Emodin anthraquinone high-yield engineering strain

Abstract

The invention discloses an emodin anthraquinone high-yield engineering strain. According to the invention, mdpG and mdpF genes of aspergillus nidulans are exogenous introduced into a chassis strain aspergillus nidulans LO8030, so that the genetically engineered bacterium for producing emodin is obtained. Furthermore, the g1234 gene is knocked out from the genetically engineered bacterium for producing the emodin, so that the yield of the emodin can be obviously improved. The decarboxylase gene is further introduced in an exogenous manner, so that the yield of the emodin can be further remarkably improved. And on the basis, exogenous introduction of mdpK, mdpC and mdpB genes is carried out, such that the chrysophanol-producing genetically engineered bacterium is obtained. Or the rugG gene from talaromyces sp.YE3016 is introduced in an exogenous manner, so that the genetically engineered bacterium for producing the muscardine hydrochloride is obtained.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to high-yield engineering strains of emodin-type anthraquinones. Background Art

[0002] Anthraquinone is a polycyclic compound containing a quinone structure in the molecule. 9,10-anthraquinone is the most common, including its products and dimers with different reduction degrees. Its main structural types can be divided into alizarin type, emodin type, reduced anthraquinone, anthraquinone dimer, etc. The reported biological activities of anthraquinone compounds include but are not limited to laxative, antibacterial, antimalarial, anti-inflammatory, anti-arthritis, diuretic, anticoagulant, neuroprotective, and anticancer activities, etc. (GRECO G, TURRINI E, et al. Marine Anthraquinones: Pharmacological and Toxicological Issues [J / OL]. Marine Drugs, 2021, 19(5) [2023-06-27]. https: / / www.proquest.com / docview / 2532172097 / abstract / 388A94C5EF944009PQ / 1. DOI: 10.3390 / md19050272.).

[0003] Emodin (chemical name: 1,3,8-trihydroxy-6-methylanthraquinone) is one of the typical representatives of anthraquinone compounds, mainly derived from the rhizomes of plants in the Polygonaceae family. In vitro and in vivo studies have shown that emodin can play roles such as promoting apoptosis, inhibiting epithelial-mesenchymal transition, cell migration, and anti-angiogenesis, and has good therapeutic effects on various cancers such as liver cancer, colon cancer, non-small cell lung cancer, and pancreatic cancer. In addition, emodin can also exert anti-inflammatory, immunomodulatory, antibacterial, antiviral, myocardial protection, and neuroprotective activities through multiple signaling pathways.

[0004] Chrysophanol (chemical name: 1,8-dihydroxy-3-methylanthraquinone) is the main active ingredient of Rheum palmatum, Polygonum multiflorum, and Polygonum cuspidatum. Current studies on the pharmacological effects of chrysophanol show that chrysophanol can further protect the nervous system of rats with ischemia-reperfusion injury by inhibiting the expression of pro-inflammatory factors; play an antioxidant role by regulating superoxide dismutase and glutathione; in addition, it also has the activity of inhibiting the metastasis and migration of oral cancer cells.

[0005] Skyrin is a homotypic symmetric dimer formed by coupling two emodin molecules through a carbon-carbon (C-C) bond, mainly derived from endophytic fungi of plants. Research has shown that skyrin has good antibacterial activity, can induce apoptosis, and can also be used as a natural dye in industries such as industry and food.

[0006] The filamentous fungus Aspergillus nidulans, as a model strain of the Aspergillus genus, is also one of the first model fungi to complete genome sequencing. Compared with chassis strains such as Escherichia coli and Saccharomyces cerevisiae, Aspergillus nidulans has eukaryotic gene intron splicing enzymes, has innate advantages and conveniences for expressing eukaryotic biosynthetic gene clusters, and is particularly suitable for expressing large secondary metabolite biosynthetic genes of eukaryotic origin.

[0007] The chassis strain Aspergillus nidulans LO8030 (CHIANG Y M, OAKLEY C E, AHUJA M, et al. An Efficient System for Heterologous Expression of Secondary Metabolite Genes in Aspergillus nidulans [J / OL]. Journal of the American Chemical Society, 2013, 135(20): 7720 - 7731. DOI: 10.1021 / ja401945a.) was obtained by modifying Aspergillus nidulans and has the following characteristics: 1) Eight major biosynthetic gene clusters were knocked out, with a blank secondary metabolite background, eliminating the influence of the host genetic background on heterologous gene expression; 2) Three auxotrophic markers pyrG-, riboB-, pyroA- and three resistance screening markers (G418, hygromycin, pyrithiamine resistance markers) can be used for genetic manipulation of multiple genes; 3) It has a ΔnkuA mutation, effectively reducing the non-homologous recombination rate; 4) It has gene loci wA and yA encoding pigments, which can be used as homologous recombination sites for foreign genes.

[0008] Emodin and chrysophanol are currently mainly extracted from plants, limited by problems such as the long growth cycle of plants, large environmental impact, and complex separation and purification processes. Skyrin is mainly separated and purified from the metabolites of endophytic fungi of plants, with low yields. Constructing cell factories through synthetic biology strategies for microbial fermentation is a promising method for efficiently and sustainably producing important natural products of plant origin. Summary of the Invention

[0009] In view of the above deficiencies in the prior art, the present invention provides an engineering strain with high yield of emodin-type anthraquinones, which can achieve low-cost, sustainable and efficient production of emodin, chrysophanol and skyrin in Aspergillus nidulans through synthetic biology methods.

[0010] The present invention first provides a genetically engineered bacterium capable of producing emodin. The mdpG and mdpF genes of Aspergillus nidulans are introduced into the chassis strain Aspergillus nidulans LO8030 to overexpress the mdpG and mdpF genes.

[0011] In the present application, for the site of gene insertion, sites commonly used in the prior art can be used, and the site selection only needs to be in a region that will not significantly affect the basic survival and reproduction of the strain. Preferably, both the mdpG and mdpF genes are inserted at the va locus of Aspergillus nidulans.

[0012] More preferably, the mdpG gene is driven by the constitutive promoter gpdAp of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans;

[0013] The mdpF gene is driven by the starch-inducible promoter amyBp of the amylase gene of Aspergillus oryzae.

[0014] In the present application, for the expression after the introduction of foreign genes, promoters commonly used and capable of being used in Aspergillus nidulans can be used. For genes that need to be expressed continuously, general constitutive promoters can be used, and for genes that need to be expressed at specific times, some inducible promoters can be used, and the specific-time expression of genes can be achieved through induction under specific conditions.

[0015] Preferably, the g1234 gene is knocked out.

[0016] Preferably, a decarboxylase gene is introduced into the chassis strain, and the decarboxylase gene is the mdpH gene derived from Aspergillus nidulans or the tpcK gene derived from Aspergillus fumigatus.

[0017] Preferably, the decarboxylase gene is inserted at the g1234 gene of Aspergillus nidulans. Inserting the decarboxylase gene at the g1234 gene disrupts the g1234 gene, synchronously achieving the knockout of the g1234 gene, so there is no need to separately knock out the g1234 gene.

[0018] The present invention also provides a genetically engineered bacterium capable of producing chrysophanol. The mdpK, mdpC and mdpB genes of Aspergillus nidulans are introduced into the genetically engineered bacterium capable of producing emodin into which the decarboxylase gene has been introduced.

[0019] Preferably, the mdpK, mdpC and mdpB genes are all inserted at the wa locus of Aspergillus nidulans.

[0020] The present invention also provides a genetically engineered bacterium for producing emodin. In the genetically engineered bacterium for producing emodin into which a decarboxylase gene has been introduced, the rugG gene derived from Talaromyces sp. YE3016 is introduced.

[0021] The present invention further provides a method for preparing emodin-type anthraquinones. When the emodin-type anthraquinone is emodin, the genetically engineered bacterium for producing chrysophanol is fermented and cultured, and pyridoxol, uracil, and uridine are added to the culture medium during fermentation and culture.

[0022] When the emodin-type anthraquinone is chrysophanol, the genetically engineered bacterium for producing chrysophanol is fermented and cultured, and pyridoxol is added to the culture medium during fermentation and culture.

[0023] When the emodin-type anthraquinone is skyrin, the genetically engineered bacterium for producing skyrin is fermented and cultured, and uracil and uridine are added to the culture medium during fermentation and culture.

[0024] Among them, the culture medium used during fermentation and culture can be a commonly used culture medium for culturing Aspergillus nidulans, such as a starch liquid medium. Among the specifically added supplementary nutrients, the supplementary concentration of pyridoxol can be 1 mg / L, the supplementary concentration of uracil can be 0.56 g / L, and the supplementary concentration of uridine can be 1.26 g / L.

[0025] In the present invention, by exogenously introducing the mdpG and mdpF genes of Aspergillus nidulans into the chassis strain Aspergillus nidulans LO8030 to overexpress the mdpG and mdpF genes, a genetically engineered bacterium for producing emodin is obtained. This genetically engineered bacterium for producing emodin can produce emodin, and the emodin yield is approximately 2.64 ± 0.11 mg / L. Further knocking out the g1234 gene in the genetically engineered bacterium for producing emodin can significantly increase the emodin yield, and the emodin yield is approximately 14.71 ± 2.22 mg / L. Further exogenously introducing a decarboxylase gene can further significantly increase the emodin yield. When the decarboxylase gene is the mdpH gene derived from Aspergillus nidulans, the emodin yield is increased to 77.04 ± 5.08 mg / L; when the decarboxylase gene is the tpcK gene derived from Aspergillus fumigatus, the emodin yield is increased to 74.93 ± 6.40 mg / L.

[0026] In the present invention, after exogenously introducing the mdpG and mdpF genes of Aspergillus nidulans into the chassis strain Aspergillus nidulans LO8030, further knocking out the g1234 gene and exogenously introducing a decarboxylase gene, and then exogenously introducing the mdpK, mdpC, and mdpB genes, a genetically engineered bacterium for producing chrysophanol is obtained. This genetically engineered bacterium for producing chrysophanol can produce chrysophanol, and the chrysophanol yield is approximately 33.79 ± 9.59 mg / L.

[0027] By introducing the mdpG and mdpF genes of Aspergillus nidulans into the chassis strain Aspergillus nidulans LO8030, further knocking out the g1234 gene, introducing a decarboxylase gene, and then introducing the rugG gene from Talaromyces sp. YE3016, a genetically engineered strain capable of producing astramycin is obtained, and this genetically engineered strain capable of producing astramycin can produce astramycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the biosynthesis route of emodin.

[0029] Figure 2 It is the plasmid map of pNN1.

[0030] Figure 3 It is the map of the digestion result of pNN1 plasmid by Not I.

[0031] Figure 4 It is the map of the verification results of the upstream and downstream interfaces and internal sequences of the transformant sNN1.

[0032] Figure 5 It is the colony morphology photos of four sNN1 transformants, where Figure 5 A in it is the front of the colony, Figure 5 B in it is the back of the colony.

[0033] Figure 6 It is the electrophoresis result map of semi-quantitative PCR products.

[0034] Figure 7 It is the HPLC result map of the fermentation metabolites of sNN1.

[0035] Figure 8 It is the mass spectrometry data map of compound 1 and emodin standard in the fermentation products of sNN1.

[0036] Figure 9 It is the standard curve of emodin.

[0037] Figure 10 It is the map of pNN6.

[0038] Figure 11 It is the comparison result map of the emodin yields of sNN4 and sNN1.

[0039] Figure 12 It is the map of pNN5.

[0040] Figure 13 It is the expression cassettes of sNN6 and sNN10.

[0041] Figure 14 It is the map of pNN9.

[0042] Figure 15It is a comparison result graph of emodin yields of sNN6, sNN10, and sNN1.

[0043] Figure 16 It is the biosynthesis route of chrysophanol.

[0044] Figure 17 It is the pNN11 spectrum.

[0045] Figure 18 It is the sNN15 expression cassette.

[0046] Figure 19 It is the phenotypic change of sNN15.

[0047] Figure 20 It is the HPLC detection result of the fermentation product of sNN15.

[0048] Figure 21 It is the standard curve of chrysophanol.

[0049] Figure 22 It is the biosynthesis route of skyrin.

[0050] Figure 23 It is the pNN19 spectrum.

[0051] Figure 24 It is the sNN24 transformant.

[0052] Figure 25 It is the HPLC detection result graph of the fermentation product of sNN24. Among them, Figure 25 a in it is the HPLC result graph, Figure 25 b in it is the ultraviolet characteristic absorption of compound 1, Figure 25 c in it is the ultraviolet characteristic absorption of compound 3.

[0053] Figure 26 It is the LC-MS detection result of the fermentation product of sNN24. Detailed implementation methods

[0054] 1. Buffer solution

[0055] Trace Elements: 2.20 g of ZnSO4·7H2O, 1.10 g of H3BO3, 0.50 g of MnCl2·4H2O, 0.16 g of FeSO4·7H2O, 0.16 g of CoCl2·5H2O, 0.16 g of CuSO4·5H2O, 0.11 g of (NH4)6Mo7O 24 ·4H2O, 5.00 g of Na4EDTA, are added in sequence to 80 mL of ddH2O for dissolution and made up to 100 ml.

[0056] 20× Nitrate Salts: Dissolve 120.0 g of NaNO3, 10.4 g of KCl, 10.4 g of MgSO4·7H2O, and 30.4 g of KH2PO4 in 1 L of ddH2O, and autoclave at 121 °C for 20 min.

[0057] 100× Supplements: Prepare by dissolving 200.0 mg of L-Histidine HCl, 1.0 g of L-Leucine, 200.0 mg of L-Tryptophan in 100 mL of ddH2O.

[0058] 20× Dropout solution: Prepare by dissolving 300.0 mg of L-Isoleucine, 1500.0 mg of L-Valine, 500.0 mg of L-Phenylalanine, 200.0 mg of L-Arginine HCl, 300.0 mg of L-Tyrosine, 300.0 mg of L-Threonine, 300.0 mg of L-Lysine, 200.0 mg of L-Methionine, 600.0 mg of Adenine hemisulfate in 1 L of ddH2O.

[0059] STC Buffer: Dissolve 218.60 g of Sorbitol (1.2 M), 0.47 g of CaCl2 (10 mM), and 10 mM Tris-HCl in ddH2O, adjust the pH to 7.5 with HCl, make up the volume to 1 L, autoclave at 121 °C for 20 min, and store at 4 °C.

[0060] PEG Solution: 60% PEG 6000, 50 mM Tris-HCl. Melt 50 mM CaCl2 (5.55 g) by microwave heating, dissolve in ddH2O, adjust the pH to 7.5 with HCl, make up the volume to 1 L, autoclave at 121 °C for 20 min, and store at 4 °C.

[0061] KCl-Citric Acid Solution: Add 8.2 g of KCl and 2.1 g of Citric Acid monohydrate (MW 210) to 50 ml of ddH2O to dissolve, adjust the pH to 5.8 with 1.1 M KOH solution (requiring a large volume), and make up the volume to 100 mL with ddH2O.

[0062] 2. Culture Medium

[0063] Glucose Minimal Media (GMM Medium): 50 mL of 20×Nitrate Salts, 1 mL of Trace Elements, 10.0 g of Glucose, dissolved in 800 mL of ddH2O, adjust the pH to 6.5, make up the volume to 1 L, add 16.0 g of agar powder, autoclave at 121°C for 20 min.

[0064] Stabilized Minimal Medium (SMM Medium): 50 mL of 20×Nitrate Salts, 1 mL of Trace Elements, 10.0 g of Glucose, 218.6 g of Sorbitol, dissolved in 500 mL of ddH2O, adjust the pH to 6.5, make up the volume to 1 L, add 16.0 g of agar powder, autoclave at 121°C for 20 min.

[0065] Top SMM Medium: Prepare SMM Medium, add 7.5 g of agar powder, make up the volume to 1 L, autoclave at 121°C for 20 min.

[0066] LMM + YE Medium: Based on LMM liquid medium, add 0.5% (w / v) of Yeast Extract, autoclave at 121°C for 20 min.

[0067] Liquid Minimal Media (LMM Medium): 50 mL of 20×Nitrate Salts, 1 mL of Trace Elements, 20.0 g of Glucose, dissolved in 800 mL of ddH2O, adjust the pH to 6.5, make up the volume to 1 L, autoclave at 121°C for 20 min.

[0068] SD Medium: 1.34 g of YNB / Yeast nitrogen base (without amino acids), 10 mL of 20×Dropout solution; 2 mL of 100×Supplements; 10 mL of 40% Glucose, dissolved in 180 mL of ddH2O, make up the volume to 200 mL. If preparing solid medium, add 4.0 g of agar powder, autoclave at 115°C for 30 min (sterilize half of the volume separately).

[0069] LB Medium: 10.0 g of Tryptone, 5.0 g of Yeast Extract, 5.0 g of NaCl, made up to 1 L with ddH2O. If preparing solid medium, add 18.0 g of agar powder, autoclave at 121°C for 20 min.

[0070] LB + Amp Medium: Prepare the stock solution of ampicillin (Amp) with a concentration of 50 mg / mL. Filter it through a sterile membrane to remove bacteria and store it at -20 °C. After sterilizing the LB medium, cool it to about 50 °C and then add 1‰ (v / v) Amp.

[0071] YG Medium: 10 g glucose, 2.5 g yeast extract, 0.5 mL Trace Elements, make up the volume to 500 mL with ddH2O. Autoclave at 121 °C for 20 min.

[0072] Starch Medium: 50 mL 20×Nitrate Salts, 1 mL Trace Elements, 20 g soluble starch. Add nutritional supplements as needed: riboflavin 2.5 mg / L, pyridoxine HCl 1 mg / L, uracil 0.56 g / L, uridine 1.26 g / L. Dissolve in 800 mL ddH2O, adjust the pH to 6.5, make up the volume to 1 L, and autoclave at 121 °C for 20 min.

[0073] Starch - Xylose Medium: 50 mL 20×Nitrate Salts, 1 mL Trace Elements, 10 g soluble starch, 10 g xylose. Add nutritional supplements as needed: riboflavin 2.5 mg / L, pyridoxine HCl 1 mg / L, uracil 0.56 g / L, uridine 1.26 g / L. Dissolve in 800 mL ddH2O, adjust the pH to 6.5, make up the volume to 1 L, and autoclave at 121 °C for 20 min.

[0074] 3. Experimental Methods

[0075] 3.1 Strain Activation and Preservation

[0076] Take 10 μL of the spore solution of the strain preserved in 25% glycerol and spot it on the GMM solid medium (add specific nutritional components as needed). Observe the growth morphology of the strain. If the growth morphology is consistent, it is a single strain; if not, the strain is not single. If the strain is not single, carefully pick the mycelium from the uncontaminated area onto a new GMM solid medium and observe the growth morphology of the strain again until the strain is a single strain.

[0077] After it grows for 3 - 5 days and there are sufficient spores, collect the spores with 0.1% tween 80 solution to obtain the spore solution, and store it in a glycerol tube with a final concentration of 25% at -80 °C.

[0078] 3.2 Fungal Genome Extraction

[0079] 1) Add 100 μL of fresh spore solution to 20 mL of LMM + YE medium and incubate statically at 37 °C for 2 days.

[0080] 2) Use a flat - headed toothpick to pick up a small amount of mycelium and place it in a biological filter cloth. Squeeze out the moisture, put it into a centrifuge tube, and let it stand at -80 °C for 4 h. Freeze - dry the pre - frozen mycelium in a freeze - dryer for 4 h, then take it out and crush the thallus into powder.

[0081] 3) Add 700 μL of LETS buffer to the thallus powder, mix well and let it stand for about 5 min.

[0082] 4) Continue to add 700 μL of DNA extraction solution (phenol: chloroform: isoamyl alcohol = 25:24:1), mix well and then let it stand for about 10 min to precipitate proteins.

[0083] 5) Centrifuge at 12000 rpm and 4 °C for 10 min, discard the precipitate, and transfer the supernatant to a new centrifuge tube.

[0084] 6) Add 1 mL of absolute ethanol to the tube, mix well and then let it stand at -80 °C for 30 min to precipitate DNA.

[0085] 7) Centrifuge at 12000 rpm and 4 °C for 10 min, discard the supernatant. Add 1 mL of 70% (v / v) ethanol to the tube to wash the flocculent DNA, centrifuge at 12000 rpm for 2 min, discard the supernatant, and let it stand for about 5 min to evaporate the ethanol.

[0086] 8) Add 40 μL of 10 mM Tris buffer (pH 8.0) to the tube and incubate in a water bath at 65 °C for 30 min to fully dissolve the flocculent DNA.

[0087] 9) Add Rnase enzyme to degrade RNA, measure the concentration of the DNA solution using Nanodrop, dilute it to about 200 ng / μL, and store it at -20 °C for use in PCR amplification.

[0088] 3.3 Plasmid Construction

[0089] 3.3.1 Design of Plasmid Map

[0090] Use SnapGene software to design the plasmid map to be constructed. The plasmid should contain the origin of replication ori / 2μ required for the Escherichia coli - yeast shuttle plasmid and Amp R / Kan RResistance marker gene, yeast uracil auxotrophic marker gene URA. If the plasmid needs to be expressed in fungi, the fungal origin of replication AMA1 needs to be added.

[0091] 3.3.2 Fragment acquisition

[0092] To obtain multiple gene purification fragments required for plasmid construction, a homologous region greater than 25 bp was designed on the primers for homologous recombination, and the primer length was approximately 59 bp. PCR amplification was performed using these primers, and the PCR reaction system and reaction program are shown in Table 1 and Table 2.

[0093] Table 1 PCR reaction system

[0094]

[0095] Table 2 PCR reaction program

[0096]

[0097]

[0098] After PCR, 1×TAE buffer was prepared using 50×TAE stock solution and placed in the electrophoresis tank. Agarose gels with different concentrations were prepared according to the DNA fragment length, and the solidified agarose gel was placed in the electrophoresis tank so that the buffer covered the top of the gel. The PCR samples were added to the gel wells, the time and voltage were set, and the power supply was started to perform electrophoresis. The electrophoresis time depends on the length of the target DNA band and the target separation effect. After electrophoresis, the gel was placed in a gel imager for observation and photography. The DNA fragments were purified and recovered using the GelExtraction kit.

[0099] 3.3.3 Transformation of Saccharomyces cerevisiae and plasmid extraction

[0100] 1) Take out two tubes of yeast competent cells (1 tube for transformation and 1 tube as a negative control), incubate in a 37°C water bath for 15 - 30 s, centrifuge at 13000 rpm for 2 min, and remove the supernatant. Mix according to the following table, where the total amount of the largest fragment for transformation should be between 200 ng and 300 ng.

[0101] 2) The yeast homologous recombination system is shown in Table 3.

[0102] Table 3 Yeast homologous recombination system

[0103] Component 1× PEG(4000) 260 μL LiAc 1.0 M 36 μL Single-stranded vector DNA (10 mg / mL) 10 μL <![CDATA[DNA + ddH2O]]> 54 μL Total volume 360 μL

[0104] 3) After adding all the components, vortex for 1 minute, incubate at 42 °C for 20 - 30 minutes, and centrifuge at 13,000 rpm for 30 seconds to remove the supernatant. Resuspend with 1 mL of ddH2O and gently pipette with a 200 μL pipette. Take 200 μL of the liquid from each plate and spread it on an SD plate. For the experimental group (yeast + DNA), use 3 - 5 plates, for the negative control (yeast + ddH2O), use 1 plate, and for the positive control (yeast + ddH2O / SD + uracil), use 1 plate. Incubate at 30 °C for 3 - 5 days. When transformants grow in the experimental group, collect the yeast transformants on the medium with 1 mL of ddH2O and transfer and inoculate them into 10 mL of liquid SD medium at 30 °C and culture overnight at 200 rpm. Centrifuge at 5000 rpm for 5 minutes, collect the cells, and extract yeast plasmids using the YeastPlasmid Kit.

[0105] 3.3.4 Escherichia coli transformation and plasmid extraction

[0106] 1) Take out two tubes of Escherichia coli competent cells. Add the plasmid DNA to be transformed (3 μL plasmid + 7 μL ddH2O) to one tube, and add 10 μL of ddH2O to the other tube as a negative control. Place on ice for 60 minutes, then heat shock at 42 °C for 90 seconds, and then place on ice for 90 seconds. Add 780 μL - 1 mL of LB liquid medium and mix well. Culture at 37 °C and 200 rpm for 40 - 50 minutes, take 200 μL and spread it on an LB resistant solid plate, and incubate inverted at 37 °C overnight. Set up the experimental group, negative control group, and positive control group and wait for the transformants to grow.

[0107] 2) After about 16 - 22 hours when the transformants grow, pick the transformants into a 1.5 mL centrifuge tube containing 500 μL of LB resistant liquid medium and culture for 3 - 4 hours. Take 50 μL of the culture solution, heat boil at 99 °C for 10 minutes, and freeze at -20 °C for 20 minutes for cell wall breaking. Use this as a template for interface verification. After successful verification, expand the culture, extract plasmid DNA, and verify whether the plasmid is successfully constructed by enzyme digestion verification or ligation fragment verification. Finally, store the correct Escherichia coli cells in a glycerol tube at -80 °C in the refrigerator.

[0108] 3) Use the AxyPrep Plasmid Miniprep Kit (Axygen) to extract Escherichia coli plasmid DNA. Store the extracted plasmid DNA at -20 °C in the refrigerator.

[0109] 3.4 Expression cassette construction

[0110] The gene expression cassette can be obtained by two methods: enzyme digestion and PCR.

[0111] 3.4.1 Construction by enzyme digestion

[0112] The restriction enzyme digestion system (50 μL) is as follows: 5 μL of Quick cut enzyme solution, 5 μL of 10× Buffer Green, 5 μg of plasmid, and supplemented with ddH2O to 50 μL. Incubate the restriction enzyme digestion system at 37 °C for 30 min. After completion, purify and recover the fragment through the Gel Extraction kit to obtain the expression cassette.

[0113] 3.4.2 Construction by PCR

[0114] After the expression plasmid is constructed, design primers to amplify the upstream homologous region - promoter - expression gene - terminator - selection marker - downstream homologous region fragment by PCR amplification. Purify and recover the fragment through the Gel Extraction kit to obtain the expression cassette.

[0115] 3.5 Preparation and transformation of LO8030 protoplasts

[0116] 1) Inoculate 1x10 8 spores into 20 mL of YG medium (add three nutrients), culture at 30 °C and 135 rpm for 14 h, filter through a sterile filter cloth to collect the mycelium, transfer the mycelium to a 100 mL sterilized conical flask, and resuspend with 8 mL of YG medium (add three nutrients).

[0117] 2) Prepare 2× protoplast solution: Mix 1.28 g of vinoflow into 10 mL of KCl - Citric Acid Solution (prepare fresh and filter through a membrane).

[0118] 3) Add 8 mL of 2× protoplast solution to the mycelium resuspended in YG medium. Incubate at 30 °C and 100 rpm for 4 h. Observe the formation of protoplasts under a microscope.

[0119] 4) Transfer 10 ml of 1.2 M (41.08 g added to 100 mL of ddH2O, filter through a membrane) sucrose solution to a 30 ml sterile Corex tube, and gently add the protoplast mixture. Centrifuge at 1800×g and 4 °C for 10 min (use a horizontal rotor). Collect the protoplasts on top of the sucrose and transfer them to a 15 mL centrifuge tube.

[0120] 5) Add an equal volume of STC Buffer to the centrifuge tube, centrifuge at 5000 rpm for 8 min at 4 °C, and discard the supernatant. Resuspend the protoplasts with STC Buffer to keep the concentration at 10 8 ~10 9 cells / mL or so, specifically depending on the transformation amount (100 μL for each transformation).

[0121] 6) Take the plasmid (total amount not less than 2 μg, 5 - 8 μg, not exceeding 30 μL) or cassette fragment (10 μg), add STC Buffer to make up to 50 μL, and pre - cool on ice for 1 h.

[0122] 7) Take the above solution (50 μL) and add it to 100 μL of protoplasts. Set up a negative control (replace the plasmid or cassette with ddH2O), gently mix, incubate on ice for 50 min, add 1.25 mL of 60% PEG solution, roll and invert gently, mix well gently, and let stand at room temperature for 20 min for full transformation.

[0123] 8) Add STC Buffer to a total volume of 6 mL, and culture the protoplasts using a double - layer plate. The lower layer is SMM medium with a 1.6% agar concentration supplemented with specific nutrients. Add 1 mL of the transformed protoplast mixture, set up 5 replicates and 1 negative control (protoplasts not transformed with gene fragments or plasmids). Then pour 8 mL of SMM medium (agar concentration of 0.8%) containing the same nutrient supplement, and horizontally disperse the upper - layer medium to mix it with the protoplast solution. The GMM plate without sorbitol can be used to test the preparation of protoplasts.

[0124] 9) After the medium solidifies, incubate statically at 37 °C for about 2 - 3 days. After the transformants appear, pick them with a toothpick to the screening plate (GMM + specific nutrient supplement) before sporulation, and incubate statically at 37 °C for 3 - 4 days.

[0125] 3.6 Verification of transformants

[0126] Pick the purified transformant spores into a culture medium containing 5 mL of LMM + YE and specific nutrient supplement, incubate statically overnight at 37 °C. The next day, extract the genomic DNA from the growing bacteria, and screen the correct transformants by PCR to verify the connection interface between the transformed fragment and the genome; if it is a plasmid expression vector, verify whether the plasmid interfaces in the genome are correct.

[0127] 3.7 Gene expression analysis

[0128] 3.7.1 Extraction of fungal RNA

[0129] 1) Use a starch liquid medium supplemented with specific additional nutrients to oscillate and ferment the Aspergillus nidulans transformant sNN1 at 30 °C and 180 rpm. Take an appropriate amount of bacteria at two time points of 3 days and 7 days. Filter the liquid with a sterile cloth and transfer it to a lysis tube containing glass beads. Add 1 mL of Trizol to the tube and mix by inverting up and down. It can be stored frozen at - 80 °C and thawed during extraction.

[0130] 2) Add 200 μL of chloroform into the cracking tube, let it stand for 30 s, shake it up and down for 15 s, let it stand at room temperature for 5 min, and centrifuge at 4 °C and 12,000 rpm for 15 min.

[0131] 3) Take 400 - 500 μL of the supernatant into a new 1.5 mL centrifuge tube, add 500 μL of RNA extraction solution, let it stand for 30 s, shake it up and down for 15 s, let it stand at room temperature for 5 min, and centrifuge at 4 °C and 12,000 rpm for 15 min.

[0132] 4) Transfer the supernatant to a new 1.5 mL centrifuge tube, add 500 μL of isopropanol to precipitate RNA, invert it several times at room temperature, let it stand for 10 min, centrifuge at 4 °C and 12,000 rpm for 10 min, and discard the supernatant.

[0133] 5) Add 1 mL of 75% ethanol prepared with Rnase-free H2O, invert it to mix well, centrifuge at 4 °C and 12,000 rpm for 10 min, discard the supernatant. The RNA precipitate is at the bottom. Invert it to let the ethanol evaporate as much as possible. Add 40 μL of Rnase-free H2O, place it on ice, incubate in a water bath at 65 °C for 5 min, quickly put it back on ice. Take 1.5 μL into a PCR tube to measure the concentration, and store the rest in a -80 °C refrigerator for later use.

[0134] 3.7.1 Reverse Transcription and Semi-Quantitative PCR

[0135] 1) Take no more than 1 μg of RNA into a PCR tube and use the RQ1 RNase-Free DNase (TaKaRa) kit to remove gDNA. The reaction system is as follows: 1 - 8 μL of RNA (<1 μg), 1 μL of 10× buffer, 1 μL of DNase, and make up to 10 μL with Rnase-free H2O.

[0136] 2) Incubate in a water bath at 37 °C for 30 min, then add 1 μL of DNase stop buffer, and incubate in a water bath at 65 °C for 10 min to inactivate DNase. Take it out and place it on ice, and take 1.5 μL into a PCR tube to measure the concentration.

[0137] 3) Take 100 ng and prepare cDNA using the PrimeScript RT reagent Kit reverse transcription kit. At the same time, set up a control group RTC, that is, replace the reverse transcriptase with an equal amount of RNase-Free H2O. The reverse transcription reaction system is as follows (10 μL): 2 μL of 5×PrimeScript Buffer (for real time), 0.5 μL of PrimeScript RT Enzyme mix I, 0.5 μL of Oligo dT Primer (50 μM), 0.5 μL of Random 6mers (100 μM), 100 ng of RNA, and make up to 10 μL with Rnase-free H2O.

[0138] 4) The reverse transcription program is to incubate in a water bath at 37°C for 15 min and then inactivate the reverse transcriptase by incubating in a water bath at 85°C for 5 s.

[0139] 5) After the reaction, use Nanodrop to detect the cDNA concentration, and store the cDNA at -20°C for later use. In semi-quantitative PCR, the template is the cDNA prepared above, and its concentration is controlled at 100 ng / μL. The fragments obtained by reverse transcription are verified by conventional PCR reactions.

[0140] 3.8 Fermentation of transformants and extraction of secondary metabolites

[0141] 1) Inoculate the fresh spore solution of the transformant into the medium supplemented with specific nutritional supplements, culture at 37°C and 180 rpm for two days to produce sufficient mycelial pellets. Take 4 mL of the above seed solution and inoculate it into 200 mL of the fermentation medium, and culture at 30°C and 180 rpm for 7 days.

[0142] 2) When extracting the fermentation products, separate the mycelial liquid and mycelium using a suction filter funnel. Extract the mycelial liquid with an equal volume of ethyl acetate 3 times, and rotary evaporate the upper organic phase at 40°C until dry. After weighing, store it in a refrigerator at 4°C in the dark. Ultrasonically assist in extracting the mycelium with an appropriate amount of methanol for 30 min, filter, rotary evaporate the liquid until dry, repeat 3 times, weigh the crude extract of the mycelium and store it in a refrigerator at 4°C in the dark.

[0143] 3.9 Analysis of fermentation products using HPLC and LC-MS

[0144] 1) Dissolve the crude extracts of the mycelial liquid and mycelium with methanol, dilute to 0.5 mg / mL, take 1 mL of the solution and filter it through a 0.22 μm organic filter membrane into an injection vial for HPLC analysis.

[0145] 2) The HPLC analysis conditions are as follows: The HPLC system model is Agilent LC1260, the analytical chromatographic column is COSMIL C18 analytical column, 250 mm × 4.60 mm, the flow rate is 1 mL / min, the column temperature is 35 °C, the detection wavelength is 254 nm, the injection volume is 20 μL, and gradient elution is carried out with 10%-100% methanol-water (v / v) for 60 min.

[0146] 3) Observe the metabolic profiles of each sample at the ultraviolet absorption wavelength of 254 nm, and preliminarily identify whether there are anthraquinone compounds. Anthraquinone compounds have characteristic absorptions at 250-300 nm and 440-450 nm.

[0147] 4) The concentration of the crude extract for LC-MS analysis is controlled at about 0.1 mg / mL, and it is also filtered through a 0.22 μm organic filter membrane into the injection vial for analysis.

[0148] 5) The LC-MS analysis conditions are as follows: The LC-MS system model is Agilent 6230 TOF / MS, the analytical chromatographic column is Agilent Eclipse Plus C18, gradient elution is carried out with 10%-100% methanol-water (v / v) for 60 min, the flow rate is 0.5 mg / mL, the injection volume is 4 μL, the column temperature is 35 °C, and the detection wavelength is 254 nm.

[0149] 6) Combine the LC-MS total ion current chromatogram TIC and the ultraviolet chromatogram, extract the possible ion peaks, and judge whether the target compound is contained in the crude extract.

[0150] Example 1: Construction of intermediate emodin high-yield strain

[0151] Figure 1 This is the biosynthetic route of emodin. The information of the three genes for emodin biosynthesis is shown in Table 1.

[0152] Table 1 Information of three biosynthetic genes of emodin

[0153]

[0154] (1) Construction of heterologous expression strain of emodin

[0155] First, construct a heterologous expression strain that produces intermediate emodin, integrate mdpG (non-reducing polyketide synthase) and mdpF (thioesterase) into the ya locus of Aspergillus nidulans LO8030 to obtain the transformant sNN1.

[0156] (2) Construction of recombinant plasmid pNN1

[0157] Construct the recombinant plasmid pNN1 through the plasmid construction method in Section 3.3 above (as Figure 2As shown in the figure, the skeletal gene mdpG and the thioesterase gene mdpF expression cassettes were obtained. Plasmid pNN1 consists of the plasmid pYX1 backbone fragment, the upstream and downstream homologous sequence fragments of yA (the sequences are shown in SEQ ID No.1 and SEQ ID No.2 respectively), the riboflavin auxotrophic marker gene AfriboB (XM_747607.1) of Aspergillus nidulans LO8030, and the gpdAp-mdpG-amyBp-mdpF fragment (the gpdAp promoter, the mdpG gene, the amyBp promoter, and the mdpF gene). There is a homologous region of about 40bp between each adjacent fragment (the homologous region is used for assembly and ligation), and the plasmid size is about 18.39kb. The plasmid pYX1 backbone fragment contains the Escherichia coli and yeast replication origin sequences ori and 2μ, the Escherichia coli screening resistance marker gene AmpR, and the yeast uracil auxotrophic marker gene URA3. gpdAp (the sequence is shown in SEQ ID No.3) and amyBp (the sequence is shown in SEQ ID No.4) are the constitutive promoter derived from the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans and the starch-inducible promoter of the amylase gene of Aspergillus oryzae respectively; mdpG and mdpF are the gene open reading frames from the secondary metabolite monodictyphenone biosynthetic gene cluster of Aspergillus nidulans and a fragment of about 200bp at the 3' end.

[0158] (3) Obtaining gene expression cassettes by enzyme digestion

[0159] For the recombinant plasmid pNN1, using the enzyme digestion method in Section 3.4.1, after digesting with Quick cut Not I for 30 minutes, two linear fragments were obtained. As Figure 3 shown, the band sizes are consistent with the design, which are 4830bp (skeletal sequence) and 13564bp (expression cassette) respectively. The expression cassette was obtained by gel extraction and recovery.

[0160] (4) Construction of mdpG and mdpF expression strains sNN1

[0161] The obtained gene expression cassette was transferred into the chassis strain Aspergillus nidulans LO8030 by the method of protoplast transformation in Section 3.5. The transformant sNN1 was screened using the riboB auxotrophic selection marker. The genomic DNA of the transformant was extracted using the method described in Section 3.2. The upstream and downstream interfaces containing the homologous region and the internal sequence of mdpG (about 1500bp each) were verified by PCR, and two correct transformants were obtained. The results are as Figure 4 shown. The growth status of the transformant is shown in Figure 5 . When the ya locus is disrupted, the spore color changes from green to yellow. Combining the auxotrophic marker can greatly improve the screening efficiency.

[0162] (5) Study on the expression of mdpG and mdpF genes

[0163] To confirm whether mdpG and mdpF are transcriptionally expressed in sNN1 and the expression time, the transformant sNN1 was fermented by shaking in a starch liquid culture medium supplemented with corresponding nutrients at 30 °C and 180 rpm. RNA in the bacterial liquid was extracted at two time points, the 3rd day and the 7th day, and reverse transcribed into cDNA. The transcriptional expression of mdpG and mdpF at different time points was detected by semi-quantitative PCR (Semi-qPCR).

[0164] Primers for the internal sequences of the two genes were designed for PCR detection, and the expected product fragment length was about 150b p or so. The internal reference gene for semi-quantitative PCR detection was the histone-coding gene H2B in Aspergillus nidulans LO8030. The PCR template for the reverse transcription RTC control group was RNA without reverse transcription, and there should be no bands to exclude the interference of uncleaned gDNA. The same semi-quantitative PCR detection was performed on the blank control strain nPMe7.2 (riboB gene inserted at the yA locus) to exclude the expression of mdpG and mdpF by the chassis strain itself. The electrophoresis results of the semi-quantitative PCR products are as Figure 6 shown.

[0165] mdpG and mdpF had transcriptional expression on the 3rd day (3d) of culture. The PCR product band of the internal reference gene H2B was single, and there were no bands in the RTC and blank control strains. This indicates that mdpG and mdpF had a certain degree of expression on the third day, suggesting the successful transcriptional expression of mdpG and mdpF.

[0166] (6) Analysis of the metabolites of sNN1

[0167] The transformant sNN1 was fermented by the method in Section 3.8, and the fermentation products of sNN1 were detected by HPLC and LC-MS by the method in Section 3.9. Pyridoxine HCl (1 mg / L), uracil (0.56 g / L), and uridine (1.26 g / L) were supplemented in the fermentation medium. HPLC detection was performed on the fermentation products, and the mobile phase conditions were 75% methanol / TFA. The results are as Figure 7 shown. Compared with the control strain nPme7.2, an obvious absorption peak of compound 1 appeared at 19 min in the metabolic profile of sNN1. Its retention time and ultraviolet characteristic absorption were consistent with those of the emodin standard. Through further LC-MS analysis( Figure 8) The [M-H]- peak m / z of Compound 1 was 269.0472, and the relative molecular mass was approximately 270, corresponding to the [M-H]- peak m / z 269.0465 (M = 270.24) of the emodin standard. Therefore, it was speculated that this compound was emodin.

[0168] In the mass spectrometry results of the fermentation products of sNN1, the ion peaks of atrochrysone carboxylic acid and endocrocin were not extracted, that is, the pre-decarboxylation products were not found. There was literature indicating that the structure of atrochrysone carboxylic acid was unstable and could undergo a certain degree of spontaneous decarboxylation, followed by spontaneous dehydration and oxidation to produce emodin. Therefore, the decarboxylation products were very likely to be spontaneously generated.

[0169] Using 0.25 mg / mL as the initial concentration, a standard curve of emodin was drawn by the serial dilution method. The concentrations and corresponding peak areas are shown in Table 2.

[0170] Table 2 Emodin concentrations and corresponding peak areas

[0171] Concentration (mg / mL) 0.25 0.125 0.0625 0.03125 0.1563 0.00781 0.00391 Peak area (mAU) 18122.6 8703.6 4344.1 2155.8 1081.0 547.6 285.8

[0172] The standard curve is shown in Figure 9 , where Y = 72292*X - 89.05, R 2 = 0.9996. From this, the emodin yield in sNN1 was calculated to be approximately 2.64 ± 0.11 mg / L. The yield was low, and further optimization was needed to improve the yield in the follow-up.

[0173] (7) Construction of high-yield emodin strains

[0174] The important precursor of polyketides, acetyl-CoA, forms malonyl-CoA under the action of acetyl-CoA carboxylase ACC, and the latter further forms various polyketides. Researchers found that SnfA (XP_001823562) in Aspergillus oryzae can phosphorylate ACC, thereby inhibiting the activity of ACC. Knocking out SnfA significantly increased the supply of malonyl-CoA (KAN E, KATSUYAMA Y, MARUYAMA J. Efficient heterologous production of atrochrysonecarboxylic acid-related polyketides in an Aspergillus oryzae host with enhanced malonyl-coenzyme A supply[J / OL]. The Journal of General and Applied Microbiology, 2020, 66(3): 195-199. DOI: 10.2323 / jgam.2019.07.001.).

[0175] Therefore, we hypothesized that there might also be an enzyme with a similar function to SnfA in Aspergillus nidulans LO8030. Using the amino acid sequence of SnfA as a probe for alignment analysis, we located that the protein encoded by the g1234 gene might perform a similar function. The identity and coverage are shown in Table 3. Knocking out the g1234 gene is expected to increase the supply of malonyl-CoA, thereby increasing the production of emodin.

[0176] Table 3 Comparison of the protein encoded by g1234 and SnfA

[0177]

[0178] Although the decarboxylation and oxidation processes of emodin precursors can proceed partially spontaneously, studies have shown that the introduction of decarboxylase can significantly increase the yield of emodin (SUN L, LIU G, LI Y. Metabolic engineering of Saccharomyces cerevisiae for efficient production of endocrocin and emodin[J / OL]. Metabolic Engineering, 2019, 54: 212-221. DOI: 10.1016 / j.ymben.2019.04.008.). Through literature research and bioinformatics analysis, we selected two decarboxylases, MdpH from Aspergillus nidulans and TpcK from Aspergillus fumigatus, and recombined them at the g1234 site (the sequence of the g1234 gene is shown in SEQ ID No. 5) to compare the effects of the two decarboxylases on the yield of emodin. The gene information of the two decarboxylases is shown in Table 4.

[0179] Table 4 Gene information of two decarboxylases

[0180] Gene Length (bp) Source GeneID mdpH 1592 Aspergillus nidulans 2875926 tpcK 429 Aspergillus fumigatus 3509609

[0181] (8) Construction of the g1234 gene knockout strain sNN4

[0182] The knockout plasmid pNN6 was constructed using the plasmid construction method in Section 3.3 ( Figure 10 ). The plasmid pNN6 consists of the backbone fragment of plasmid pNN1, the upstream and downstream homologous sequence fragments of g1234 (the sequences are shown in SEQ ID No. 6 and SEQ ID No. 7 respectively), the auxotrophic marker gene AfpyroA (XM_748734.1) of Aspergillus nidulans LO8030, and the β-six marker excision system fragment. There is a homologous region of about 40 bp between each adjacent fragment, and the plasmid size is about 12.5 kb.

[0183] The knockout cassette was obtained by digestion with SmaI. The specific operation is described in Section 3.4.1. The g1234 knockout cassette was transferred into the sNN1 protoplasts to construct the knockout strain sNN4. The method for preparing protoplasts is described in detail in Section 3.5. Eight correct transformants were obtained, and the transformants with better growth status were selected for subsequent fermentation detection.

[0184] (9) Analysis of the fermentation product yield of sNN4

[0185] The transformant sNN4 was fermented using the method in Section 3.8, and the fermentation products of sNN4 were detected by HPLC using the method in Section 3.9. Uracil (0.56 g / L) and uridine (1.26 g / L) were added to the fermentation medium.

[0186] Using Figure 9 the emodin standard curve in Section Figure 11 3 to quantitatively detect emodin in the fermentation products of sNN4. As shown in

[0187] (10) Construction of decarboxylase-expressing strains sNN6 and sNN10

[0188] The expression plasmid pNN5 ( Figure 12 ) was constructed using the plasmid construction method in Section 3.3. Plasmid pNN5 consists of the backbone fragment of plasmid pNN1, the upstream and downstream homologous sequence fragments of g1234, the auxotrophic marker gene AfpyroA of Aspergillus nidulans LO8030, gpdAp-mdpH, and the β-six marker excision system fragment. There is an approximately 40-bp homologous region between each adjacent fragment, and the plasmid size is approximately 15.5 kb.

[0189] The expression cassette was obtained by digestion with SmaI. The specific operation is described in Section 3.4.1. In addition to the target gene, the expression cassette also contains the β-six marker excision system to achieve the recycling of the selection marker. The expression cassette was integrated into the g1234 locus of sNN1 by the method of protoplast transformation (see Figure 13 ). After obtaining the correct transformant, xylose was used to induce the excision of the marker, and the mdpH gene-expressing strain sNN6 was obtained.

[0190] The expression plasmid pNN9 ( Figure 14 ) was constructed using the plasmid construction method in Section 3.3. Plasmid pNN9 consists of the backbone fragment of plasmid pNN1, the upstream and downstream homologous sequence fragments of g1234, the auxotrophic marker gene AfpyroA of Aspergillus nidulans LO8030, gpdAp-tpcK, and the β-six marker excision system fragment. There is an approximately 40-bp homologous region between each adjacent fragment, and the plasmid size is approximately 14.3 kb.

[0191] The expression cassette was obtained by digestion with SmaI. The specific operation is described in Section 3.4.1. The expression cassette was integrated into the g1234 locus of sNN1 by the method of protoplast transformation (see Figure 13 ). After obtaining the correct transformant, xylose was used to induce the excision of the marker, and the tpcK gene-expressing strain sNN10 was obtained.

[0192] (11) Analysis of the fermentation product yields of sNN6 and sNN10

[0193] The transformants sNN6 and sNN1Q were fermented using the method in Section 3.8, and the fermentation products of sNN6 and sNN1Q were detected by HPLC using the method in Section 3.9. The fermentation medium was supplemented with 1 mg / L pyridoxine HCl, 0.56 g / L uracil, and 1.26 g / L uridine.

[0194] use Figure 9 The standard curve of rheum officinale was used to quantitatively detect rheum officinale in the fermentation products of sNN6 and sNN10, such as Figure 15 As shown, the rhein production of sNN6 was about 77.04±5.08 mg / L, and that of sNN10 was about 74.93±6.40 mg / L. The introduction of decarboxylase significantly increased the production of rhein. The ability of MdpH and TpcK to increase the production of rhein was comparable. Thus, a high-yield strain of intermediate rhein was successfully constructed. Subsequently, sNN6 will be used as the starting strain to express subsequent modification enzyme genes.

[0195] Example 2: Construction of heterologous chrysophanol production strain

[0196] (1) Chrysophanol biosynthesis gene information

[0197] Chrysophano1 is another very important intermediate compound of the emodin family. The hydroxyl group at position 6 of emodin is removed to form chrysophanol. Chrysophanol has antibacterial effects on a variety of bacteria and has biological activities such as promoting intestinal function, promoting nerve excitation and muscle paralysis (MATTOS-SHIPLEY KMJ de, SIMPSON T J. The'emodinfamily'of fungal natural products-amalgamating a century of research with recent genomics-based advances [J / OL]. Natural Product Reports, 2022 [2022-11-16]. https: / / pubs.rsc.org / en / content / articlelanding / 2022 / np / d2np00040g.

[0198] DOI: 10.1039 / D2NP00040G.). The biosynthetic pathway of chrysophanol is as follows Figure 16As shown, after the formation of intermediate emodin, under the action of reductases MdpK, MdpC and dehydratase MdpB, the hydroxyl group at position 6 is removed to form chrysophanol. Therefore, we inserted the three genes mdpK, mdpC, and mdpB (gene information is shown in Table 5) into the wa locus of the high-yield strain sNN6 of intermediate emodin to construct a heterologous production strain of chrysophanol.

[0199] Table 5 Information of three biosynthetic genes of chrysophanol

[0200] Gene Length (bp) Gene function GeneID mdpK 798 Reductase 74896169 mdpC 925 Ketoreductase 2875920 mdpB 692 Catalyze dehydration at positions 6 and 7 to form a double bond 75896174

[0201] (2) Construction of gene expression cassette

[0202] The expression plasmid pNN11 ( Figure 17 ) was constructed using the plasmid construction method in Section 3.3. The plasmid pNN11 consists of the backbone fragment of plasmid pNN1, the upstream and downstream homologous sequence fragments of wa (the sequences are shown as SEQ ID No.11 and SEQ ID No.12 respectively), the auxotrophic marker gene AppyrG (EU817656.1) of Aspergillus nidulans LO8030, and the gpdAp-mdpK-AmyBp-mdpC-gpdAp-mdpB fragment (which are the gpdAp promoter, mdpK gene, amyBp promoter, mdpC gene, gpdAp promoter and mdpB gene respectively). There is a homologous region of about 40 bp between each adjacent fragment, and the plasmid size is about 14.8 kb. The expression cassette was obtained by digestion with NotI. The specific operation is shown in Section 3.4.1.

[0203] (3) Construction of expression strain sNN15

[0204] The expression cassette was integrated into the wa locus of sNN6 by the method of protoplast transformation (see Figure 18 ). Since the wa locus was disrupted, the spore color changed from yellow to white, as Figure 19 shown. A total of 8 correct transformants were obtained.

[0205] (4) Analysis of metabolites of sNN15

[0206] The transformant sNN15 was fermented using the method in Section 3.8, and the fermentation product of sNN15 was detected by HPLC using the method in Section 3.9. Pyridoxine HCl (1 mg / L) was supplemented in the fermentation medium. The fermentation product was detected by HPLC. The mobile phase was 65% methanol / TFA. The results are as Figure 20 shown. Compared with the control strain sNN6, an absorption peak of compound 2 appeared at 45 min. The ultraviolet absorption indicated that the structure was an anthraquinone compound. By comparing with the retention time and ultraviolet characteristic absorption of the standard product, this compound was determined to be chrysophanol.

[0207] Using the double dilution method, an emodin standard curve was plotted with an initial concentration of 0.25 mg / mL. The concentrations and corresponding peak areas are shown in Table 6.

[0208] Table 6 Chrysophanol concentration and corresponding peak area

[0209] Concentration (mg / mL) 0.25 0.125 0.0625 0.03125 0.1563 0.00781 0.00391 Peak area (mAU) 11599.1 6301.2 2402.0 1759.0 896.6 514.9 7.6

[0210] The standard curve is shown in Figure 21 , where Y = 46464*X + 204.5, R 2 = 0.9964. From this, the chrysophanol production in sNN15 was calculated to be approximately 33.79 ± 9.59 mg / L. Thus, the heterologous production strain sNN15 of chrysophanol was successfully constructed.

[0211] Example 3: Construction of a heterologous production strain of skyrin

[0212] (1) Information on skyrin biosynthetic genes

[0213] Skyrin is an anthraquinone dimer compound with antibacterial and antitumor biological activities found in many fungi. This compound is formed by linking two emodin molecules through a C5-C5' bond. Han et al. (HAN Y B, BAI W, DING C X. Intertwined Biosynthesis of Skyrin and Rugulosin A Underlies the Formation of Cage-Structured Bisanthraquinones[J / OL]. Journal of the American Chemical Society, 2021, 143(35): 14218-14226. DOI: 10.1021 / jacs.1c05421.) identified the rug gene cluster in Talaromyces sp. YE3016, in which the rugG gene was identified as a cytochrome P450 monooxygenase responsible for catalyzing the formation of skyrin from two molecules of emodin (as Figure 22 ), and the function of this gene was characterized through gene knockout and heterologous expression in Aspergillus oryzae. Thus, we envisioned expressing rugG (gene information is shown in Table 7) in the intermediate emodin high-yielding strain sNN6 to obtain a heterologous production strain of skyrin.

[0214] Table 7 Information on skyrin biosynthetic genes

[0215] Gene Length (bp) Gene function GenBank rugG (cDNA) 1548 P450 monooxygenase MZ286349.1

[0216] (2) Construction of gene expression vectors

[0217] Construct the expression plasmid pNN19 using the plasmid construction method described in Section 3.3 Figure 23 ), and the plasmid pNN19 consists of the plasmid pNN1 backbone fragment, the Aspergillus nidulans LO8030 auxotrophic marker gene AfpyroA, the xylose-inducible promoter XylP (the sequence is shown in SEQ ID No. 8), the rugG cDNA fragment (synthesized), the Aspergillus autonomous replicon AMA1 (the sequence is shown in SEQ ID No. 9), and the trpC terminator fragment (the sequence is shown in SEQ ID No. 10). There is a homologous region of about 40 bp between each adjacent fragment, and the plasmid size is about 13.8 kb.

[0218] (3) Construction of the expression strain sNN24

[0219] Transfer the plasmid pNN19 into the intermediate emodin-producing strain sNN6 using the method described in Section 3.5 to obtain the rugG-expressing strain sNN24. A total of 8 correct transformants were obtained, as shown in Figure 24 shown.

[0220] (4) Analysis of the metabolites of sNN24

[0221] Ferment the transformant sNN24 using the method described in Section 3.8, and perform HPLC detection on the fermentation products of sNN24 using the method described in Section 3.9. Supplement 0.56 g / L of uracil and 1.26 g / L of uridine in the fermentation medium. Perform HPLC detection on the fermentation products. The mobile phase conditions are 10%-100% methanol / TFA. The results are as shown in Figure 25 shown. Compared with the control strain sNN6, an absorption peak of compound 3 appeared at about 55 min in sNN24, and the ultraviolet absorption indicated that the structure was an anthraquinone compound.

[0222] Perform LC-MS detection on the fermentation products, as shown in Figure 26 shown, indicating that the molecular ion peak of compound 3 is [M+H] + = 539, [M+Na] + = 561, and the molecular weight of the compound is 538, which is consistent with the molecular weight of skyrin. It is basically determined that the compound is skyrin.

[0223] Table 8 Compound 3 1 1H-NMR results

[0224] Position δH (mult, J, Hz) Literature value 2，2′ 7.04(s) 7.09(s) 4，4′ 7.31(s) 7.31(s) 7，7′ 6.68(s) 6.78(s) 11.11′ 2.35(s) 2.37(s)

[0225] Table 9 Compound 3 13 13C-NMR results

[0226] Position δC Literature value C-1，1′ 162.03 162 C-2,2′ 121.5 121.4 C-3,3′ 149.17 149.1 C-4,4′ 123.13 123.1 C-4a, 4a′ 134.25 134.2 C-5.5′ 125.09 125.1 C-6,6′ 164.75 164.7 C-7,7′ 107.33 107.3 C-8,8′ 164.35 164.1 C-8a, 8a′ 109.5 109.1 C-9,9′ 190.33 190.1 C-9a, 9a′ 112.9 113.1 C-10,10′ 186.1 186.1 C-10a, 10a′ 131.6 131.5 CH3-11, 11′ 21.76 21.7

[0227] Approximately 20 mg of Compound 3 was obtained by preparative HPLC separation and purification, and its structure was further confirmed by 1H-NMR and 13C-NMR. The results are shown in Table 8 and Table 9. It was confirmed that the structure of this compound is skyrin. Thus, the heterologous production strain sNN24 of skyrin was successfully constructed.

Claims

1. A genetically engineered bacterium producing emodin, characterized in that, The mdpG and mdpF genes of Aspergillus nidulans were introduced into the chassis strain Aspergillus nidulans LO8030 to overexpress the mdpG and mdpF genes.

2. The genetically engineered bacterium for producing emodin according to claim 1, characterized in that, Both the mdpG and mdpF genes were inserted at the va locus of Aspergillus nidulans.

3. The genetically engineered bacterium for producing emodin according to claim 2, characterized in that, The mdpG gene was driven by the constitutive promoter gpdAp of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene; The mdpF gene was driven by the starch-inducible promoter amyBp of the Aspergillus oryzae amylase gene.

4. The genetically engineered bacterium for producing emodin according to claim 1, characterized in that, The g1234 gene was knocked out.

5. The genetically engineered bacterium for producing emodin according to claim 1, wherein A decarboxylase gene was introduced into the chassis strain, and the decarboxylase gene was the mdpH gene from Aspergillus nidulans or the tpcK gene from Aspergillus fumigatus.

6. The genetically engineered bacterium for producing emodin according to claim 5, wherein The decarboxylase gene was inserted at the g1234 gene of Aspergillus nidulans.

7. A genetically engineered bacterium producing chrysophanol, characterized in that, The mdpK, mdpC, and mdpB genes of Aspergillus nidulans were introduced into the genetically engineered bacterium for producing emodin described in claim 5 or 6.

8. The genetically engineered bacterium for producing chrysophanol according to claim 7, characterized in that, The mdpK, mdpC, and mdpB genes were all inserted at the wa locus of Aspergillus nidulans.

9. A genetically engineered bacterium producing Tianjing, characterized in that, The rugG gene from Talaromyces sp. YE3016 was introduced into the genetically engineered bacterium for producing emodin described in claim 5 or 6.

10. A preparation method of emodin anthraquinone, characterized in that, When the emodin-type anthraquinone is emodin, the genetically engineered bacterium for producing chrysophanol described in any one of claims 1 to 6 was fermented and cultured, and pyridoxol, uracil, and uridine were added to the culture medium during fermentation and culture; When the emodin-type anthraquinone is chrysophanol, the genetically engineered bacterium for producing chrysophanol described in claim 7 or 8 was fermented and cultured, and pyridoxol was added to the culture medium during fermentation and culture; When the emodin-type anthraquinone is skyrin, the genetically engineered bacterium for producing skyrin described in claim 9 was fermented and cultured, and uracil and uridine were added to the culture medium during fermentation and culture.

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