nuclear magnetic resonance characterization including¹The spectrum of the H is shown in the specification,¹³c spectrum, COSY spectrum, HMBC spectrum, HSQC spectrum, ROESY spectrum (as shown in FIGS. 5-10).

The results show that: the relative molecular mass of the bethilin measured by high-resolution mass spectrometry is 638.0831;

of the betigraine¹H NMR(800MHz,Acetone-d₆)δ：7.36–7.23(2H,m,H-25,H-26),5.71(1H, d,J＝4.2Hz,H-42),4.95(1H,s,H-3),4.50(1H,dt,J＝12.3,4.5Hz,H-19),4.05(1H,d,J＝1.3 Hz,H-38),3.71(3H,s,H-41),3.33(2H,d,J＝3.4Hz,H-7),2.92(1H,ddd,J＝19.1,11.4,7.2Hz, H-17),2.65(1H,ddd,J＝19.7,6.8,1.4Hz,H-17),2.34(1H,dtd,J＝13.3,11.7,6.8Hz,H-18),2.12 (1H,dddd,J＝13.8,7.2,5.2,1.4Hz,H-18),1.64(3H,s,H-40)；

Of the betigraine¹³C NMR(201MHz,Acetone-d₆)δ:202.57(C-23),187.29(C-12),186.84(C-4), 183.46(C-20),181.37(C-16),170.12(C-43),158.22(C-27),157.41(C-9),156.09(C-11),155.62 (C-24),144.79(C-1),130.49(C-25),127.78(C-26),116.59(C-2),114.88(C-8),114.43(C-22), 112.88(C-21),106.60(C-10),103.19(C-5),101.79(C-13),86.98(C-14),71.63(C-19),60.61 (C-38),59.26(C-37),53.24(C-41),49.65(C-6),44.77(C-3),40.35(C-7),28.50(C-17),25.89 (C-18),18.71(C-40)。

(ii) high-resolution mass spectrometry,¹h NMR and¹³c NMR is consistent with the publication "Structures of the beta, the yellow toxins produced by Cercospora beta", and thus the structure is confirmed to be Bedford;

in addition, by complementing the two-dimensional nuclear magnetic spectrum, the structure of bedigold was further confirmed (as shown in fig. 11).

(3) Taking 1L of the fermentation liquid obtained in the step (2) of the example 2, extracting by the method in the step (3) of the example 2 for 3 times, performing rotary evaporation drying on the extraction liquid after extraction, redissolving the obtained sample by using 20mL of methanol, detecting by using a Waters 2695 liquid chromatograph, wherein the sample loading volume is 50 mu L, performing peak area integration on a detection signal diagram according to the ultraviolet absorption value at 254nm, and substituting into a standard curve (shown in figure 12), wherein the calculated yield of the Bedford forest is 5 mg/L.

The procedure of example 3 was repeated, and the yield of purified bethia was 2.5mg/L, as determined by purifying bethia in 1L of the fermentation broth, dissolving the obtained high-purity bethia in 20mL of methanol, detecting the product by a Waters 2695 liquid chromatograph with a sample loading volume of 50 μ L, integrating the peak area in the detection signal chart based on the uv absorption value at 254nm, and substituting the integrated peak area into the standard curve.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> a method for preparing bethest forest

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cgtgcacagc tgatggagaa acatctttcg caaggcacac atgcaatgct ggccgtccgt 3420

gcgaaagaag aagccattgt cgcagcgatt gacgggcctc caggagaggc atacgagttc 3480

tcttgccgca atggcgaaca gcgaaacgtt ctcggaggca ccgttgctca aatccaggcg 3540

gcgaaagccg cgcttgaggc taagaagatt cgatgccagt acttggacac cccgatggca 3600

ttccacactg gccaagtcga tccgattctg cccgagctct tgcaggtcgc tgcggcatgc 3660

ttcatccagg atccccaaat tcctgtcatc tccccagcat atggcaaagt gatcaggtct 3720

gccaaggact tccgaccaga gtacttcacc catcactgcc gcagttctgt caacatggtc 3780

gatgctctcc aaagtgcagt cgaagaaggc ttacttgaca agaacattat tggcctcgag 3840

attggccctg gcccggtcgt cacgcagttc gtcaaggaag ctgtgggcac aaccatgcag 3900

acctttgcgt ccatcaataa agacaaagac acatggcagc tcatgacgca agcgctagct 3960

aagttctatc ttgcaggtgc cagtgtcgag tggtcgcgct atcacgaaga cttccctgga 4020

gctcagaaag tccttgagct cccggcttat ggctggacct tgaaaaacta ctggctgcaa 4080

tatgtcaacg actggtctct gaggaagggc gatccagccg tggttgttgc cgcgtcaaat 4140

ttggaactct cttcgtcgat acacaaggtc ataaccaaca cgatcaccgc caacagcgac 4200

ggcgagcttg tcgtggacgc ggacctcagt cgtgaggacc tgcatcccat ggttcagggg 4260

catcaggttt acggtgttcc actgtgcaca ccttccgtgt acgcggatat cgctctgaca 4320

ctcggcgagt atattcgaca ggtcatcaag ccaggcgagg ttgcacagac atccgttgaa 4380

gtagcagaga tgaacatcca aagcgcactg gtggctaaca acacgggcag agtgcaactc 4440

cttcgcacgt atgccaagtt tgaccccaag gcccaggtag cgtcatgcac gttctctagt 4500

atcaaggtta gtcgtgaggg tcatcagctc attatggatt ccatgctaat tttgactagg 4560

aggacggcag tggtgtagtc gagcagcatg ctaattgcaa gatccggttc ggcagtctcg 4620

agaaggagaa gaccgcgctc aagagtgctg cactagctgc ccaagccagg atggccgctc 4680

tcaaaacaca agttggccag gatgacaaca cctaccgctt cagcaaaggc atgatttaca 4740

agatgattgg ccaattagct gacttcgatg agaagtatcg cggcctctgc gcgatcacac 4800

tcgacaacga cgccatggaa gcctcgggca aagtatcatt caagggcatt ccaaacgagg 4860

gcaaattcca ctctagcccg gcttatctcg acgcgctgtc gcaacttggc ggattcgtca 4920

tgaacgcgaa cgaggtcagt agcgatggta gacagcacat tgtagattgc gatgctaact 4980

ctttctgttc cttagggtgt ggatcttgag aaagaagtct ttgtcaatca cggctggggt 5040

tccatgcgtt tcttcgccgc tctggatcca gcaatgactt actacactca tgtgaagatg 5100

acccaaggca aagacaaatt gtggactggc gatgtcttga tcttcgacga caagcaagca 5160

ttgatcggca ttgtcggggg agtggcgttg cagggcgtgc ccaagcgact tatgcattac 5220

attgttacag ctgccaacaa gaaagcttcc ggcccgccga cagagaagaa aacttctggt 5280

ccgccagtcg aaaagaaagc cagcgcgcct gttgcgccca caaggccagc aatccagcgt 5340

aagaatgctt cgattcctcc acctgcaacc caagtgactc cgcaaaacaa gaccatcaag 5400

acgccaagtg tgtcggcact gatagccccg gccctcgaga ttgttagcga ggagatcggg 5460

atgccaatcg acgagctcaa ggatgatatc gacttcactg atgctggtct tgacagtctg 5520

ctctccttgg taatcagcag tcgcatgcgg gaccagctgg gcatcgaatt cgaatccgcg 5580

cagttcatgg agattggatc tatcggtgga ctcaaggagt tcttgaccag gctcagtccc 5640

ccagtagcag tcgccgttgc cactgccgtg gaaattgtca aggaggaagc gctcacctca 5700

ttgggagagc ttactgatcc cagccctaac gagatcggca ctgtctggcg cgatgccctc 5760

aagatcctgt ctgaagagag tgggctcact gatgaggagt tgaccgatga cacaagtttc 5820

gccgacgtgg gcgttgatag tctcatgagt cttgtgatca ccagtcgcct acgggacgaa 5880

ttggacatcg acttcccgga ccgagcatta ttcgaagaat gccaaactat atttgacctt 5940

cgcaagaggt tctctgggtc aacggaaagc ttcgactcga cgacgaccaa gcctagcgct 6000

ggtgatgcga cgccacctct gaccgattcc agcgcgccat ctccgccctc ctccgagttc 6060

gatggcgaga cgccgatgac tgatctggac gaggtgttcg attctccccc agcgcaaaag 6120

aggataccat ccccgcccaa aggacgaatc ccgcctgcat ggtcgatgta tctgcaaggc 6180

tcacagaagc ggtcgaagga gattcttttc ttgtttccag acggcgctgg cgccgcaact 6240

tcttacttgt ctttacctcg tttgggtgaa gacattggcg tagtcgcctt caattcgcct 6300

ttcatgaagt aagttgtctc tacagaggtc aaagtgacag gaactaacac atcactcctg 6360

taggacaccg cacaagtttg ctgatcatac cttaccggac gtcatcgcgt cctatgtaga 6420

aggcattcga ggccgtcaag cgcaaggccc atatcatctg ggcggttggt ctgctggtgg 6480

cattctggcc tatgccgttg cccaagaact cattgcagct ggcgaggagg tttccacact 6540

cctcctcatc gactcgcctt cgccaaccaa aggcctggat cgcctaccaa cacgattctt 6600

cgatcactgc acgaacgttg gactctttgg aacagagctc tcaagaggca gtggaggtcc 6660

caacaagacg cccgaatggc tgatgcctca cttcagagct agcatcgaac tgctacacga 6720

ctaccacgct cctcctatga agctcggcaa caagacgaaa gtcatggtca tctgggccgg 6780

cgaatgtgca tttgatggcg tccactatgc tcacataccg ccatctgcag gcgataccga 6840

tgaagacact gaaggcatga agttcctgac agagaagagg aaagatttcg gggccacaga 6900

atgggcaagt ctgttcccgg gcactgatgt tgacgcgaga gttgtggaga gcgagcatca 6960

cttcagcatg atgcgtgatt ctggtgcaca gatgcttgtt gagcacatgc gagattgttt 7020

ggggattgtt ccatca 7036

<210> 2

<211> 1500

<212> DNA

<213> Artificial sequence

<400> 2

cgctaagctt gtatgcagct ggcacagagt agttgacctg tgagaccgag gtcagcatga 60

tctgatttct ctgactcctt cttcaaagga gaagggagtt cactcaccat ccatctagcc 120

caacctaaat agctttcatc gcgagcgaac aaggcggaaa tgcgactgtg gcggacttgg 180

ttggcctcgg gctcttccag gaatccattg catatggcca tgcgacagat gcttctcaat 240

tgcataggcg gcacgctggc ttttctcgca aggtctgtgt agctgatcgt tccgctatgt 300

ggaaggtggt ccaatatgtt gagttccacc agccagcgaa aacaggttaa atgttgatac 360

tgatacagct tagatcaaga caccttggcg cgactaatcg aacttacgcc gttttgtaga 420

tgctccagat actcttgagg tcggatcgac aactgcaaga ttcttgtcgc tgcctctgcc 480

agctgcgcac gagcggcttg agtttgagca tcgcagttgt ctaaaggatc cttcggcaac 540

gacgcgttga ggtcttgaac atccttgcga ctcttgagag atttgagcag ctcctgggcg 600

ttggccgata cggcctccaa ggacgcctca agatcgcgtt ggaactgcat catgtgtgac 660

aaatgtgtgt gattacagta gatgttagca ttgagaccag gacgtttcga tcataagaac 720

cggcgacgga gctcaagtag cagacattcg tagcaggggg acaagttggc tgtgcacagt 780

cgccgcaaac caaattgagc gtgctgtttc cctatgttgt gtttcccggg ccgcttgtac 840

gcaaagtccc cgtcctctgg tgatatgtca cgtgaccgtg cataggacat atgctgttcc 900

atgctcgcgc gcttccatgt ggcgaatgtg ttgattgcct gtcgcacgtg gagactgggc 960

atggcctgtc tcgaggtctc aatcatcgtg tgcaggctaa gtcgccgggc ctcttgcgct 1020

aggagctgtc tcgcatactg acactttggg tatcacggcc ggtcgtatcg acgatcatca 1080

gcttcaatgg tgacaaagca attgctgcca ctcagtgtgc atctggtggc taggaaaagg 1140

aaaacgctgt aacagagtca tcgagcggta gagagcattc actcacagaa cctgagcttg 1200

gcgtcatgag ggcatcgatt ccgctctttc gagaatctct gtcgactcag tgtacgagga 1260

acaaacgccg aatgtgtcga acggctactt tcttaggctt aaaacggcgc gcgtaagcta 1320

tgcatgccat agcgaccggc gattgactcg acctacggtt agcatgggga tatagatgcg 1380

catacactgt cacgagctcg tgcccagctg tatctctgcc atctcatctg cacttccgtt 1440

cttcaacata cctctcttga tcgcttcaac acattgtcac tctttttgac atcccacacc 1500

<210> 3

<211> 1500

<212> DNA

<213> Artificial sequence

<400> 3

tgatagcgct agatttaggt gttggagttc gactgtaatg aaattttgaa tgttcttttg 60

aatgtaactc cggcttagcc acaggcacat ggcttggtct acccgtaatt tctcgaacat 120

cctttccgga gtctctccat caacacacct ttgactccat caccttatgg actcccgagc 180

catttcttcc acacaccatg ctggttctgc atgatagcgt ttcccagaag cataatgaac 240

tctgttcgac attctcgaga ttcaaacgca gtggcgatgg tcggtctcgg caacagcgtt 300

caccgatccc agctcggtga acgtcgctct tacacctcgt agtctccctt cgccgtatcc 360

acgaaacagt actcctttct cggtgattgt ctcgcggtat gcgacgaagc accgtaagcg 420

ggcacaatgg ccgactcggt gtcacgacta gcgctcgcga agccatatca tattcagctc 480

atagcatcat cattcgactt cacaattgca attagcaagt ctctcatcaa tcaaaatgcc 540

taacagaatc gaagcggaca atctctttga gctcacggca gagctggtct cagcctcctc 600

aaaacttcac aagtttctag accagaagaa ccttccgcag ccatcgtttg atgctccagc 660

tccatcggtg gctctcaaca cagccaacaa gccgtattat gatgcgagaa gtgcaattgt 720

agaggctgct gagcaactca ttcgcctggt ccgtggacct cgcgatactc ttctcgctct 780

gtccttcgaa cactgcgcta cggcgtcgtt gcaggtcagt cgggccaaag ttgtttccac 840

tttcatccca actaatacct tttacaggtt atcttcaagt acaaattcgc aaatcatatc 900

ccacttcatg gctccacaac gtatagcaaa attgccgaag cagttggaga tggtgtgaca 960

acagctctcg ttgagcgcac gatacaacat tgcgcttctt ttggcctctt cgagaccatt 1020

cctggaggct atgttactca caatgctact tcatcactac tggtcaccga tcctgatctc 1080

gaagcctgga tgtatctctc ggcggtgata gcctacccag ctggcgcagc tatccctaag 1140

gctgtagagc agtatggcgt ttcccatgaa gcagacgagg caggctatgg tgccagtata 1200

aatcgcaaaa tcgcacaatt ccagcggttc cgcgagcctg atggaaagaa ggatcacgag 1260

atgtttgcga gagcgatgcg tggtattgcg gctggtggtg cgtacgactt ccgccatgca 1320

gtcgatggcg gatacccttg gcacctcctc gcagagggcg caggccacct ggttgtggat 1380

gttggtggag gtcccggcca cgtcgccatg gcgctcgcgg agaagtaccc aagcttgcgt 1440

ttccaagtcc aagatctgcc cgagaccgtc caagtgggag cgaagaattg ccctgagcac 1500

Claims (10)

1. A genetically engineered bacterium is characterized in that the genetically engineered bacterium is obtained by knocking out a polyketide synthase CTB1 gene on a host cell genome by taking cercospora JNU001 with CCTCC NO: M2017842 as a host cell.

2. The genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the poly-coding ketosynthase CTB1 gene is represented by SEQ ID No. 1.

3. A method for preparing beth-ghrelin, which is characterized in that the beth-ghrelin is prepared by fermentation of the genetically engineered bacterium of claim 1 or 2.

4. The method according to claim 3, wherein the genetically engineered bacterium according to claim 1 or 2 is inoculated into a fermentation medium for liquid fermentation culture to prepare a fermentation broth, and the fermentation broth is extracted to prepare the bedigold.

5. The method of claim 4, wherein the liquid fermentation culture is carried out for 8-15 days at 25-30 ℃ and 100-150 rpm.

6. The method of claim 5, wherein the fermentation medium comprises: 15-25 g/L glucose, 1-3 g/L soybean peptone, 0.5-2 g/L sodium acetate, 1-10 mg/L L-phenylalanine, 50-200 mg/L sodium benzoate, 80-200 mg/L potassium dihydrogen phosphate, 0.5-2 mg/L biotin, 3-10 mg/L calcium nitrate, 0.5-2 mg/L pyridoxal phosphate, 0.5-2 mg/L calcium pantothenate, 0.5-2 mg/L thiamine hydrochloride, 2-10 mg/L manganese chloride, 2-10 mg/L ferric chloride, 0.5-2 mg/L copper nitrate, 0.5-5 mg/L magnesium sulfate, and 1-5 mg/L zinc sulfate.

7. The method according to claim 6, wherein the extraction of the fermentation broth is carried out by using a mixed solution of dichloromethane and methanol at a volume ratio of (4: 1) to (10: 1) at a rotation speed of: 100rpm to 300rpm, and the extraction time is 0.5 to 1 hour.

8. The method of claim 7, further comprising a purification step of preliminary purifying the prepared bethilin by thin layer chromatography and preparing the bethilin pure product using semi-preparative liquid chromatography.

9. Use of the genetically engineered bacterium of claim 1 or 2 in the preparation of bedigoline or a product containing bedigoline.

10. The use of claim 9, wherein the product is a pharmaceutical or chemical.

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