CN113388527B - A strain of high-yield physcion Aspergillus kawachii (Aspergillus chevalieri) BYST1 - Google Patents
A strain of high-yield physcion Aspergillus kawachii (Aspergillus chevalieri) BYST1 Download PDFInfo
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- CN113388527B CN113388527B CN202110649166.6A CN202110649166A CN113388527B CN 113388527 B CN113388527 B CN 113388527B CN 202110649166 A CN202110649166 A CN 202110649166A CN 113388527 B CN113388527 B CN 113388527B
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- WLXGUTUUWXVZNM-UHFFFAOYSA-N anthraglycoside A Natural products C1=C(C)C=C2C(=O)C3=CC(OC)=CC(O)=C3C(=O)C2=C1OC1OC(CO)C(O)C(O)C1O WLXGUTUUWXVZNM-UHFFFAOYSA-N 0.000 title claims abstract description 38
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- 241001065417 Aspergillus chevalieri Species 0.000 title claims description 15
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- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 claims description 6
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
- A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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Abstract
The invention discloses a physcion high-yield strain, and simultaneously provides an acquisition mode of the strain and a physcion yield analysis method, the physcion high-yield strain provided by the invention is Aspergillus chevalieriBYST1, the preservation unit is China center for type culture Collection, the preservation number is CCTCC2021285, and the preservation date is 26 months at 3 months in 2021. The invention provides an obtaining mode of the strain and the yield analysis of physcion, which mainly comprises the following steps: (1) separating and purifying the strain; (2) fermenting, separating and identifying the main component physcion; (3) liquid phase quantifying the yield of physcion; (4) nine different culture mediums are used for shake flask fermentation to evaluate the yield difference of the physcion. The strain is fermented in a liquid PDB culture medium, the yield of physcion is 28mg/L, and the current report shows the highest yield.
Description
Technical Field
The invention belongs to the technical field of biology, and provides an physcion high-yield strain.
Background
Physcion, also known as cinnabar B; a non-ketonic group; 1, 8-dihydroxy-3-methoxy-6-methylanthraquinone is golden yellow needle crystal. The physcion can inhibit the germination and growth of fungi and can induce crops to generate anti-stress defense reaction; the bactericidal composition has excellent control effect on powdery mildew of most crops, and also has better control effect on downy mildew, gray mold, anthracnose and the like; the physcion has extremely low toxicity to human and livestock, is environment-friendly, and is particularly suitable for biological preparations produced by green and organic vegetables.
At present, physcion is autonomously developed into a novel plant source bactericide by China, has huge effect on disease control and good market economic benefit, but the method deviates from the green economic concept, has large demand on plants, more residues and generally lower yield, and the main raw material glucose of the microbial fermentation method belongs to renewable resources, so the cost is lower and the product yield is higher.
Disclosure of Invention
The invention provides a physcion high-yield strain named Aspergillus chevalieri BYST1 aiming at the technical defects, and the preservation unit is as follows: china Center for Type Culture Collection (CCTCC); and (4) storage address: wuhan university in Wuhan, China; the preservation date is as follows: 26 months 3 in 2021; the preservation number is as follows: CCTCC M2021285. The strain is symbiotic bacteria of odontotermes formosanus, the yield of the physcion produced by shaking flask fermentation is 28mg/L, and the strain has important significance for the production of pesticide after the day.
Insect symbiotic bacteria are a special class of microorganisms and are a source of many new active natural products. The invention relates to a method for preparing physcion from Aspergillus chevalieri BYST1 strain separated from odontotermes formosanus, which comprises the following steps:
(1) preparation of BYST1 strain fermentation liquor: the strain BYST1 is inoculated in an ME (raw malt 20g, cane sugar 20g, peptone 1g, 1000mL water) liquid culture medium, and the seed liquid obtained after 3d of shaking culture is inoculated in other culture media for mass fermentation for 7d to obtain fermentation liquid under the conditions of 28 ℃ and 180 r/min.
(2) Separation and purification of metabolites: concentrating the organic layer of fermentation liquor of Aspergillus chevalieri BYST1 strain after ethyl acetate extraction under reduced pressure to obtain crude extract. And performing gradient elution by using dichloromethane and methanol at the volume ratio of 100:0, 100:1, 100:2, 100:4 and 100:8 as a mobile phase, and combining the analysis results of the same components to obtain four components Fr1-Fr 4. Fr1 was repacked on a silica gel column and graded with a petroleum ether/ethyl acetate system, petroleum ether: ethyl acetate eluted at 5:1(v/v) to give Compound A1(320 mg).
(3) Identification of metabolites: using mass spectrometry (ESI-MS) and nuclear magnetic resonance (ESI-MS)1H-NMR、13C-NMR) to identify the structure of the compound A1, and determining that the compound is physcion, wherein the structure is as follows:
after the fermentation medium is further screened, the yield of the physcion can reach 28mg/L, which is the highest reported at present.
In conclusion, the invention obtains the high-yield physcion strain Aspergillus chevalieri BYST1, and the yield of physcion reaches 28mg/L by using a shake flask experiment, which is the highest reported at present. In view of the excellent effect of preventing and treating powdery mildew and other plant pathogenic bacteria, the physcion has good application value, and especially has the potential of developing into high-yield physcion microorganism chassis cells.
Description of the drawings:
FIG. 1 is a chemical structural formula of physcion which is a main product of fermentation of Aspergillus chevalieri BYST1 provided by an embodiment of the invention.
FIG. 2 shows the colony morphology of the strain Aspergillus chevalieri BYST1 provided in the examples of the present invention.
FIG. 3 is a HPLC check chart of fermentation products of strains provided by examples of the present invention in liquid PDB medium.
FIG. 4 is a graph of the standard curve of physcion established by HPLC as provided in the examples of the present invention.
FIG. 5 is a bar graph of the production of physcion by the strains provided by the examples of the present invention in different liquid media.
Detailed Description
The invention is further explained below with reference to specific embodiments.
Example 1 isolation, purification and characterization of symbiotic species of odontotermes formosanus masst 1(Aspergillus chevalieri BYST 1):
the black-wing odontotermes formosanus adopted from Lanxi Zhejiang, from newly excavated termite nest, taking out soldier ants (20) with sterile forceps, placing in a sterile centrifuge tube, adding 1mL of sterile PBS buffer solution with pH of 7.4, and shaking to obtain rinsing liquid; diluting with sterile water to 10-1、10-2、10-3Three concentration gradients. 0.2mL of each concentration gradient diluent is respectively taken and coated on a PDA solid culture medium, and inverted culture is carried out in a thermostat at 28 ℃. After the colony grows out, picking a small amount of spores from the edge of the colony, transferring the spores to a new PDA culture medium, repeating the transfer to obtain a single colony, and storing the single colony BYST1 to a high-gradient test tube for later use.
Extracting the genome DNA of the sample by using a fungus genome DNA kit, and performing PCR amplification by using a fungus universal primer ITS1 sequence. And the PCR product was sequenced by Shanghai Bioengineering technology services, Inc. The obtained sequence was analyzed by NCBI data nucleotide blast tool alignment to identify the strain as Aspergillus chevalieri.
The PDA culture medium comprises the following components: peeling and chopping 200g of potatoes, boiling to obtain potato juice, adding 20g of glucose, adding 15-20 g of agar, adding water to 1000mL, standing at 121 ℃, and sterilizing for 20 min. Transferring the fungus Aspergillus chevalieri to a PDA slant test tube for standby. The fungus BYST1 is preserved by the following culture collection unit: china Center for Type Culture Collection (CCTCC); and (4) storage address: wuhan university in Wuhan, China; the preservation date is as follows: 26 months 3 in 2021; the preservation number is as follows: CCTCC M2021285.
Example 2 liquid fermentation and activation of the symbiotic fungus Aspergillus chevalieri BYST1 of odontotermes formosanus: the strain Aspergillus chevalieri BYST1 prepared according to the method of example 1 is inoculated into malt liquid culture medium (20.0 g/L of raw malt, 20.0g/L of cane sugar, 1.0g/L of peptone and 1000mL) after being cultured for four days, seed liquid is prepared after being cultured for three days at constant temperature and constant speed (28 ℃ and 180rpm) of a shaking table, the seed liquid is inoculated into 9 different culture media, the culture media are cultured for 7 days at 28 ℃ and 180r/min of shaking table, the collected fermentation liquid is extracted by ethyl acetate in equal volume for three times, and then the organic layer is concentrated under reduced pressure to obtain 10g of crude extract.
Example 3 isolation and purification of metabolite of the symbiotic fungus Aspergillus chevalieri BYST1 of odontotermes formosanus: the crude extract prepared as described in example 2 was loaded dry, packed wet into a column, subjected to gradient elution with dichloromethane/methanol in volume ratios of (100:0, 100:1, 100:2, 100:4, 100:8) in order to obtain four different fractions Fr1-Fr4, and Fr1 was reloaded into a silica gel column, sequentially loaded with petroleum ether: gradient elution of ethyl acetate system, petroleum ether: ethyl acetate 5:1(v/v) gave a yellow powder, which was designated A1. And finally, carrying out structure identification on the compound by combining a plurality of spectrum technologies, and finally determining that A1 is physcion, wherein the spectrum data are as follows:
compound a1 (physcion): orange needle crystals in chloroform, ESI-MS M/z 285.0760[ M + H ]]+The molecular formula is presumed to be C16H12O5。1H-NMR(CDCl3):δH 2.45(3H,s,3-CH3),3.94(3H,s,6-OCH3),6.68 (1H,d,J＝2.5Hz,H-7),7.07(1H,d,J＝1.0Hz,H-2),7.35(1H,d,J＝2.6Hz,H-5),7.61(1H,d, J＝1.0Hz,H-4),12.10(1H,s,-OH),12.30(1H,s,-OH)；13C-NMR(CDCl3)δC165.42(C-8), 108.40(C-5),162.73(C-1),107.00(C-7),121.48(C-4),148.64(C-3),124.70(C-2),166.77(C-6), 191.00(C-9),182.18(C-10),135.49(C-4a),110.49(C-8a),113.91(C-9a),133.44(C-10a),22.36 (-CH3),56.29(-OCH3)。
Example 4: yield optimization of physcion
The seed liquid obtained by the culture is subjected to high performance liquid chromatography to respectively examine the fermentation capacity of 9 liquid culture media (potato culture medium, malt culture medium, SDB, oat culture medium, Chashi culture medium, Gauss No. 1 culture medium, Takashio culture medium, YPB culture medium and seed culture medium).
(1) The formulation of the nine media was as follows:
PDB: potato, 200.0 g/L; glucose, 20.0 g/L; 1000mL of water.
ME culture medium, raw malt, 20.0 g/L; sucrose, 20.0 g/L; peptone, 1.0 g/L; 1000mL of water.
SDB medium is peptone, 10.0 g/L; glucose, 40.0 g/L; 1000mL of water.
Oat culture medium (OA) of pure oat, 30.0 g/L; 1000mL of water.
Chase Medium (CZB): NaNO3,3.0g/L；KH2PO4,1.0g/L；MgSO4·7H2O,0.5g/L；KCL, 0.5g/L；FeSO4·7H2O,0.01 g/L; glucose, 30.0 g/L; 1000mL of water.
Gao 1 medium (GA 1): soluble starch, 20.0 g/L; NaCl,0.5 g/L; KNO3,1.0g/L； K2HPO4·3H2O,0.5g/L；MgSO4·7H 20,0.5g/L；FeSO4·7H2O,0.01 g/L; 1000mL of water.
Takashio medium: KH (Perkin Elmer)2PO4,1.0g/L；MgSO4·7H2O,1.0 g/L; glucose, 20 g/L; tryptone, 1.0 g/L; 1000mL of water.
YPB medium: yeast extract, 1.0 g/L; KH (Perkin Elmer)2PO4,6.0g/L；NaH2PO4,4.0g/L；NH4OH,1.0 g/L; 1000mL of water.
Seed medium (ZZB): glucose, 60.0 g/L; peptone, 20.0 g/L; KH (Perkin Elmer)2PO4,2.0g/L； MgSO4·7H2O,1.0 g/L; 1000mL of water.
(2) Preparation of an physcion standard solution: the physcion was accurately weighed and sequentially prepared as a gradient standard solution of 250/8,250/5,250/4,250/3,250/2,250. mu.g/mL.
(3) Chromatographic conditions are as follows: thermo Scientific U3000; column ZORBAX Eclipse Plus C18 Rapid Resolution HD 2.1 x 100mm 1.8-micro; UV 254; eluting with a mobile phase of methanol and 1% acetic acid water solution at a volume ratio of 85:15 for 20 min; the flow rate was 0.2 mL/min.
(4) Drawing a standard curve: drawing a standard curve shown in figure 4 according to the relationship between the peak area and the concentration of the standard substance(y＝0.3322x，R2＝0.9999)。
(5) Treatment of the test samples: inoculating the activated BYST-1 thalli into a seed culture medium, preparing a seed solution, inoculating the seed solution into the 9 culture media in an inoculation amount of 1:10, inoculating 3 bottles of each culture medium, and then placing the culture medium at 28 ℃; shaking the shaking table at constant temperature, fermenting for 7 days, extracting 9 kinds of fermentation broth with different culture media with ethyl acetate for 4 times, mixing ethyl acetate layers, concentrating, evaporating to dryness to obtain crude extract, dissolving with methanol completely, and recording the volume of the solution. And calculating the content of the physcion in the fermentation liquid of different culture mediums by using a standard curve.
The results show that: in the fermentation liquid of nine kinds of test liquid culture mediums, except that physcion can not be detected in the fermentation liquid of oat culture medium, physcion can be detected in other 8 kinds of culture mediums (as shown in figure 5), wherein the yield of PDB, ME and SDB is above 15mg/L, and the yield in the fermentation liquid of PDB is the highest and reaches 28.0 mg/L.
Finally, it is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications (especially the construction of the high-yield physcion-producing microbial underpan cells from the bacterium) which can be directly derived or suggested by one of ordinary skill in the art from the present disclosure are to be considered as the protection scope of the present invention.
Claims (2)
1. Symbiotic fungi Aspergillus awamori (Aspergillus chevalieri) BYST1, the strain is preserved in China Center for Type Culture Collection (CCTCC) at 26 months 3 and 2021, and the preservation number of the strain is CCTCC M2021285.
2. Use of the aspergillus kawachii (a) as claimed in claim 1Aspergillus chevalieri) The method for producing physcion by BYST1 is characterized by comprising the following steps:
1) the strain Aspergillus kawachii cultured for four days is addedAspergillus chevalieri) BYST1 is inoculated in malt liquid culture medium, the shaking table is kept at constant temperature and constant speed of 28 ℃ and at constant speed of 180rpm, and the culture is carried out for three days to obtain the productSeed liquid;
2) inoculating the seed liquid into a PDB and ME liquid culture medium, culturing at 28 ℃ for 180r/min in a shaking table for 7d to obtain a fermentation liquid;
3) filtering the fermentation liquor obtained in the step 2), extracting the filtrate with ethyl acetate, and performing vacuum concentration and drying to obtain a crude extract;
4) loading the crude extract obtained in the step 3) by a dry method, filling the crude extract into a column by a wet method, and performing gradient elution by adopting dichloromethane/methanol, wherein the volume ratio of the two is 100:0, 100:1, 100:2, 100:4 and 100:8 in sequence to obtain four different components Fr1-Fr 4;
5) the 3 rd eluate fraction Fr1 obtained was repacked on a silica gel column and successively purified by passing through petroleum ether: gradient elution of ethyl acetate system, petroleum ether: eluting with ethyl acetate 5:1(v/v) to obtain a brown powder, and identifying the structure of the brown powder by mass spectrum and nuclear magnetic resonance to determine that the compound is physcion, and the structural formula is as follows:
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