Marine fungus fusarium keratinoides and application thereof

Abstract

The invention discloses a marine fungus fusarium keratinoides and application thereof. The fusarium keratinoides capable of efficiently producing the perindoprine is screened from marine sediments, and the content of the produced perindoprine can reach 561.7 mu g / L after the fusarium keratinoides is fermented in a GPY liquid culture medium, so that possibility and a foundation are provided for breeding strains suitable for industrial large-scale production of the perindoprine in the future; mass spectrum, hydrogen spectrum and carbon spectrum data of the separated perindoprine are completely consistent with perindoprine data reported in literatures.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering, and in particular to a marine fungus, *Fusarium oxysporum*, and its applications. Background Technology

[0002] Perlolyrine (PL) is one of the effective active alkaloids of the traditional Chinese medicine Ligusticum chuanxiong. Its chemical structure is 1-(5-hydroxymethyl-2-furanyl)-9H-pyrido[3,4-b]indole, belonging to the β-carboline alkaloid class. Its structural feature is a pyridoindole core connected to a furan ring via carbon bonds, with the core skeleton of pyrido[3,4-b]indole being the main active group. Modern pharmacological studies have shown that perlolyrine has multi-target and multi-pathway effects, exhibiting significant activity in cardiovascular protection, anti-fibrosis, and neuroprotection.

[0003] Ligusticum chuanxiong is primarily derived from the traditional Chinese medicine Ligusticum chuanxiong. It also exists in other plants such as ryegrass and Polygala tenuifolia, but at much lower levels than in Ligusticum chuanxiong. The content of natural ligusticum chuanxiong in Ligusticum chuanxiong is relatively low (below 0.1%). Extracting ligusticum chuanxiong from this traditional Chinese medicine often results in underutilization of the medicinal material and is costly. Furthermore, the long plant growth cycle and complex extraction process make ligusticum chuanxiong production challenging. Extracting ligusticum chuanxiong from the secondary metabolites of microorganisms offers advantages such as low cost, rapid propagation, and a relatively simple extraction process. Moreover, to date, only a few microorganisms have been reported to produce ligusticum chuanxiong; therefore, this invention provides the possibility and foundation for future breeding of strains suitable for large-scale industrial production of ligusticum chuanxiong.

[0004] The ocean contains abundant biological resources. In order to adapt to the special living environment of the ocean, marine organisms have evolved genes, enzymes and metabolic pathways that are different from those of terrestrial organisms. They can produce metabolites with novel chemical structures and unique biological activities, which have become a rich source of drug lead compounds and have a very broad development prospect. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a marine fungus, Fusarium keratinosa.

[0006] Another object of the present invention is to provide the application of the above-mentioned marine fungus Fusarium keratinosa.

[0007] The objective of this invention is achieved through the following technical solution: A strain of Fusarium oxysporum ( Fusarium keratoplasticum (), derived from deep-sea sediment samples collected in the South China Sea, was obtained through enrichment culture, separation, and purification, and is named as Fusarium keratoplasticum LWJ2314.

[0008] The aforementioned Fusarium keratogenis, with accession number GDMCC 67094, was deposited on October 13, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The colony morphology of the aforementioned Fusarium keratogenes on PDA plates is white mycelium, which can cover the entire plate in about 5-7 days.

[0010] The nucleotide sequence of the ITS of the aforementioned Fusarium oxysporum is shown in SEQ ID NO.1.

[0011] The aforementioned Fusarium keratinosa can produce perlolyrine.

[0012] The structural formula of the chuanxiongdole is shown in Formula I:

[0013] Formula I The application of the aforementioned Fusarium keratinosa in the production of ligustrazine.

[0014] A method for producing chuanxiong indole includes the following steps: Fusarium oxysporum was inoculated into a liquid culture medium for fermentation. After the culture was completed, the mycelium and fermentation broth were separated. The fermentation broth was extracted with ethyl acetate to obtain extract A. The separated mycelium was dried and extracted with methanol. After concentration, extract B was obtained. Extracts A and B were combined and purified to obtain ligustrazine.

[0015] The liquid culture medium is GPY liquid culture medium.

[0016] The formula for the GPY liquid culture medium is: 15g glucose, 10g peptone, 2g yeast extract, and 20g sea salt. L - Tryptophan 2g, water 1000mL.

[0017] The fermentation culture conditions are as follows: cultured at 27–29°C for 40–50 days.

[0018] The purification process involved separation using silica gel column chromatography followed by semi-preparative HPLC purification.

[0019] The present invention has the following advantages and effects compared with the prior art: The inventors screened a cuticular Fusarium strain capable of efficiently producing ligustrazine from marine sediments. After fermentation in GPY liquid medium, the ligustrazine content produced reached 561.7 μg / L, providing a possibility and foundation for future breeding of strains suitable for large-scale industrial production of ligustrazine. The isolated ligustrazine was identified by high-resolution mass spectrometry and nuclear magnetic resonance, and its structure was confirmed to be completely consistent with the ligustrazine reported in the literature. Attached Figure Description

[0020] Figure 1 The colony morphology of Fusarium oxysporum and the structure of its metabolite ligustrol are shown in Example 1.

[0021] Figure 2 The fungus in Example 1 Fusarium keratoplasticum GDMCC 67094 phylogenetic tree.

[0022] Figure 3 This is the mass spectrum of chuanxiongdole isolated from the fermentation product of the strain in Example 2.

[0023] Figure 4 This is the UV absorption spectrum of ligustrazine obtained from the fermentation product of the strain in Example 2.

[0024] Figure 5 The 1H NMR spectrum of ligustrazine isolated from the fermentation product of the strain in Example 2 ( 1 H-NMR spectrum.

[0025] Figure 6 The carbon spectrum of ligustrazine isolated from the fermentation product of the strain in Example 2 (…). 13 C-NMR spectrum. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0027] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0028] Example 1: Isolation and Identification of Strains 1.1 Strains Isolation The specific steps for screening bacterial strains from deep-sea sediment samples collected from the South China Sea are as follows: Under aseptic conditions, 2 g of sediment sample was inoculated into freshly prepared 3% sea salt PDB medium (200 g peeled fresh potato, 20 g anhydrous glucose, 30 g crude sea salt, 1000 mL distilled water). The medium was incubated statically at room temperature in the dark for 2 days, and then shaken at 180 rpm for 2 hours on a shaking plate. 100 µL of the culture solution was evenly spread onto freshly prepared 3% sea salt PDA medium (200 g peeled fresh potato, 20 g anhydrous glucose, 30 g crude sea salt, 16 g agar powder, 1000 mL distilled water) with a diameter of 90 mm. The medium was incubated at 28°C for 7 days, and observed daily. After the fungal colony formed, hyphae from the edge of the fresh fungal colony were picked and inoculated into freshly prepared PDA medium. The above operation was repeated until a single colony was observed, and a marine fungus, *Fusarium oxysporum*, was isolated.

[0029] 1.2 Strain Identification The purified fungi were inoculated into the center of freshly prepared PDA medium and cultured at 28 °C for 3 days, or until the fungal colonies occupied 2 / 3 of the PDA medium. Fungal hyphae were scraped off with a bamboo stick or a 1000 µL pipette tip, placed in a mortar, and ground under liquid nitrogen. Fungal DNA was extracted according to the instructions of the UNlQ-10 column-based fungal genomic DNA extraction kit (Shanghai Sangon Biotech, Shanghai, China). The extracted fungal DNA was amplified by PCR using universal fungal primers ITS 1F and ITS 4, and the premixed solution was prepared using SanTaq Plus PCR Mix (Shanghai Sangon Biotech, Shanghai, China). The amplified sequence was sent to Shanghai Biotech Co., Ltd. for sequencing. The obtained ITS sequence is shown in SEQ ID NO. 1. BLAST comparison of the sequencing results showed that the ITS sequence of the marine fungus *Fusarium oxysporum* had 99.8% identity with GenBank entry NR_130690.1, and therefore it was identified as... F. keratoplastic .

[0030] Subsequently, the ITS sequences of fungal species in the Fusarium genus in GenBank were retrieved and compared with the sequences obtained from sequencing. Using the integrated bioinformatics analysis software phylosuite, the processed sequences were aligned sequentially using the MAFFT program, the trimAI program was used to remove redundant sequences, and the IQtree program was used to construct a phylogenetic tree from the removed sequences. The resulting phylogenetic tree is shown below. Figure 2 As shown.

[0031] This strain was deposited on October 13, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 67094. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0032] Example 2: Strain culture and identification of metabolites 2.1 Cultivation and large-scale fermentation of Fusarium oxysporum 2.1.1 Strain resuscitation The culture strains stored in the preservation bank are taken out, thawed at room temperature, and inoculated onto PDA plates containing 3% sea salt. They are then incubated at 28°C for 72 hours until a large number of white mycelia grow. For subculturing: a small amount of mycelia is picked up from the plate using an inoculation needle and streaked onto another PDA plate to expand the culture. The plate is then incubated at 28°C for 72 hours. Once the mycelia have completely covered the PDA plate, it is ready for large-scale fermentation.

[0033] 2.1.2 Fermentation by bacterial strain Using an inoculation needle, a piece of bacterial cell approximately 1cm × 1cm in size was picked up from the cultured strain and inoculated into a glass flask containing 400mL of GPY liquid medium. A total of 120L of fermentation was carried out, and the room temperature was controlled at approximately 28℃. The culture was then allowed to stand at room temperature for 45 days.

[0034] PDA tablet formula: 200g potato, 20g glucose, 30g sea salt, 1000mL water.

[0035] GPY culture medium formula: glucose 15g, peptone 10g, yeast extract 2g, sea salt 20g. L - Tryptophan 2g, water 1000mL.

[0036] 2.2 Extraction of secondary metabolites After fermentation, the mixture was filtered through gauze to separate the fermentation broth and mycelium. The fermentation broth was extracted with ethyl acetate at a volume ratio of 1:1, for a total of three extractions. All extracts were combined and concentrated under reduced pressure by rotary evaporation to obtain a paste, thus yielding the ethyl acetate extract of the fermentation broth. The mycelium was air-dried and then extracted with methanol at a weight ratio of 1:10 (mycelium weight to methanol volume), for one day. This extraction was repeated twice. The methanol extracts were combined and concentrated under reduced pressure by rotary evaporation to obtain the methanol extract of the mycelium. The combined ethyl acetate extract of the fermentation broth and the methanol extract of the mycelium constitute the total extract of secondary metabolites.

[0037] 2.3 Isolation and purification of metabolites The total extract was separated using normal silica gel column chromatography with gradient elution of petroleum ether to ethyl acetate at a volume ratio of 10:0 to 0:10. The TLC results were combined into seven fractions. Fractions with polarity between the petroleum ether to ethyl acetate volume ratios of 10:3 and 3:10 were selected for TLC analysis with a petroleum ether-ethyl acetate ratio of 1:1. Fluorescence was observed at 254 nm, and fractions exhibiting strong fluorescence were selected for color development using potassium bismuth iodide reagent. Spots showing orange-red color were then combined. The combined fractions were purified using semi-preparative high-performance liquid chromatography to obtain ligustrazine with high purity. The ligustrazine content produced by fermentation with the *Fusarium oxysporum* strain described in this invention can reach 561.7 μg / L.

[0038] Semi-preparative high-performance liquid chromatography (HPLC) conditions: Column: Phenomenex C18 column, 250 × 10.00 mm; gradient elution mode was used, with formic acid water (0.1%, v: v) as mobile phase A and methanol as mobile phase B, with a mobile phase ratio of B% = 45%, flow rate 2 mL / min, detection wavelengths of 254 nm and 365 nm, and retention time of ligustrazine 11.7 min.

[0039] 2.4 Structural identification of the product The structure of chuanxiongdole isolated in section 2.3 was identified by LCMS-IT-TOF, UV, HPLC and NMR analysis.

[0040] The obtained ligustrazine was a pale yellow powder that dissolved in methanol to produce a yellow-green color. Its molecular weight was analyzed using LC-MS-IT-TOF, and the molecular ion peak of this compound was visible in the spectrum. m / z 265.1029 [M+H] + (The calculated value is C) 16 H 12 N2O2H + The molecular formula can be determined to be C(265.0972). 16 H 12 N₂O₂ has an unsaturation degree of 12. From... 1 H NMR and 13 Analysis of the 12C NMR spectroscopy data revealed the presence of an aromatic ring within the compound molecule, containing seven biphasic methyl groups and two triplet methyl groups. The UV spectroscopy showed maximum absorption peaks at 273 nm and 291 nm, while the characteristic absorption peak of a benzene ring is typically at 254 nm. This suggests that the red shift of the benzene ring absorption peak may be due to the conjugation effect or electronic effect of substituents on the benzene ring. 1 H-NMR spectral data, chemical shift δ 8.27(1H, d, J = 7.9 Hz), 7.29 (1H, t,J = 7.4 Hz), 7.61 (1H, t, J = 7.1 Hz), 7.78 (1H, d, J = 8.2 Hz) is speculated to be on the benzene ring 1 H signal, and based on peak splitting, it is inferred to be an ortho-disubstituted benzene ring, chemical shift δ 8.39(1H, d, J = 5.1 Hz), 8.07 (1H, d, J = 5.1 Hz) 1 The H signal is presumed to be a pyridine ring signal, chemical shift. δ 7.23 (1H, d, J = 3.3 Hz), 6.60 (1H, d, J = 3.3 Hz) is presumed to be an olefin signal, chemical shift δ 4.69(2H, d, J = 5.9 Hz) can be inferred to be an oxygen-bound methylene signal. According to 13 C-NMR spectral data show that this compound has 16 carbon signals and chemical shifts. δ 121.11 (C-4b), 122.10 (C-5), 120.18 (C-6), 128.89 (C-7), 112.90 (C-8), and 141.42 (C-8a) are characteristic carbon signals of the benzene ring, with chemical shifts. δ 133.63 (C-1), 138.67 (C-3), 114.11 (C-4), 129.92 (C-4a), and 130.96 (C-9a) are characteristic carbon signals of the pyridine ring, with chemical shifts... δ 152.62 (C-2'), 110.11 (C-3'), 109.54 (C-4'), and 157.23 (C-5') are characteristic carbon signals of the furan ring.

[0041] In summary 1 H-NMR, 13 C-NMR spectral information indicates that this compound has an ortho-disubstituted benzene, pyridine ring, and furan ring structure, and also combines... 1 Chemical shift in H-NMR spectrum δ The active hydrogen at 11.22 (1H, s) suggests that the compound has a β-carbaline structure. After reviewing the literature, the mass spectra of this compound... 1 H-NMR, 13 The C-NMR spectral data were completely consistent with the data of ligustrol reported in the literature, so the compound was identified as ligustrol.

[0042] Fusarium oxysporum from marine sediments ( Fusarium keratoplasticum Physical constants and spectral data of the produced ligustrazine: pale yellow powder, pale yellow needle-like crystals; molecular formula C 16 H 12 N2O2; HRESIMS m / z 265.1029 [M+H] + (The calculated value is C) 16 H 12 N2O2H + , 265.0972); UV(MeOH) λ max (Abs.) 380(0.309), 367(0.295), 291(0.485), 273(0.441), 253(0.454), 237(0.549),205(0.834)nm; 1 H NMR (400MHz, DMSO-) d 6) δ = 11.22 (1H, s, NH-9), 8.39 (1H, d, J = 5.1Hz, H-3), 8.27 (1H, d, J = 7.9 Hz, H-5), 8.07 (1H, d, J = 5.1 Hz, H-4), 7.78 (1H,d, J = 8.2 Hz, H-8), 7.61 (1H, t, J = 7.1 Hz, H-7), 7.29 (1H, t, J = 7.4 Hz, H-6), 7.23 (1H, d, J = 3.3 Hz, H-3'), 6.60 (1H, d, J = 3.3 Hz, H-4'), 5.49 (1H, t, J =6.1 Hz, OH-6'), 4.69 (2H, d, J = 5.9 Hz, H-6'); 13 C NMR (100MHz, DMSO-) d 6) δ= 157.23(C-5'), 152.62(C-2'), 141.42(C-8a), 138.67(C-3), 133.63(C-1),130.96(C-9a), 129.92(C-4a), 128.89(C-7), 122.10( C-5 ), 121.11 ( C-4b ), 120.18 ( C-6 ), 114.11 ( C-4 ), 112.90 ( C-8 ), 110.11 ( C-3' ), 109.54 ( C-4' ), 56.44 ( C-6' ).

[0043] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Fusarium oxysporum, characterized by: The sample was obtained from deep-sea sediments collected in the South China Sea, and was enriched, cultured, separated, and purified. Its name is... Fusarium keratoplasticum LWJ2314, with accession number GDMCC 67094, was deposited on October 13, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The *Fusarium oxysporum* according to claim 1, characterized in that: The colony morphology of the aforementioned Fusarium keratogenes on PDA plates is white mycelium.

3. The *Fusarium oxysporum* according to claim 1, characterized in that: The nucleotide sequence of the ITS of the aforementioned Fusarium oxysporum is shown in SEQ ID NO.

1.

4. The *Fusarium oxysporum* according to claim 1, characterized in that: The aforementioned Fusarium keratinosa can produce ligustrazine.

5. The use of Fusarium oxysporum according to any one of claims 1 to 4 in the production of ligustrazine.

6. A method for producing chuanxiong indole, characterized in that... Includes the following steps: Fusarium oxysporum was inoculated into a liquid culture medium for fermentation. After the culture was completed, the mycelium and fermentation broth were separated. The fermentation broth was extracted with ethyl acetate to obtain extract A. The separated mycelium was dried and extracted with methanol. After concentration, extract B was obtained. Extracts A and B were combined and purified to obtain ligustrazine.

7. The method for producing chuanxiong indole according to claim 6, characterized in that: The liquid culture medium is GPY liquid culture medium.

8. The method for producing chuanxiong indole according to claim 6, characterized in that: The fermentation culture conditions are as follows: cultured at 27–29°C for 40–50 days.

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