Fusarium oxysporum for saline-alkali soil and application of fusarium oxysporum

Abstract

The invention provides a saline-alkali soil Fusarium oxysporum and application thereof, belongs to the technical field of development and utilization of microbial resources, and aims to solve the problems of high cost, low repeatability, lack of wild strains capable of efficiently synthesizing higher alcohols and the like in a higher alcohol chemical synthesis process. The invention provides a strain capable of efficiently fermenting and synthesizing various higher alcohols such as isoamyl alcohol, 2-methyl butanol, n-hexyl alcohol, n-propyl alcohol, sec-octyl alcohol, isobutanol, alpha-acorus calamus alcohol, phenethyl alcohol and the like, as well as ethyl acetate, isoamyl acetate, 2, 2, 4-trimethyl-1, 3, 4-triazole-1, 3-dione, 2, 3, 4-triazole-1, 3-dione and 2, 3, 4-triazole-1, 3-dione by utilizing PDA culture medium components. The invention relates to a salt-resistant, acid-resistant and alkali-resistant fungus strain KKFV1024 (Fusarium aromatic) of ester compounds such as 1, 3-pentanediol diisobutyrate, hexyl acetate, 2-methylbutyl acetate, ethyl phenylacetate and the like. The strain provided by the invention not only can effectively synthesize various higher alcohols and ester compounds, but also is short in fermentation time, low in cost, simple to operate and reusable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial resource development and utilization, and particularly relates to a halophilic Fusarium odoratissimum and application thereof. BACKGROUND

[0002] Higher alcohols, also known as higher fatty alcohols, refer to a mixture of monohydric alcohols containing 3 or more carbon atoms, including isoamyl alcohol, n-propanol, isobutyl alcohol, isoamyl alcohol, n-pentanol and n-hexanol, etc. Higher alcohols are widely used in food, medicine and manufacturing industries as biofuels, chemical raw materials and food additives. For example, a hair cosmetic composition added with higher alcohols (content 0.1%) such as cetyl alcohol, oleyl alcohol and stearyl alcohol can effectively inhibit hair damage (CN114760979B). An aerosol composition added with higher alcohols such as lauryl alcohol, myristyl alcohol and stearyl alcohol can effectively enhance the smooth use feeling and good manageability of hair (CN115666500B). A nine-grain fragrant liquor and its brewing formula and method are invented (CN107937212A), in which higher alcohols synthesized by microorganisms in the liquor starter can increase the flavor of the liquor. At present, the methods for preparing higher alcohols include methanol carbonylation, olefin hydration and synthesis gas catalysis, etc. For example, a method for continuously catalytically converting higher alcohols is invented (CN117645528B), in which ethanol is used as raw material, and aldehyde ester compounds are used as catalysts, which effectively reduces the complexity of subsequent separation of higher alcohols. A series catalytic method for improving the catalytic efficiency of CO2 hydrogenation for preparing higher alcohols is invented (CN119500145A), which can effectively reduce the selectivity of byproduct hydrocarbons and significantly increase the recovery rate of higher alcohols. A multi-stage programmed temperature method is used to couple C8-C16 higher alcohols in ethanol aqueous phase (CN116283492B), in which different intermediate products can continuously couple under the most suitable temperature conditions for their own continuous reaction. However, the chemical synthesis process has limitations such as high raw material cost, low repeatability and expensive catalyst price. Compared with the chemical synthesis process, the microbial fermentation synthesis process of higher alcohols has unique advantages.

[0003] Fusarium sp. belongs to Ascomycota, and has good environmental adaptability and stress resistance. At present, the researches on Fusarium sp. mainly focus on eliminating or alleviating the plant diseases caused by Fusarium, ignoring the potential application value of its metabolites. It is worth noting that the secondary metabolites of Fusarium sp. are rich in terpenes, polyketides, aromatic hydrocarbons and alkaloids and other compounds, which have a wide range of biological activities such as antiviral, antibacterial and antitumor, and plant protection and growth promotion. For example, a Fusarium oxysporum strain SYfx123.3 can convert yellow ginger to yucca ketone under the culture condition containing yellow ginger, with the announcement number CN119753074A. A Fusarium oxysporum strain FOZJ2024 has excellent killing effect on Melaleuca longipennis, mealybug, Spodoptera exigua and Solenopsis invicta, with the announcement number CN119842502A. A Fusarium sp. NX1 can promote the growth of blueberry seedlings in cooperation with Alternaria sp. CS9, with the announcement number CN119490914A. In addition, it has been reported that Fusarium sp. can effectively synthesize higher alcohols. For example, a Fusarium solani HCO1 can quickly induce linaloe tree to synthesize chromone and linaloe tetrol, with the announcement number CN117801960B. However, the strains of Fusarium sp. that can directly utilize sugar compounds to ferment and synthesize higher alcohols are relatively scarce. Therefore, the purpose of the present application is to find wild type fungi that can be practically applied to ferment and synthesize higher alcohols and have good stress resistance, and to provide new strains for high-efficiency fermentation production of higher alcohols. SUMMARY

[0004] In view of the problems in the prior art, such as high cost and low repeatability in chemical synthesis of higher alcohols, and the lack of wild type strains that can efficiently synthesize higher alcohols, the present application provides a salt-tolerant, acid-tolerant and alkali-tolerant fungal strain KKFV1024 that can utilize PDA medium components to efficiently ferment and synthesize a variety of higher alcohols such as isoamyl alcohol, 2-methylbutanol, n-hexanol, n-propanol, sec-octanol, isobutyl alcohol, alpha-canalol and phenethyl alcohol, and ester compounds such as ethyl acetate, isoamyl acetate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, hexyl acetate, 2-methylbutyl acetate and phenylacetic acid ethyl ester. The strain provided by the present application can not only effectively synthesize various higher alcohols and ester compounds, but also has the advantages of short fermentation time, low cost, simple operation and repeatability.

[0005] The technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a saline-alkali soil Fusarium redolens, the preservation name of the Fusarium redolens (KKFV1024) is Fusarium redolens KKFV1024, the ITS sequence of the Fusarium redolens is the accession number PV653231 in NCBI, and the Fusarium redolens is preserved in China Center for Type Culture Collection on June 30, 2025, and the preservation number is CCTCC NO: M20251493.

[0007] KKFV1024 is obtained by being separated and purified from saline-alkali soil in a lake area of Koko Salt Lake, and the fungus is identified as belonging to Fusarium redolens through morphology and molecular biology.

[0008] The KKFV1024 strain has the following characteristics: (1) can grow in PDA medium with a concentration of 0.0-1.5 mol / L NaCl; (2) can grow in PDA medium with a pH value of 3-12; (3) in PDA liquid medium, at least one of higher alcohols such as isoamyl alcohol, 2-methyl-1-butanol, n-hexanol, ester compounds or small molecule compounds can be synthesized within 3-5 days of fermentation culture.

[0009] The above characteristics can show that the KKFV1024 grows rapidly (3d to plateau), has salt tolerance (0-1.5 mol / L NaCl) and strong alkali and acid tolerance (can grow in pH value 3-12), and can utilize a wide range of carbon sources (such as starch and cellulose) and nitrogen sources (including organic nitrogen and inorganic nitrogen).

[0010] In a second aspect, the present application provides application of the Fusarium redolens in preparation of higher alcohols, and the Fusarium redolens synthesizes higher alcohols by fermentation in PDA medium.

[0011] CG-MS shows that KKFV1024 can effectively synthesize higher alcohols such as isoamyl alcohol (relative content / peak area is 44,526,192.00±969,459.53), 2-methylbutanol (20,491,658.33±97,524.80), n-hexanol (1,317,114.00±30,975.48), n-propanol (3,645,436.00±106,162.57), sec-octanol (2,732,149.33±30,726.08), isobutanol (2,104,102.00±109,263.13), alpha-calamenol (757,442.20±43,151.08) and phenethyl alcohol (487,233.00±73,653.61) after 3 days of fermentation in PDA medium.

[0012] In a third aspect, the present application provides a use of the Fusarium redolens in the preparation of ester compounds, wherein the Fusarium redolens is used to ferment and synthesize the ester compounds in PDA medium.

[0013] The ester compounds include, but are not limited to, ethyl acetate (7,519,704.67±134,968.77), isoamyl acetate (1,118,552.00±96,472.91), 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (642,943.30±427,551.98), hexyl acetate (15,608.33±7,165.80), 2-methylbutyl acetate (100,300.16±3,495.26), and ethyl phenylacetate (75,719.01±3,243.00), etc.

[0014] In a fourth aspect, the present application provides a use of the Fusarium redolens in the preparation of small molecule compounds, wherein the Fusarium redolens is used to ferment and synthesize the small molecule compounds in PDA medium.

[0015] The small molecule compounds include, but are not limited to, isoamyl aldehyde (3,645,535.00±171,743.63), phenylacetaldehyde (1,726,459.00±249,106.00), sec-octanone (1,854,681.33±294,084.60), 2-heptanone (109,368.71±14,668.91), and caryophyllene (242,606.80±10,905.17).

[0016] Advantages of the present application:

[0017] The present application discloses a Fusarium redolens KKFV1024 (Fusarium redolens) from the native soil of Qinghai-Tibet Plateau for the first time, which has strong stress resistance and can utilize a wide range of carbon sources and nitrogen sources. The KKFV1024 can obtain a variety of higher alcohols and ester compounds by being inoculated into PDA medium for fermentation, and has low cost, simple operation and reusability, thereby providing a strain resource for microbial fermentation synthesis of higher alcohols. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0019] Figure 1 Growth characteristic curve of KKFV1024.

[0020] Figure 2 Colony morphology (A1 and A2), mycelium morphology (B1, B2, B3) and cell morphology (C1, C2, C3, C4) of KKFV1024.

[0021] Figure 3 Phylogenetic analysis of KKFV1024 and its closely related species.

[0022] Figure 4 Compound classification pie chart of KKFV1024 fermentation supernatant. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application.

[0024] The test materials used in the following examples are all purchased from a conventional biochemical reagent store, unless otherwise specified.

[0025] Example 1 Growth characteristics and morphological characteristics of KKFV1024

[0026] PDA liquid medium (potato infusion powder 12 g / L and glucose 20 g / L, original pH) 250 mL was configured, 1 g of Koko Salt Lake region saline soil was added, and after static culture at 28°C for 2 d (shaking once every 12 h), 50 μL of dilution was coated on PDA solid medium (potato infusion powder 12 g / L, glucose 20 g / L and agar powder 14 g / L, original pH), and the dilution ratio was 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 , each group had 3 repeats. Single colonies were selected, and a pure culture fungus strain Fusarium redolens KKFV1024 was obtained through multiple rounds of separation. Blank PDA liquid medium was set, KKFV1024 was inoculated, the inoculation amount was 1%, and the optical density value OD 600 was measured every 8 h, and the culture was incubated to the plateau phase, and a growth curve was drawn. PDA liquid medium (n=3 / group) of different NaCl concentration gradient groups (0.0-3.0 mol / L, interval 0.5) and pH gradient groups (3.0-12.0, interval 1.0) was set, KKFV1024 was inoculated at an amount of 1%, and the optical density value OD 600, and a growth characteristic curve was drawn. A blank PDA solid medium was prepared, and KKFV1024 was inoculated with an inoculating needle. The culture was incubated at 28°C until the mycelium filled the culture dish. The morphology of the KKFV1024 colonies was recorded. KKFV1024 colonies were picked and stained with lactic acid phenol cotton blue stain. The morphology of KKFV1024 cells was observed under an optical microscope (400×). The cells were fixed using the glutaraldehyde-osmium acid double fixation method, and the microscopic morphology of the bacterial cells was observed using a scanning electron microscope (500×, 2,000×, and 5,000×).

[0027] The growth characteristic curve results show that Figure 1 Fusarium redolens KKFV1024 grew rapidly and entered the plateau phase after 3 days ( Figure 1 In addition, KKFV1024 has good salt tolerance (growth salinity 0.0-1.5 mol / L NaCl) and a wide pH tolerance range (growth pH 3-12, Figure 1 B and C). The morphological results showed that ( Figure 2 ): Colony morphology of strain KKFV1024 ( Figure 2 A1) is fluffy, white, has a special fragrance, and spreads; the back of the culture medium is dark pink, the bacteria can synthesize pigments, and the hyphae grow embedded in the solid culture medium ( Figure 2 Middle A2). High magnification microscope shows that KKFV1024 is filamentous ( Figure 2 B3), a large number of fusiform aerial spores and some mycelial conidia were observed ( Figure 2 B1) and blastospores ( Figure 2 Middle B2). Scanning electron microscopy showed that KKFV1024 had a smooth surface and a large number of short ciliary branches ( Figure 2 C1); blastospores are smooth, swollen spherical with pores on the surface and are about 15 μm long ( Figure 2 In addition, there are pores on the branch surface of KKFV1024 ( Figure 2 C3), the culture medium components accumulated in each branch, which may be a potential strategy for KKFV1024 to quickly obtain nutrients ( Figure 2 (C4).

[0028] Example 2 Physiological and biochemical identification results and phylogenetic tree of KKFV1024

[0029] The purified strain was activated in PDA liquid medium and cultured statically at 28°C for 3 days. Carbon and nitrogen source utilization tests and physiological and biochemical identifications were performed according to the instructions of the microbial biochemical identification kit.

[0030] The genomic DNA of KKFV1024 was extracted by fungal genome extraction kit and ITS gene full-length PCR amplification was performed. The primers were ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3').

[0031] PCR reaction system (30 μL): 2x PCR Mix 15 μL, DNA template (10-20 ng / μL) 1 μL, upstream and downstream primers (10 μmol / L) 2 μL each, ddH2O 10 μL. PCR reaction conditions: 95℃ 5 min; 94℃ 30 s, 55℃ 30 s, 72℃ 1 min, 33 cycles; 72℃ 5 min.

[0032] The PCR purification product was sequenced by Sanger method, and the sequencing results were spliced and arranged by Lasergene (v7.1) software package. The spliced sequence was compared and analyzed by NCBI database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The phylogenetic tree was constructed by MEGA (v11.0) software, using Neighbor-joining method, bootstrap was 1,000.

[0033] The physiological and biochemical identification results showed that KKFV1024 could utilize arginine, lysine, peptone, yeast extract, (NH4)2SO4, NH4Cl, KNO3, NaNO3, glucose, rhamnose, sucrose, soluble starch, melibiose, maltose, starch, cellulose as the only carbon and nitrogen source for growth, and could not utilize inositol, sorbitol, lactose and ornithine. The β-galactosidase, urease, H2S and citrate utilization tests were positive, and the V-P test, indole test and gelatin liquefaction test were negative. It could produce acid. ITS gene sequence alignment analysis showed that strain ZB109 had the highest similarity (100.00%) with F. redolens QK-1 (MW911468.1). The phylogenetic tree was constructed by MEGA (v.11.0) software ( Figure 3 ), and it was preliminarily determined that strain KKFV1024 (PV653231.1) was homologous to Fusarium strain evolution, and belonged to Ascomycota Imperficti Sphaeropsidales Discellaceae Fusarium F. redolens.

[0034] Table 1 Physiological and biochemical characteristics of Fusarium redolens KKFV1024

[0035]

[0036]

[0037] Example 3: GC-MS analysis of main components in KKFV1024 fermentation broth

[0038] Using PDA liquid medium, inoculating 1% of KKFV1024 seed liquid, static culture at 28℃ for 3d and 5d, centrifuging at 12,000r / min to obtain supernatant, using headspace solid phase microextraction method (HS-SPME) to extract KKFV1024 fermentation broth metabolites and using GC-MS for detection. Mass spectrometry conditions: mass spectrometry quadrupole temperature 150℃, ion source temperature 230℃; electron energy 70eV; mass spectrometry scanning range m / z 20~650amu. Programmed temperature conditions: 40℃ (3min), 4℃ / min to 220℃, holding for 10min. Injection conditions: split injection; mass spectrometry inlet temperature is 280℃; injection volume is 1.0mL; constant flow rate 1.0mL / min; carrier gas is high-purity helium (purity ≥99.999%). Metabolites are annotated and identified by NIST Chemistry WebBook database (https: / / webbook.nist.gov / chemistry / ; NIST, 98L), mass spectrum (MS) and retention time. Excluding noise, chromatographic column outflow, halide-containing compounds and BSTFA compound peaks in the identification process, peaks of the same compound are combined. Each sample is repeated 3 times to evaluate the repeatability of the experiment.

[0039] After the original data were preprocessed by outlier filtering, missing value filtering, missing value filling and data standardization, 224 compounds were obtained, of which 192 were qualitative substances. Mainly involving 10 types of compounds (amino acids, organic acids, sugars, alcohols, ketones, aldehydes, phenols, hydrocarbons, heterocyclic compounds and other compounds) Figure 4), including organic oxygen compounds (Organic oxygen compounds, 25.521%), benzenoids (Benzenoids, 19.271%), lipids and lipid-like molecules (Lipids and lipid-like molecules, 13.021%), organoheterocyclic compounds (Organoheterocyclic compounds, 10.938%), organic acids and derivatives (Organic acids and derivatives, 7.292%), hydrocarbons (Hydrocarbons, 5.729%), organohalogen compounds (Organohalogen compounds, 1.562%), organosulfur compounds (Organosulfur compounds, 1.042%), phenylpropanoids and polyketides (Phenylpropanoids and polyketides, 1.042%), and organic nitrogen compounds (Organic nitrogen compounds, 0.520%).

[0040] The results of the change in the content of compounds in the fermentation broth of Fusarium redolens KKFV1024 showed (Table 2): the highest relative content / peak area was isoamyl alcohol (44,526,192.00±969,459.53, 3d), followed by 2-methylbutanol with a relative content / peak area of 20,491,658.33±97,524.80, and the third highest relative content / peak area was n-hexanol with a relative content / peak area of 1,317,114.00±30,975.48, all of which belong to higher alcohols. In addition, the fermentation broth of KKFV1024 also contained ethyl acetate (7,519,704.67±134,968.77, 3d), isoamyl acetate (1,118,552.00±96,472.91), and dibutyl phthalate (1,080,219.77±327,947.44) and other special odor ester compounds with high relative content / peak area. It is worth noting that after KKFV1024 fermentation stabilized in the stationary phase (3d), it can still ferment to synthesize higher alcohols, such as isoamyl alcohol with a relative content / peak area of 47,317,332.00±554,328.59 (P<0.05) at 5d, 2-methylbutanol with a relative content / peak area of 24,322,047.00±262,226.44 (P<0.001), and n-hexanol with a relative content / peak area of 26,451,007.67±399760.17 (P<0.001), indicating that KKFV1024 has the ability to stably ferment to synthesize higher alcohols.

[0041] Table 2 Changes in the contents of compounds in the fermentation broth of Fusarium redolens KKFV1024

[0042]

[0043]

[0044] Note: The table is the relative content of the top 30 known compounds; ***: P < 0.001; ** : P < 0.01; *: P < 0.05; ns: P ≥ 0.05.

[0045] The above description is merely preferred specific embodiments of the present application, the protection scope of the present application is not limited to this, any simple change or equivalent replacement of the technical solutions within the technical range disclosed by the present application, which can be obviously obtained by those skilled in the art, shall belong to the protection scope of the present application.

Claims

1. A saline-alkali soil fusarium, characterized in that The fragrant Fusarium is named Fusarium redolensKKFV1024 and was deposited in the China Center for Type Culture Collection on June 30, 2025, with the preservation number CCTCCNO: M20251493.

2. The Fusarium odoriferum according to claim 1, wherein The accession number of the ITS sequence of Fusarium odoratum in NCBI is PV653231.

3. The Fusarium odoriferum according to claim 1, wherein The fragrant Fusarium is salt-tolerant, acid-tolerant and alkali-tolerant Fusarium.

4. The Fusarium odoriferum according to claim 3, wherein The salinity tolerance of the fragrant Fusarium is 0.0-1.5 mol / L.

5. The Fusarium odoriferum according to claim 3, wherein The pH tolerance of the Fusarium odoratum is 3-12.

6. The use of Fusarium odoratum in the preparation of higher alcohols and / or ester compounds and / or small molecule compounds according to claim 1, characterized in that: The fusarium odoriferum is fermented in a PDA culture medium to synthesize higher alcohols and / or ester compounds and / or small molecule compounds.

7. The use according to claim 6, characterized in that The higher alcohols include isopentanol, 2-methylbutanol, n-hexanol, n-propanol, sec-octanol, isobutanol, α-calendulol and phenylethanol.

8. The use according to claim 6, characterized in that The ester compounds include ethyl acetate, isoamyl acetate, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, hexyl acetate, 2-methylbutyl acetate, and ethyl phenylacetate.

9. The use according to claim 6, characterized in that The small molecule compounds include isovaleraldehyde, phenylacetaldehyde, sec-octanone, 2-heptanone and caryophyllene.

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