Aspergillus alliaceus strain with high yield of chloroxine and application thereof

Abstract

The application provides a chloroguanidin-producing strain, and the strain is Aspergillus candidus ZWAC-1, the preservation number of which is CGMCC No.40506, and the yield of chloroguanidin produced by fermentation of the strain in a PDB culture medium can reach 500 mg / L, thereby providing a basis for production of chloroguanidin and other flavonoid compounds.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a high-chloroxylenin-producing Aspergillus bleachingensis strain and its application. Background Technology

[0002] Flavonoids are a large class of natural products mainly found in plants, possessing diverse structural types and broad biological activities. They play important physiological roles in plant growth, regulation, and defense. The biosynthetic pathways of flavonoids in plants are well understood. First, L-phenylalanine undergoes action by phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-hydroxycinnamoyl-CoA ligase (4CL) to form p-coumaroyl-CoA. Then, under the action of chalcone synthase (CHS, III PKS), it condenses with three molecules of malonyl-CoA to form chalcone. Chalcone isomerase (CHI) catalyzes the formation of configuration-specific flavanones from chalcone. Finally, under the action of flavonoid synthase (FNS), a flavonoid skeleton is formed. Based on this skeleton, modifications such as hydroxylation, methylation, and glycosylation are performed to form flavonoids with diverse structural types and biological activities. Currently, there are many functional foods and drugs related to flavonoids on the market, such as anthocyanins and quercetin with anti-inflammatory and anti-aging effects, soy isoflavones with phytoestrogenic functions, and silymarin with hepatoprotective effects.

[0003] Chlorflavonin, also known as chlorflavonine (CAS: 23363-64-6), is a flavonoid compound containing chlorine atoms produced by fungi. In this invention, the applicant, through the isolation of fungal resources, discovered a fungus capable of producing high levels of chlorflavonin, identified as *Aspergillus candidus*. The discovery of this strain provides a foundation for the production of chlorflavonin and other flavonoid compounds. Summary of the Invention

[0004] On the one hand, the present invention provides a chloramphenicol-producing strain, namely Aspergillus candidus ZWAC-1, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 21, 2023, with accession number CGMCC No. 40506.

[0005] On the other hand, the present invention also provides the application of the above-mentioned strain in the preparation of chlorfluazurin.

[0006] On the other hand, the present invention also provides a method for preparing chlorfluazurin, the method comprising the step of fermenting the above-mentioned strain.

[0007] Furthermore, the method also includes the step of separating chloramphenicol from the fermentation broth.

[0008] Furthermore, the method also includes the step of centrifuging the fermentation broth to obtain the supernatant.

[0009] In one embodiment, the fermentation temperature is 20°C-35°C, preferably 25°C-30°C, and more preferably 28°C.

[0010] In one embodiment, the fermentation time is 2-20 days, preferably 5-15 days, more preferably 8-14 days.

[0011] In one embodiment, the fermentation is carried out at 150 rpm to 300 rpm, preferably 180 rpm to 250 rpm.

[0012] In one embodiment, the fermentation medium is selected from PDB, BPY, or PPM media.

[0013] In this invention, the PDB, BPY, or PPM culture medium can be a self-prepared culture medium or a commercially available culture medium.

[0014] In one embodiment, the PDB medium (potato culture medium) is a commercially available culture medium.

[0015] In one embodiment, the BPY culture medium comprises peptone, beef extract, NaCl, yeast extract, and glucose.

[0016] In one embodiment, the PPM culture medium comprises glucose, sucrose, yeast extract, peptone, and NaCl. Attached Figure Description

[0017] Figure 1 HPLC analysis of secondary metabolites of Aspergillus bleachingus ZWAC-1 and structure of chlorfluazurin.

[0018] Figure 2 HRESIMS and NMR spectra of chlorfluazine; among which Figure 2 (A) High-resolution mass spectrometry of chlorfluazine (m / z 377.0434 [MH)) - ,calcd.for C 18 H 14 ClO7, 377.0434); Figure 2 (B) Chloramphenicol 1 H NMR spectrum (DMSO-d6, 600MHz); Figure 2 (C) Chloramphenicol 13C10 NMR spectrum (DMSO-d6, 150MHz).

[0019] Figure 3 The yield curves of chloramphenicol from Aspergillus bleachingii ZWAC-1 in different culture media: a) chloramphenicol yield curve in PDB medium; b) chloramphenicol yield curve in BPY medium; c) chloramphenicol yield curve in PPM medium. Detailed Implementation

[0020] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0021] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions or conditions for filamentous fungi, or as recommended by the manufacturer. Percentages and parts are by weight. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] Example 1: Isolation and Identification of Aspergillus bleachingii Strains

[0023] Prepare the culture medium according to the following formula:

[0024] PDA plates: 39 g / L potato broth PDA powder (BD Company product, catalog number: 633840), with the remainder being deionized water, autoclaved at 121°C for 20 minutes, and then cooled to approximately 60°C to prepare plates.

[0025] 1.1 Samples used for strain resource isolation were collected from marine sediment samples in Aoshan Bay, Qingdao. First, the surface of the marine sediment was washed with sterile water to remove attached algae and seaweed. Then, it was sprayed with 75% alcohol for surface disinfection for 1 minute and placed in an ultra-clean workbench for 20 minutes under external light.

[0026] 1.2 The marine mud sample was thoroughly ground in a sterile mortar. The resulting homogenate was then diluted 10 times, 100 times, and 1000 times with sterile water, and spread onto PDA plates containing streptomycin (25 mg / mL) and penicillin (25 mg / mL).

[0027] 1.3 After plating, the plates were placed in an incubator at 28°C and incubated for 1-3 weeks until colonies were clearly visible. Different morphological colonies were transferred to new antibiotic-containing PDA plates, and single strains were obtained through multiple passages.

[0028] 1.4 A small amount of isolated and purified mycelium was used to extract the genome using a nucleic acid extractor. The genome was used as a template for PCR with ITS universal primers. The obtained ITS sequence was sequenced and submitted to NCBI for sequence alignment. The strain numbered ZWAC-1 was identified as Aspergillus candidus by ITS.

[0029] The strain *Aspergillus candidus* ZWAC-1 was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with the telephone number 010-64807355; accession number: CGMCCNo.40506.

[0030] Example 2: Determination of chloramphenicol content in Aspergillus bleachingii ZWAC-1

[0031] Prepare the culture medium according to the following formula:

[0032] SM medium: 10% glucose, 1% sucrose, 0.5% yeast extract, 0.5% peptone, 0.1% Na2CO3, with the remainder being deionized water. Autoclave at 121°C for 20 minutes, and inoculate and culture after cooling to approximately 30°C.

[0033] PDB medium: 24 g / L potato broth (PDB dry powder, BD product, catalog number: 7114771), with the remainder being deionized water. Autoclave at 121°C for 20 minutes, then cool to approximately 30°C for inoculation.

[0034] BPY medium: 10 g / L peptone, 5 g / L beef extract, 5 g / L NaCl, 5 g / L yeast extract, 5 g / L glucose, with the remainder being deionized water. Autoclave at 121°C for 20 minutes, then cool to approximately 30°C for inoculation.

[0035] PPM medium: 15% glucose, 2.5% sucrose, 0.5% yeast extract, 0.5% peptone, 0.1% NaCl, with the remainder being deionized water. Autoclave at 121°C for 20 minutes, and inoculate after cooling to approximately 30°C.

[0036] 2.1 The Aspergillus bleachingii ZWAC-1 (CGMCC No.40506) strain was placed on a PDA plate for static culture. The spores were washed with sterile water and inoculated into SM medium. The seed culture was cultured at 28℃ and 220 rpm. The seed culture was then inoculated into PDB, BPY and PPM mediums and fermented at 28℃ and 220 rpm for 14 days.

[0037] 2.2 Take 1 mL of fermentation broth and add it to methanol solution. Shake for 30 min, centrifuge, and filter the supernatant through a 0.22 μm filter. Take 15 μL for HPLC analysis. Analytical method: Mobile phase A (95% water + 5% acetonitrile + 0.05% formic acid), mobile phase B (100% acetonitrile + 0.05% formic acid), gradient elution: 0-0.58 min 100%-80% A, 0.58-4.05 min 80%-40% A, 4.05-5.79 min 40% A, 5.79-6.37 min 40%-0% A, and 6.95-7.53 min 100% A, flow rate 0.6 mL / min, detection wavelength 345 nm. Analytical column: Eclipse Plus C18 RRHD (50 mm × 2.1 mm, 1.8 μm). The retention time of chlorfluazurin is approximately 3.9 minutes (e.g., ...). Figure 1 (as shown); the structure of chlorfluazine was determined by HRESIMS and NMR (e.g., as shown). Figure 2 (As shown).

[0038] 2.3 Analysis of chlorfluazurin yield at different fermentation times revealed that the yield reached 500 mg / L in PDB medium, 200 mg / L in BPY medium, and 300 mg / L in PPM medium (e.g., ...). Figure 3 (As shown). The highest reported yield of chloramphenicol in Aspergillus bleachingus is 25 mg / L (Appl Microbiol. 1970, 19, 718-720), while the chloramphenicol yield in Aspergillus bleachingus ZWAC-1 is higher than the highest reported yield. The discovery of this strain provides a basis for the production of chloramphenicol and other flavonoids.

[0039] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art can make various modifications and variations on this basis. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A chloramphenicol-producing strain, wherein the strain is *Aspergillus glehanii* (… Aspergillus candidus ZWAC-1 was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40506.

2. The use of the strain described in claim 1 in the preparation of chlorfluazurin.

3. A method for preparing chlorfluazurin, the method comprising the step of fermenting the strain of claim 1.

4. The method according to claim 3, characterized in that, The method also includes the step of separating chloramphenicol from the fermentation broth.

5. The method according to claim 3, characterized in that, The fermentation temperature is 20℃-35℃.

6. The method according to claim 3, characterized in that, The fermentation time is 2 to 20 days.

7. The method according to claim 3, characterized in that, The fermentation was carried out at 150 rpm-300 rpm.

8. The method according to claim 3, characterized in that, The fermentation medium is selected from PDB, BPY or PPM medium.

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