Preparation method of anthraquinone medicine

Abstract

The invention discloses a preparation method of anthraquinone drugs, which comprises the following steps: by taking CCTCC NO: M 2017842 cercospora JNU001 as an original strain, knocking out a polyketide synthase CTB1 gene on the genome of the original strain, then carrying out single-point mutation on a BTG5 gene to obtain a cercospora JNU001 delta CTB1 / BTG5-T318C mutant strain, and fermenting the mutant strain to obtain six anthraquinone drugs. According to the present invention, the aureospora sp. Strain is modified through the gene editing strategy to produce the anthraquinone drug, the prepared anthraquinone drug has characteristics of various production types, simple separation and low cost, and the application of the anthraquinone drug is substantially expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a method for preparing anthraquinone drugs. Background Art

[0002] Anthraquinone natural products, a class of natural products with an anthraquinone parent ring structure, exhibit anti-inflammatory, antithrombotic, laxative, and antimicrobial activities. With the deepening of research into the pharmacological activities of anthraquinone compounds, their clinical applications are becoming increasingly widespread. Anthraquinone compounds are particularly effective in treating cardiovascular and cerebrovascular diseases such as myocardial infarction and cerebral infarction, where they are often used in combination with anti-inflammatory and antithrombotic agents. The mild, long-lasting laxative effect of anthraquinone compounds, combined with minimal side effects, points the way for the future development of effective diuretics.

[0003] Since the main sources of anthraquinone natural products are extracted from traditional Chinese medicines such as rhubarb, knotweed, polygonum multiflorum, cassia seed, aloe, and madder, in recent years, there have been increasing reports on the pharmacological activities of anthraquinone compounds, and clinical research has also received increasing attention. However, due to the limited source of botanical Chinese medicines, there are shortcomings such as slow growth cycles and unstable raw material sources. In addition, traditional extraction processes are complex and use large amounts of organic solvents, all of which restrict the application of anthraquinone compounds. In addition, in traditional Chinese medicines, only a few high-content anthraquinones, such as emodin, chrysophanol, and aloe-emodin, have been found in recent years. Anthraquinone compounds with lower content, such as hydroxyemodin, hydroxyhelminthes, and helminthes, are expensive and have scarce sources.

[0004] Although there are a small number of fungi in nature that can produce anthraquinone drugs, most of them can only produce common anthraquinone drugs such as emodin and chrysophanol. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defect that the types of anthraquinone drugs produced by existing strains are limited.

[0006] In order to achieve the above-mentioned object, the present invention provides a genetically engineered bacterium, which uses Cercospora JNU001 with CCTCCNO: M 2017842 as a starting strain, knocks out the polyketide synthase CTB1 gene on the genome of the starting strain, and then performs a single-point mutation on the BTG5 gene to obtain the Cercospora JNU001ΔCTB1 / BTG5-T318C mutant strain.

[0007] Optionally, the nucleotide sequence encoding the polyketide synthase CTB1 gene is shown as SEQ ID NO.1, and the nucleotide sequence encoding the BTG5 gene is shown as SEQ ID NO.2.

[0008] The present invention also provides a method for preparing anthraquinone drugs, wherein the anthraquinone drugs are prepared by fermentation using the genetically engineered bacteria as described in any one of the above.

[0009] Optionally, the genetically engineered bacteria described in any one of the above items are inoculated into a fermentation medium for liquid fermentation to prepare a fermentation broth, and the fermentation broth is extracted to prepare the anthraquinone drug.

[0010] Optionally, the liquid fermentation culture has a culture time of 6 to 10 days, a culture temperature of 22° C. to 28° C., and a rotation speed of 110 rpm to 140 rpm.

[0011] Optionally, the fermentation medium comprises: 16-24 g / L glucose, 1.5-2.5 g / L soy peptone, 0.8-1.2 g / L sodium acetate, 2-8 mg / L L-phenylalanine, 80-120 mg / L sodium benzoate, 100-180 L potassium dihydrogen phosphate, 0.8-1.2 mg / L biotin, 4-8 mg / L calcium nitrate, 0.8-1.2 mg / L pyridoxal phosphate, 0.8-1.2 mg / L calcium pantothenate, 0.8-1.2 mg / L thiamine hydrochloride, 3-8 mg / L manganese chloride, 3-8 mg / L ferric chloride, 0.8-1.2 mg / L copper nitrate, 1-3 mg / L magnesium sulfate, and 1-3 mg / L zinc sulfate.

[0012] Optionally, the fermentation broth is extracted using ethyl acetate at a rotation speed of 200 rpm to 300 rpm and an extraction time of 0.5 h to 1 h.

[0013] Optionally, the method further comprises a purification step, wherein the purification step is to prepare the anthraquinone drug using semi-preparative liquid chromatography.

[0014] Optionally, the preparative column of the semi-preparative liquid chromatography is an XDB C18 column with a size of 4.6 mm × 250 mm, the mobile phase is acetonitrile and water, and 0.1% formic acid is added to each, and the elution conditions are 40% to 70% acetonitrile gradient elution, the time is 30 min, and the flow rate is 4 mL / min.

[0015] The present invention also provides a use of any of the above-mentioned genetically engineered bacteria in the preparation of anthraquinone drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least:

[0017] (1) The present invention uses Cercospora JNU001 with CCTCC NO: M 2017842 as the starting strain, and knocks out the key gene CTB1 for cercosporin synthesis through gene knockout technology to obtain a Cercospora JNU001ΔCTB1 mutant strain, thereby eliminating the influence of background products; further, a single-point mutation is performed on the gene BTG5 to obtain a mutant strain, thereby obtaining a Cercospora JNU001ΔCTB1 / BTG5-T318C mutant strain, and the Cercospora JNU001ΔCTB1 / BTG5-T318C mutant strain is fermented to obtain 6 anthraquinone drugs with almost no other impurities.

[0018] (2) The anthraquinone drugs obtained by the preparation method of the present invention have high purity, low cost, and simple preparation method, which greatly expands the application of anthraquinone drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the frontal form of the Cercospora JNU001ΔCTB1 / BTG5-T318C of the present invention.

[0020] Figure 2 This is the reverse form of the Cercospora JNU001ΔCTB1 / BTG5-T318C of the present invention.

[0021] Figure 3 This is the liquid phase analysis result of the metabolic profile of Cercospora JNU001ΔCTB1 / BTG5-T318C of the present invention.

[0022] Figure 4 The emodin prepared by the present invention 1 H spectrum.

[0023] Figure 5 The emodin prepared by the present invention 13 C spectrum.

[0024] Figure 6 The chrysophanol prepared by the present invention 1 H spectrum.

[0025] Figure 7 The chrysophanol prepared by the present invention 13 C spectrum.

[0026] Figure 8 The helminthes prepared by the present invention 1 H spectrum.

[0027] Figure 9 The helminthes prepared by the present invention 13 C spectrum.

[0028] Figure 10 The hydroxy-emodin prepared by the present invention1 H spectrum.

[0029] Figure 11 The hydroxy-emodin prepared by the present invention 13 C spectrum.

[0030] Figure 12 The hydroxychrysophanol prepared by the present invention 1 H spectrum.

[0031] Figure 13 The hydroxychrysophanol prepared by the present invention 13 C spectrum.

[0032] Figure 14 The hydroxyl helminthesporin prepared by the present invention 1 H spectrum.

[0033] Figure 15 The hydroxyl helminthesporin prepared by the present invention 13 C spectrum.

[0034] Figure 16 Schematic diagram of the structures of six anthraquinone drugs prepared in the present invention. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0036] There are many strains of anthraquinone-producing bacteria in nature, but they all suffer from the drawback of producing a limited variety and low yields. Most of these strains can only produce common anthraquinone drugs such as emodin and chrysophanol. The applicant team previously studied the biosynthetic pathways of wild-type Cercospora metabolites and discovered that Cercospora has the potential to synthesize a variety of anthraquinone drugs. Therefore, they genetically modified it to produce anthraquinone drugs, prepared crude anthraquinone drugs through microbial fermentation, and purified the crude products using semi-preparative liquid chromatography based on the chemical polarity of various anthraquinone drugs. This enabled the preparation of a series of anthraquinone drugs with lower yields in traditional Chinese medicines, including emodin and chrysophanol, such as helminthes, hydroxy-emodin, aloe-emodin, and hydroxy-emodin. This also enabled the large-scale production of emodin and chrysophanol.

[0037] The starting strain of this application, Cercospora JNU001, is deposited in the China Center for Type Culture Collection with the accession number CCTCC NO: M 2017842.

[0038] The gene CTB1, a member of the cercosporin biosynthesis gene cluster that mediates polyketide synthase, was knocked out to block the cercosporin synthesis pathway, which is unrelated to the synthesis of anthraquinone drugs, thereby altering the metabolic flux within Cercospora and enhancing the biosynthesis of anthraquinone compounds. The BTG5 gene was then mutated again to generate a mutant strain, designated Cercospora JNU001ΔCTB1 / BTG5-T318C. BTG5 is a gene that mediates P450 expression and catalyzes the dimerization reaction between anthraquinone and xanthone. Monomeric xanthone is formed through anthraquinone catalysis. Therefore, modifying key sites in BTG5 inhibits the downstream catalytic metabolic flux of anthraquinone, enabling the efficient accumulation of anthraquinone compounds and achieving the goal of targeted anthraquinone synthesis.

[0039] The mutalytic enzyme involved in the present invention was purchased from Guangdong Institute of Microbiology. The plasmid pGD-GFP involved in the present invention was disclosed in the paper “Discovery of the Biosynthetic Pathway of Beticolin 1 Reveals a Novel Non-hemeIron-dependent Oxygenase for Anthraquinone Ring Cleavage”.

[0040] The culture medium used in the present invention is as follows:

[0041] Resistance screening medium: 10 g / L glucose, 200 g potato, 15-20 g agar, 50 mg / L hygromycin B, 50 mg / LG418.

[0042] S-7 solid medium: 15-20 g / L agar, 20 g / L glucose, 2 g / L soy peptone, 1 g / L sodium acetate, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, and 2.5 mg / L zinc sulfate.

[0043] S-7 liquid fermentation medium: 20 g / L glucose, 2 g / L soy peptone, 1 g / L sodium acetate, 5 mg / L L-phenylalanine, 100 mg / L sodium benzoate, 136 mg / L potassium dihydrogen phosphate, 1 mg / L biotin, 6.5 mg / L calcium nitrate, 1 mg / L pyridoxal phosphate, 1 mg / L calcium pantothenate, 1 mg / L thiamine hydrochloride, 5 mg / L manganese chloride, 2 mg / L ferric chloride, 1 mg / L copper nitrate, 3.6 mg / L magnesium sulfate, 2.5 mg / L zinc sulfate.

[0044] The present invention provides a method for culturing Cercospora JNU001: The Cercospora JNU001ΔCTB1 mutant is inoculated onto an S-7 plate, grown at 25°C for 5-10 days, sealed with parafilm, and stored in a refrigerator at 4°C. 1 L of S-7 liquid culture medium is added to a 2-L shake flask, sterilized at 115°C for 15 minutes, cooled, and then used for later use. The plate is then punched with a hole punch to obtain 5 mm x 5 mm stipes. Three stipes are then placed in 1 L of S-7 liquid culture medium and cultured in a shaker at 25°C, 135 rpm, and continuous light for 8-10 days.

[0045] The buffer used in the present invention is as follows:

[0046] Infiltration buffer: 1.42 g sodium dihydrogen phosphate and 144 g magnesium sulfate were dissolved in deionized water and diluted to 1000 mL double-distilled water; 0.276 g sodium dihydrogen phosphate and 29 g magnesium sulfate were dissolved in deionized water and diluted to 200 mL double-distilled water. The two prepared solutions were mixed and adjusted to pH 5.8.

[0047] STC buffer: 218.6 g sorbitol, 0.47 g calcium chloride, dilute to volume with 1000 mL double-distilled water, filter sterilize, and store in a refrigerator at 4°C.

[0048] PEG6000 buffer: 250 g PEG6000, 11.098 g calcium chloride, 44.73 g potassium chloride, dilute to volume with 1000 mL double-distilled water, filter sterilize, and store in a refrigerator at 4°C.

[0049] Example 1. Construction of the Cercospora JNU001ΔCTB1 mutant

[0050] The specific steps are as follows:

[0051] (1) Protoplast preparation:

[0052] Cercospora JNU001 was inoculated onto S-7 solid medium and cultured in an incubator at 25°C for 5-6 days. Mycelia was then cultured in 100 mL of S-7 liquid medium for 5 days. The mycelia was removed and ground in a sterile mortar and pestle. The mycelia was added to 100 mL of S-7 liquid medium and cultured for another 18 hours. An appropriate amount of mycelia was collected and centrifuged at 6000g for 10 minutes. The mycelia was washed twice with infiltration buffer and the supernatant discarded. 15 mL of infiltration buffer containing 0.5 g of lytic enzyme was added, and the suspension was incubated at 30°C and 120 rpm for 2-3 hours to prepare a suspension. The suspension was collected by filtration through 6 layers of gauze, gently added an equal volume of STC buffer, and centrifuged at 5000g for 10 minutes. The supernatant was discarded. Two volumes of STC buffer were added, and the suspension was centrifuged at 6000g for 5-8 minutes. The supernatant was discarded, and the suspension was resuspended in an appropriate amount of STC buffer and aliquoted.

[0053] (2) Construction of gene knockout plasmid:

[0054] The upstream sequence (nucleotide sequence shown in SEQ ID NO.3) and downstream sequence (nucleotide sequence shown in SEQ ID NO.4) outside the reading frame of the CTB1 gene were found from the complete genome of Cercospora JNU001. 1500 bp were selected for cloning, and the hygromycin resistance gene was inserted into the upstream and downstream sequences. The vector fragment of plasmid pGD-GFP was ligated to construct a complete plasmid.

[0055] (3) Cloning of exogenous fragments: including upstream sequences, hygromycin B resistance gene and downstream sequences. The cloned fragment sequence is about 20 μg and is purified for later use.

[0056] (4) Transformation of exogenous fragments: Add 200 μL of STC buffer to 200 μL of protoplasts to dissolve the exogenous fragments prepared in step (3), mix gently, and incubate on ice for 1 hour. Add 1.25 mL of PEG6000 buffer, swirl gently to mix, and incubate at room temperature for 30 minutes. Add 5 mL of PDB medium containing 1.2 mol / L sorbitol and incubate for 12 hours. Spread on a plate containing antibiotics.

[0057] (5) Transformant screening: After the transformants grow, the genome is extracted, PCR verification is performed, and correct transformants are screened to prepare Cercospora JNU001ΔCTB1 with the CTB1 gene knocked out.

[0058] Example 2: Construction of the Cercospora JNU001ΔCTB1 / BTG5-T318C mutant

[0059] The specific steps are as follows:

[0060] (1) Protoplast preparation:

[0061] Cercospora JNU001ΔCTB1 was inoculated on S-7 solid culture medium, and the preparation method was the same as in Example 1.

[0062] (2) Construction of gene knockout plasmid:

[0063] The BTG5 gene reading frame was found in the whole genome of Cercospora JNU001. The 1500bp sequence upstream of the start codon of the reading frame (the nucleotide sequence is shown in SEQ ID NO.5) and the 2000bp sequence downstream of the start codon (the nucleotide sequence is shown in SEQ ID NO.6) were selected. The T318 site was mutated to obtain a T318C mutation site. The geneticin resistance gene was then inserted into the upstream and downstream sequences, and the vector fragment of the plasmid pGD-GFP was connected to construct a complete plasmid.

[0064] (3) Cloning of exogenous fragments:

[0065] Same as Example 1

[0066] (4) Transformation of exogenous fragments:

[0067] Same as Example 1

[0068] (5) Transformant screening:

[0069] The same method as in Example 1 was used to finally obtain the Cercospora JNU001ΔCTB1 / BTG5-T318C mutant strain.

[0070] Example 3: Fermentation preparation and liquid phase analysis of anthraquinone drugs

[0071] The specific steps are as follows:

[0072] (1) The Cercospora JNU001ΔCTB1 / BTG5-T318C prepared in Example 2 was inoculated onto S-7 solid culture medium, grown at 25°C for 5-10 days, sealed with a parafilm, and stored in a refrigerator at 4°C.

[0073] (2) 1 L of S-7 liquid culture medium was prepared in a 2 L shake flask, sterilized at 115 ° C for 15 min, and cooled for use; the plate cultured with Cercospora JNU001ΔCTB1 / BTG5-T318C obtained in step (1) was punched with a hole punch to obtain 5 mm × 5 mm stipes, 3 pieces of which were put into the prepared 1 L of S-7 liquid culture medium, and placed in a shaker at 25 ° C, 135 rpm, continuous light and reconstituted, and cultured for 8-10 days to prepare a fermentation liquid containing mycelium.

[0074] (3) The mycelium and the fermentation broth in the fermentation broth containing mycelium collected in step (2) were separated using a Buchner funnel. After separation, 50% by volume of ethyl acetate was added to the harvested fermentation broth. The mixture was stirred and extracted with a magnetic stirrer for 1 hour, and the extraction was repeated three times. The organic phase was separated and collected using a separatory funnel. The ethyl acetate solvent was rotary evaporated at 37° C. to prepare a solid crude anthraquinone compound.

[0075] (4) 1-5 mg of the compound obtained in step (3) was dissolved in methanol. The purity of the crude solid product was tested using a Waters 2695 liquid chromatograph, using mobile phases A: acetonitrile (containing 0.1% formic acid) and B: water (containing 0.1% formic acid). The liquid phase conditions were gradient elution, mobile phase A 5%-85%, elution time 50 min, and detection wavelength 200 nm-600 nm. Figure 3 As shown, in the UV detection signal diagram, different components were marked using liquid chromatography software.

[0076] Example 4: Separation and purification of anthraquinone drugs

[0077] The specific steps are as follows:

[0078] The crude solid product prepared in step (3) of Example 3 was dissolved in methanol to a concentration of 5 mg / mL after dissolution. It was filtered through a 0.22 μm PTFE filter and set aside. A Waters 2775 liquid chromatograph was used for preparation, with a diode array detector at wavelengths of 254 nm and 280 nm. The semi-preparative chromatographic column used was an Agilent Eclipse XDB-C18 with a size of 9.4 mm × 250 mm. The mobile phases used were acetonitrile and water, with 0.1% formic acid added to each. The semi-preparative chromatographic column was installed on a Waters 2775 liquid phase and equilibrated with 40% acetonitrile at a flow rate of 4 mL / min. After equilibration, the sample was loaded, with a sample volume of 600 μL each time. The liquid phase conditions were gradient elution, with a gradient elution of 40% to 70% acetonitrile for 30 minutes. Different fractions were collected after the peak appeared. The collected samples were mixed, and the acetonitrile and water were rotary evaporated at 37°C to obtain 6 different anthraquinone compounds.

[0079] Example 5: Characterization and analysis of 6 anthraquinone drugs

[0080] The specific steps are as follows:

[0081] Take 1-5 mg of each of the different anthraquinone drugs prepared in Example 4, dissolve them in 600 μL of deuterated solvent, and perform nuclear magnetic resonance characterization, including 1 H spectrum, 13 C spectrum (such as Figure 4-15 As shown). 1 H NMR (600 MHz, DMSO-d6) δH 11.96 (s, 1H), 11.87 (s, 1H), 7.31 (s, 1H), 7.01 (s, 1H), 6.98 (s, 1H), 6.49 (s, 1H), 2.34 (s, 3H) ppm, emodin 13 CNMR (151MHz, DMSO-d6) δ189.94, 181.46, 165.98, 164.86, 161.81, 148.56, 135.31, 133.01, 124.42, 120.79, 113.57, 109.19, 108.26, 21.94ppm; 1 HNMR (600MHz, Chloroform-d) δH 12.09 (s, 1H), 11.97 (s, 1H), 7.79 (dd, J = 7.4, 1.0 Hz, 1H), 7.68–7.55 (m, 2H), 7.27 (d, J = 9.0 Hz, 1H), 7.07 (s, 1H), 2.45 (s, 3H) ppm, chrysophanol13 CNMR13C NMR (151MHz, Chloroform-d)δ187.75,177.16,157.96,157.66,144.59,132.18,121.88,121.58,119.79,119.59,116.59,115.16,111.11,108.97,17.50ppm; 1 HNMR (600MHz, Chloroform-d) δH 12.93 (s, 1H), 12.24 (s, 1H), 12.06 (s, 1H), 7.62 (s, 1H), 7.22 (s, 1H), 7.19 (s, 1H), 7.04 (s, 1H), 2.41 (s, 3H) ppm, helminthine 13 C NMR (151MHz, Chloroform-d) δ189.59, 185.57, 161.83, 157.24, 156.58, 148.10, 132.20, 128.60, 128.50, 123.53, 119.80, 112.95, 111.77, 111.49, 21.27ppm. 1 H NMR and 13 CNMR is consistent with the public document "Discovery of the Biosynthetic Pathway of Beticolin 1 Reveals a Novel Non-heme Iron-dependent Oxygenase for Anthraquinone Ring Cleavage".

[0082] Hydroxy-emodin 1 H NMR (600 MHz, DMSO-d6) δ 12.10 (d, J = 19.4 Hz, 2H), 11.40 (s, 1H), 7.67 (s, 1H), 7.27 (s, 1H), 7.16 (d, J = 2.3 Hz, 1H), 6.63 (d, J = 2.3 Hz, 1H), 5.57 (t, J = 5.8 Hz, 1H), 4.62 (d, J = 5.6 Hz, 2H) ppm, hydroxy-emodin 13 C NMR (151 MHz, DMSO-d6) δ 192.1, 182.0, 166.1, 165.0, 161.9, 153.4, 135.7, 133.5, 121.3, 117.6, 114.6, 109.6, 109.3, 108.5, 62.5, 40.4 ppm. 1 H NMR spectrum and rhamnolide1 Comparison of H NMR spectra shows that there are two hydrogen signals of CH2 at 4.6ppm and a hydroxyl signal at 5.5ppm, which proves that the structure is hydroxy-emodin. 1 H NMR (600 MHz, DMSO-d6) δ 11.95 (s, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.75–7.71 (m, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 5.60 (t, J = 5.8 Hz, 1H), 4.64 (d, J = 5.7 Hz, 2H) ppm, aloe-emodin 13 C NMR (151 MHz, DMSO-d6) δ 192.2, 182.0, 162.1, 161.8, 154.2, 137.8, 133.9, 133.6, 124.9, 121.2, 119.8, 117.6, 116.4, 115.0, 62.5 ppm. 1 H NMR spectrum and chrysophanol 1 Comparison of H NMR spectra shows that there are two hydrogen signals of CH2 at 4.6ppm and a hydroxyl signal at 5.6ppm, which proves that the structure is aloe-emodin. 1 H NMR (600 MHz, DMSO-d6) δ 12.12 (br, 3H), 7.77 (s, 1H), 7.42 (s, 2H), 7.31 (s, 1H), 5.62 (s, 1H), 4.64 (s, 2H) ppm, hydroxyl helminthine 13 CNMR (151MHz, DMSO-d6) δ 190.1, 186.6, 162.3, 157.7, 157.3, 154.0, 133.5, 130.5, 129.9, 121.4, 117.2, 115.1, 113.2, 113.0, 62.5ppm. 1 HNMR spectrum and helminthine 1 Comparison of the HNMR spectra showed that there were two hydrogen signals of CH2 at the 4.6 ppm position and a hydroxyl signal at the 5.6 ppm position, thus proving that the structure was hydroxyl helminthesporin.

[0083] In summary, the present invention uses Cercospora JNU001 with CCTCC NO: M 2017842 as a starting strain, knocks out the polyketide synthase CTB1 gene on the genome of the starting strain, and then performs a single-point mutation on the BTG5 gene to obtain the Cercospora JNU001ΔCTB1 / BTG5-T318C mutant strain. After fermentation, the mutant strain produces six anthraquinone drugs, namely, emodin, chrysophanol, helminthesine, hydroxy-emodin, aloe-emodin, and hydroxy-helminthesine. The Cercospora strain is transformed by a gene editing strategy to produce anthraquinone drugs. The prepared anthraquinone drugs have a wide variety of production and are simple to separate, which greatly expands the application of anthraquinone drugs.

[0084] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria uses Cercospora JNU001 with CCTCC NO: M2017842 as a starting strain, knocks out the polyketide synthase CTB1 gene on the starting strain genome, and then performs a single-point mutation on the BTG5 gene to obtain the Cercospora JNU001ΔCTB1 / BTG5-T318C mutant strain.

2. The genetically engineered bacterium according to claim 1, wherein The nucleotide sequence encoding the polyketide synthase CTB1 gene is shown in SEQ ID NO.1, and the nucleotide sequence encoding the BTG5 gene is shown in SEQ ID NO.

2.

3. A method for preparing anthraquinone drugs, characterized in that: The anthraquinone drug is prepared by fermentation using the genetically engineered bacteria according to any one of claims 1 or 2.

4. The preparation method according to claim 3, wherein The genetically engineered bacteria according to any one of claims 1 or 2 are inoculated into a fermentation medium for liquid fermentation to prepare a fermentation broth, and the fermentation broth is extracted to prepare the anthraquinone drug.

5. The preparation method according to claim 4, wherein The liquid fermentation culture has a culture time of 6 to 10 days, a culture temperature of 22° C. to 28° C., and a rotation speed of 110 rpm to 140 rpm.

6. The preparation method according to claim 4, wherein The fermentation medium comprises: 16-24 g / L glucose, 1.5-2.5 g / L soy peptone, 0.8-1.2 g / L sodium acetate, 2-8 mg / L L-phenylalanine, 80-120 mg / L sodium benzoate, 100-180 L potassium dihydrogen phosphate, 0.8-1.2 mg / L biotin, 4-8 mg / L calcium nitrate, 0.8-1.2 mg / L pyridoxal phosphate, 0.8-1.2 mg / L calcium pantothenate, 0.8-1.2 mg / L thiamine hydrochloride, 3-8 mg / L manganese chloride, 3-8 mg / L ferric chloride, 0.8-1.2 mg / L copper nitrate, 1-3 mg / L magnesium sulfate, and 1-3 mg / L zinc sulfate.

7. The preparation method according to claim 4, wherein The fermentation liquid is extracted using ethyl acetate at a rotation speed of 200 rpm to 300 rpm and an extraction time of 0.5 h to 1 h.

8. The preparation method according to claim 7, wherein The method further comprises a purification step, wherein the purification step is to prepare the anthraquinone drug using a semi-preparative liquid chromatography method.

9. The preparation method according to claim 8, wherein The preparative column of the semi-preparative liquid chromatography method is an XDBC18 column with a size of 4.6 mm × 250 mm. The mobile phases are acetonitrile and water, each with 0.1% formic acid added. The elution conditions are a gradient elution of 40% to 70% acetonitrile, a time of 30 min, and a flow rate of 4 mL / min.

10. Use of the genetically engineered bacteria according to any one of claims 1 or 2 in the preparation of anthraquinone drugs.

Images