A recombinant strain of Aspergillus niger for degrading vomitoxin and its application

Abstract

A recombinant Aspergillus niger strain that degrades vomitoxin and its application belong to the field of genetic engineering. This invention addresses the lack of efficient methods for degrading vomitoxin in existing technologies by providing a recombinant Aspergillus niger strain that degrades vomitoxin for co-expression. FsTRI12 Using *Aspergillus niger* strains as recipient bacteria, an intracellular gene expression vector was constructed by selecting the vomitoxin acetyltransferase gene fragment, and homozygous recombinant *Aspergillus niger* strains were screened. The recombinant *Aspergillus niger* strains provided by this invention can be used to degrade vomitoxin, offering a new option for effectively solving the problem of vomitoxin contamination in grain raw materials and feed, reducing grain loss, and ensuring food and feed safety, and has potential industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a recombinant strain of Aspergillus niger that degrades vomitoxin and its application. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by fungi (such as Fusarium, Alternaria, Aspergillus, and Penicillium) that infect plant products during field or storage. These include aflatoxin, zearalenone, ochratoxin, deoxynivalenol, and patulin. Among these, deoxynivalenol (DON), also known as deoxynivalenol or deoxyfuricin, is widely found in cereal grains such as wheat, barley, and corn, and their products, making it the most widely distributed mycotoxin globally.

[0003] Currently, methods for degrading vomitoxin mainly include physical adsorption, chemical degradation, and biodegradation. Among these, physical adsorption (such as using adsorbents), while simple to operate, cannot completely remove the toxin; chemical degradation (such as oxidant treatment) may produce harmful byproducts; and biodegradation (such as utilizing microorganisms) is considered an environmentally friendly and efficient method, but it still has the following problems:

[0004] Glycosylation: Glycosylation reactions transfer sugar molecules (such as glucose) to DON molecules, forming glycosidic compounds (such as DON-3-glucoside, D3G), thereby reducing their biological activity. Plant UDP-glucosyltransferase (UGT) typically converts DON to DON-3-glucoside (D3G). While D3G is less toxic than DON, it can be reconverted to DON under certain conditions (such as the action of gut microbiota), leading to a recovery in toxicity. Current technologies lack sufficient research on the interaction mechanisms, catalytic sites, and reaction kinetics between UGT and DON, resulting in a lack of in-depth understanding of the structure-function relationship of UGT, making it difficult to improve its catalytic efficiency through genetic engineering.

[0005] Deepoxidation: Deepoxidation disrupts the toxic core structure of DON by hydrolyzing its epoxy group (COC). Epoxide hydrolase (EH) may only partially hydrolyze the epoxy group, leaving residual epoxy fragments that remain toxic; deepoxidation may generate various metabolites, the toxicity of which is not fully understood. For example, deepoxidation of DON may generate DOM-1 (deepoxidized DON), but DOM-1 may be further converted into derivatives with unknown toxicity under the action of intestinal microorganisms.

[0006] Carbonylation: Carbonylation reactions alter the chemical properties of DON by introducing a carbonyl (C=O) group. Carbonylation may generate more toxic intermediates. For example, DON can be converted to DON-8-ketone during oxidative carbonylation, a product that is approximately 20% more toxic to hepatocytes than DON alone. Furthermore, oxidation reactions can generate free radicals, triggering chain reactions such as lipid peroxidation. Carbonylation reactions typically require noble metal catalysts (such as palladium and platinum); for example, the oxidative carbonylation of amines requires palladium catalysts, which are expensive and susceptible to sulfide poisoning, leading to catalyst deactivation.

[0007] Therefore, those skilled in the art are eager to develop a method for efficiently degrading vomitoxin, to solve the problem of vomitoxin pollution, and to prevent harm to livestock and human health. Summary of the Invention

[0008] To address the lack of efficient methods for degrading vomitoxin in the prior art, this invention provides a recombinant Aspergillus niger strain that degrades vomitoxin and its applications.

[0009] One of the objectives of this invention is to provide a form of Aspergillus niger that degrades vomitoxin. Aspergillus niger The recombinant strain of *Aspergillus niger* was prepared by: ligating the vomitoxin acetyltransferase gene fragment with the p-6-g vector fragment to obtain a recombinant plasmid; introducing the recombinant plasmid into AGL1 competent cells using a freeze-thaw method to obtain *Agrobacterium* transformants; co-culturing the *Agrobacterium* transformants with *Aspergillus niger* strain Fs, transferring the mixture to a membrane, and subculturing for 3-5 days to screen for homozygous *Aspergillus niger* recombinant strains; the *Aspergillus niger* strain Fs is a co-expressing... FsTRI12 Genetically modified Aspergillus niger strain.

[0010] Preferably, the vomitoxin acetyltransferase gene fragment includes Ff1TRI201、Fcu2TRI101 or Fcr2TRI101 Any one of them; the Ff1TRI201 The gene nucleotide sequence is shown in SEQ ID NO.5. Fcu2TRI101 The gene nucleotide sequence is shown in SEQ ID NO.6. Fcr2TRI101 The gene nucleotide sequence is shown in SEQ ID NO.7.

[0011] Preferably, the culture medium used for co-culture is PDA+AS medium.

[0012] Preferably, the culture medium used for the subculture is PDA liquid medium.

[0013] The second objective of this invention is to provide the application of the above-mentioned recombinant Aspergillus niger strain in the degradation of vomitoxin.

[0014] The third objective of this invention is to provide a method for degrading vomitoxin, the method comprising the following steps: fermenting the above-mentioned recombinant Aspergillus niger strain to obtain a fermentation suspension of the recombinant Aspergillus niger strain; adding the above-mentioned fermentation suspension to a substrate for degrading vomitoxin for degradation.

[0015] Preferably, the fermentation treatment lasts for 8 days, and the degradation treatment is carried out at a temperature of 25-35°C, a pH value of 5-6, and a time of 24 hours.

[0016] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a recombinant Aspergillus niger strain that degrades vomitoxin, for co-expression FsTRI12 The *Aspergillus niger* strain Fs was used as the starting strain, and the DON acetyltransferase gene was selected. Ff1TRI201 , Fcu2TRI101 or Fcr2TRI101 Intracellular gene expression vectors were constructed, and homozygous recombinant strains of *Aspergillus niger* were screened. This invention provides a method for degrading vomitoxin using the aforementioned recombinant strains of *Aspergillus niger*. The fermentation suspensions of the three recombinant strains (PFFS, PUFS, and PRFS) provided by this invention were optimized for vomitoxin degradation conditions, determining the optimal fermentation time to be 8 days, the optimal degradation temperature to be 25-35℃, the optimal pH to be 5-6, and the optimal time to be 24 hours. Under the optimal reaction conditions, the vomitoxin degradation rates of the three recombinant strains of *Aspergillus niger* (PFFS, PUFS, and PRFS) provided by this invention were 72.37%, 60.93%, and 74.51%, respectively; among them, the recombinant strain PFFS showed a higher degradation rate compared to the single-expression strain. FsTRI12 The control strain (PFT) increased the vomitoxin degradation rate by 7.06 times, indicating that the recombinant Aspergillus niger strain provided by this invention, utilizing... FsTRI12 Synergistic effect with acetyltransferase significantly improves the acetylation detoxification efficiency of bacterial suspension.

[0017] The recombinant Aspergillus strain provided by this invention can be used to degrade vomitoxin, providing a new option for effectively solving the problem of vomitoxin contamination in grain raw materials and feed, reducing grain loss, and ensuring food and feed safety, and has potential industrial application prospects. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the recombinant plasmid construction in Example 1; a represents p-6-g- Fcr-His Schematic diagram of recombinant plasmid construction; b represents p-6-g- Fcu-His Schematic diagram of recombinant plasmid construction; c represents p-6-g- Ff-His Schematic diagram of recombinant plasmid construction;

[0019] Figure 2This is a graph showing the results of double enzyme digestion identification in Example 1; M is the DL15000 Marker; lane 1 is p-6-g- Fcr- His Lane 2 is p-6-g- Fcu-His Lane 3 is p-6-g- Ff-His ;

[0020] Figure 3 This is a graph showing the PCR identification results of bacterial culture in Example 1; M is the DL5000 Marker; lane 0 is the blank control; 01 + 02 + and 03 + Lanes 1, 2, and 3 represent the plasmid controls for each plasmid; lanes 1, 2, and 3 represent Agrobacterium-mediated transformation p-6-g. -Fcr-His p-6-g- Fcu-His and p-6-g- Ff-His ;

[0021] Figure 4 This is a graph showing the genomic PCR identification results of the recombinant strain in Example 1; M is the DL5000 Marker; lane 0 is the blank control; 01 - and 02 - Negative controls for the starting strains were distributed in each lane; 01 + 02 + 03 + and 04 + Each lane contains a plasmid positive control; lane 1 is PFFS; lane 2 is PRFS; lane 3 is PUFS; lane 4 is PFT.

[0022] Figure 5 This is a graph showing the Western blot results of intracellular protein detection in the recombinant strain in Example 1; M represents Protein Marker; 01 - Lane 1 is designated Fs; Lane 2 is designated PFFS; Lane 3 is designated PUFS;

[0023] Figure 6 The graph shows the effect of fermentation time on the degradation of DON by the recombinant strain in Example 2.

[0024] Figure 7 This is a graph showing the effect of degradation temperature on the degradation of DON by the recombinant strain in Example 2;

[0025] Figure 8 This is a graph showing the effect of degradation pH on the degradation of DON by the recombinant strain in Example 2;

[0026] Figure 9 This is a graph showing the effect of degradation time on the degradation of DON by the recombinant strain in Example 2;

[0027] Figure 10 The figure shows the DON degradation detection results of the recombinant strain fermentation suspension in Example 2. All data in the figure are expressed as mean ± standard deviation. Different letters indicate significant differences in degradation rates between strain samples. p <0.05. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0029] The starting strain used in this invention to prepare Aspergillus niger strain Fs was purchased from Zhaodong Richeng Enzyme Preparation Co., Ltd., with the accession number Aspergillus niger CICC2462 and the laboratory name Aspergillus niger TH-2.

[0030] Example 1: Preparation and identification of a recombinant Aspergillus niger strain that degrades vomitoxin

[0031] 1. Preparation and identification of recombinant plasmids

[0032] The gene fragment of vomitin acetyltransferase Ff1TRI201 (The nucleotide sequence is shown in SEQ ID NO.5) Fcu2TRI101 (The nucleotide sequence is shown in SEQ ID NO.6) and Fcr2TRI101 (The nucleotide sequence is shown in SEQ ID NO.7) was ligated with the p-6-g vector fragment and the His histidine tag to obtain the recombinant plasmid, which was named p-6-g- Ff-His p-6-g- Fcu-Hi s and p-6-g- Fcr-His ,like Figure 1 As shown.

[0033] After plasmid extraction from E. coli transformants, the three recombinant plasmids obtained above were identified by double restriction endonuclease digestion with Nhe I and Hind III. The plasmid extraction steps for the E. coli transformants were as follows: (1) Collect bacterial cells: Take 1.5 mL of overnight culture, centrifuge (12,000 rpm, 1 min), and discard the supernatant; (2) Resuspend: Add 100 μL Solution I (containing RNase A), and resuspend the precipitate by pipetting; (3) Lysis: Add 200 μL Solution II, immediately and gently invert to mix for 5-10 seconds (the solution becomes clear and viscous), and incubate on ice for 2-5 min; (4) Neutralize: Add 150 μL Solution III, immediately and gently invert to mix for 10 seconds (white flocculent matter appears), and incubate on ice for 5-10 min; (5) Precipitate DNA: Centrifuge (12,000 rpm, 1 min) and resuspend the precipitate by pipetting (12,000 rpm, 1 min), and discard the supernatant; (6) Resuspend DNA: Add 100 μL Solution I (containing RNase A), and resuspend the precipitate by pipetting (12,000 rpm, 1 min), and incubate on ice for 5-10 min; (7) Precipitate DNA: Centrifuge (12,000 rpm, 1 min), and resuspend the precipitate by pipetting (12,000 rpm, 1 min), and discard the supernatant ... (6) Washing: Centrifuge (12,000 rpm, 10 min, 4℃), discard the supernatant; add 500 μL 70% ethanol, centrifuge (12,000 rpm, 5 min, 4℃), discard the supernatant, invert and air dry for 5-10 min (do not dry completely); add 30-50 μL TE buffer or sterile water to dissolve.

[0034] like Figure 2 As shown, the enzyme digestion results presented two DNA bands as expected, preliminarily confirming p-6-g- Ff-His p-6-g- Fcu-His and p-6-g- Fcr-His All recombinant plasmids were successfully constructed. Sequencing and alignment of the three recombinant plasmids were performed using vector-specific primers P1 (nucleotide sequence shown in SEQ ID NO.1) and P2 (nucleotide sequence shown in SEQ ID NO.2). Sequence alignment analysis showed that the inserted gene sequences were correct, further confirming the successful construction of the three recombinant plasmids.

[0035] 2. Agrobacterium-mediated transformation and identification of recombinant plasmids

[0036] The above p-6-g- was prepared using a freeze-thaw method. Ff-His p-6-g- Fcu-His and p-6-g- Fcr-His Recombinant plasmids were introduced separately AGL1 Competent cells were used to obtain Agrobacterium transformants. These transformants were then plated on LB+Rif+Kan plates and cultured at 28°C for 48 h to screen for single clones, yielding p-6-g- Ff-His p-6-g- Fcu-His and p-6-g- Fcr- His Agrobacterium-mediated transformation.

[0037] Using the above three Agrobacterium transformant colonies as PCR templates, PCR verification was performed using glaA homologous arm primers P1 and P2, and glaA514 homologous arm primers P3 (nucleotide sequence shown in SEQ ID NO.3) and P2.

[0038] PCR identification results as follows Figure 3 As shown, the transformant band is consistent with the positive control, indicating that p-6-g- Ff-His p-6-g- Fcu-His and p-6-g- Fcr-His The recombinant plasmid was successfully transformed into Agrobacterium. AGL1 In the middle, p-6-g- was successfully obtained Ff-His p-6-g- Fcu-His and p-6-g- Fcr-His Agrobacterium-mediated transformation.

[0039] 3. Preparation and Identification of Recombinant Aspergillus niger Strains

[0040] (1) Preparation of Aspergillus niger strain Fs:

[0041] a) FsTRI12 Preparation of recombinant plasmids

[0042] The nucleotide sequence is shown in SEQ ID NO.8 FsTRI12 The gene was sent to GenScript Biotech Co., Ltd. for codon optimization and synthesis to obtain the target gene recovery product. Recombinant vector construction was performed using a T4 DNA ligase catalytic system with the following specific parameters: 4.5 μL of linearized vector fragment, 3 μL of target gene recovery product, 2 μL of 5× ligation reaction buffer, and 0.5 μL of T4 DNA ligase. The reaction volume was brought to 10 μL and ligation was performed at 16℃ for 12 h. The ligation product was purified by ethanol precipitation to obtain... FsTRI12 Recombinant plasmid.

[0043] b) Preparation of Agrobacterium competent cells

[0044] 200 μL of AGL1 cells preserved in glycerol were inoculated into 10 mL of YEB medium containing Rif (50 μg / mL) and cultured with shaking at 28℃ and 200 rpm for 16–18 h. The cells were then transferred to fresh YEB medium containing Rif (50 μg / mL) at a 1:25 (v / v) inoculation rate and cultured with shaking at 28℃ and 200 rpm for 3.5 h. 1.5 mL of the bacterial culture was transferred to a pre-chilled centrifuge tube, incubated at 4℃ on ice for 30 min, centrifuged at 8000 rpm for 8 min, and the supernatant was discarded, retaining the bacterial pellet. 1 mL of pre-chilled 50 mM CaCl2 solution (0.278 g CaCl2 to 100 mL) was added to the bacterial pellet for gentle resuspending, and the pellet was stored at 4℃. Finally, the bacterial pellet was resuspended in 200 μL of pre-chilled 20 mM CaCl2 solution (0.111 g CaCl2 to 100 mL) for gentle resuspending to obtain competent cells, and stored at 4℃.

[0045] c) Freeze-thaw transformation of Agrobacterium tumefaciens

[0046] Add 1-5 μL of the solution obtained in a) to the competent cells obtained in b). FsTRI12 Recombinant plasmid DNA was gently tapped to mix, and the mixture was incubated on ice for 30 min. The mixture was then incubated on ice at -4°C for 5 min, followed by freezing in liquid nitrogen for 8 min. Immediately after incubation, the mixture was placed in a 37°C water bath for 5 min, and 800 μL of YEB liquid medium was added. The mixture was then incubated at 28°C and 200 rpm with shaking for 2-4 h. 50 μL of the bacterial culture was spread onto LB agar plates containing 100 mg / L Kan and 50 mg / mL Rif. The plates were incubated upside down at 28°C for 2-3 days. Single colonies were observed to obtain Agrobacterium transformants.

[0047] d) Co-culture of Agrobacterium and Aspergillus niger TH-2

[0048] The Agrobacterium transformants obtained in c) were inoculated into YEB+Kan (100 μg / mL) medium and cultured with shaking at 28℃ and 200 rpm for 16-18 h. One mL of the first-activated Aspergillus niger TH-2 culture was transferred to YEB medium containing AS (200 μM) and Kan (100 μg / mL) and cultured for another 2-4 h. The third-day mycelium of Aspergillus niger TH-2 was collected, homogenized in sterile PDA, and allowed to stand for 10 min to obtain a Aspergillus niger TH-2 mycelial homogenate. 450 μL of the second-activated Agrobacterium niger culture was centrifuged to collect the precipitate, and then mixed with 600 μL of the supernatant from the above Aspergillus niger TH-2 mycelial homogenate. 50 μL of this mixture was spread onto a PDA plate (containing 200 μM AS) covered with cellophane. After co-culturing at 28℃ inverted for 48 h, the cellophane was transferred to a plate containing cefotaxime sodium (200 μg / mL) and hygromycin (400 μg / mL). The screening medium (μg / mL) was incubated at 30℃ for 24 h, the membrane was removed, and the culture continued for 5-8 days until resistant colonies appeared.

[0049] e) Screening of homologous recombinant strains

[0050] Select the target colonies from the resistant plates in d) and inoculate them into PDA liquid medium containing double antibiotics (cefotaxime sodium 200 μg / mL + hygromycin B 400 μg / mL); after incubation at 30℃ in a shaker for 3-5 days, when the mycelial biomass reaches the experimental requirements, collect the mature mycelia for genomic DNA extraction.

[0051] Using the extracted genome as a template, ddH2O (blank control), DNA from the starting strain (Aspergillus niger TH-2) (negative control) and... FsTRI12 The recombinant plasmid (positive control) was amplified by PCR using primers P1 (nucleotide sequence shown in SEQ ID NO.1) and P2 (nucleotide sequence shown in SEQ ID NO.2). The characteristic bands of the amplification products were analyzed by electrophoresis, and positive Aspergillus niger strains Fs were obtained by screening.

[0052] (2) Preparation of recombinant strains of Aspergillus niger

[0053] The p-6-g- obtained above Ff-His p-6-g- Fcu-His and p-6-g- Fcr-HisAgrobacterium transformants were co-cultured with the above-mentioned Aspergillus niger strain Fs (PDA+AS medium). The resistant colonies that grew after the transfection were passaged in PDA liquid medium for 3-5 days. The cells were collected by centrifugation and genomic DNA was extracted. PCR verification was performed using p-6-g homologous arm primers P4 (nucleotide sequence shown in SEQ ID NO.4) and P2. Genotypic recombinant Aspergillus niger strains were screened and named PFFS, PRFS, and PUFS, respectively.

[0054] The p-6-g- obtained above Ff-His The recombinant plasmid was co-cultured with Aspergillus niger TH-2 (PDA+AS medium). The resistant colonies that grew after transfection were passaged in PDA liquid medium for 3-5 days. The cells were collected by centrifugation, and genomic DNA was extracted. PCR verification was performed using p-6-g homologous primers P4 (nucleotide sequence shown in SEQ ID NO.4) and P2 to screen for separately expressed plasmids. Ff1TRI201 The control strain (with the nucleotide sequence shown in SEQ ID NO.5) was named PFT.

[0055] PCR verification, as follows Figure 4 As shown, when the amplification products of the experimental group show a complete match with the positive control group, it indicates that the genotype homozygous recombinant strains of Aspergillus niger (PFFS, PRFS, PUFS) and the control strain (PFT) have been successfully screened.

[0056] The target protein levels in the cell lysates of the PFFS, PRFS, and PUFS recombinant strains obtained above were detected. The bacterial cells were broken up using a chemical lysis method. The fermentation supernatant of the three homozygous recombinant strains PFFS, PRFS, and PUFS with His tags, as well as the starting strain (Aspergillus niger strain Fs) after 10 days of fermentation treatment, was analyzed by Western blot.

[0057] The results are as follows Figure 5 As shown, the target protein was successfully detected in the cell lysates of the PFFS, PRFS, and PUFS recombinant strains.

[0058] Example 2: Application of recombinant Aspergillus niger strains in the degradation of vomitoxin

[0059] The PFFS, PRFS, PUFS, and PFT recombinant strains obtained in Example 1 were fermented for 8 days to obtain Aspergillus niger recombinant strain fermentation suspensions. The above fermentation suspensions were added to the vomitoxin substrate to be degraded for degradation treatment. The degradation treatment was carried out at a temperature of 25-35℃, a pH of 5-6, and a time of 24 h.

[0060] 1. Optimization of methods for degrading vomitoxin (DON) by recombinant strains

[0061] (1) Effect of fermentation time on the degradation of DON by recombinant strains

[0062] The DON substrate concentration was set at 8 ppm, and the degradation capacity of the recombinant strain fermentation suspension was evaluated by the reduction of DON substrate in the reaction system.

[0063] The specific experimental steps are as follows:

[0064] (a) Preparation of bacterial suspension: Take 1 mL of the fermentation broth of the recombinant strain from day 5 to day 10, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the centrifuged bacterial cells 3 times with PBS buffer, and resuspend to the original bacterial volume to obtain the bacterial suspension.

[0065] (b) Set the total volume of the DON degradation reaction to 2 mL, which includes 1 mL of bacterial suspension and 160 μL of 100% DON.

[0066] μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer;

[0067] (c) After incubating the reaction system in a constant temperature water bath at 30℃ for 24 h, the enzyme was inactivated by boiling at 100℃ for 10 min to terminate the reaction. Then, the solid-liquid separation was performed by centrifugation at 12000 rpm for 10 min at room temperature. The supernatant was collected and then filtered through a 0.22 μm filter membrane. The filtered sample was then subjected to liquid chromatography analysis, and the liquid chromatography method was the same as that for the fermentation supernatant.

[0068] The optimal fermentation time for the DON-degrading recombinant strain was determined as follows: Figure 6 As shown, the DON degradation efficiency of recombinant strains PFFS, PRFS, and PUFS showed a significant increasing trend during the first 8 days of fermentation. On day 8, the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS reached their highest levels, at 65.85%, 56.83%, and 69.30%, respectively. After 8 days of fermentation, the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS showed different decreasing trends. This indicates that obtaining the recombinant strain fermentation suspension at day 8 of fermentation yielded the best DON degradation effect.

[0069] (2) Effect of degradation temperature on the degradation of DON by recombinant strains

[0070] The DON substrate concentration was set at 8 ppm, and the degradation capacity of the recombinant strain fermentation suspension was evaluated by the reduction of DON substrate in the reaction system.

[0071] The specific experimental steps are as follows:

[0072] (a) Preparation of bacterial suspension: Take 1 mL of the fermentation broth of the recombinant strain on day 8, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the centrifuged bacterial cells 3 times with PBS buffer, and resuspend to the original bacterial volume to obtain the bacterial suspension.

[0073] (b) Set the total volume of the DON degradation reaction to 2 mL, which includes 1 mL of bacterial suspension and 160 μL of 100% DON.

[0074] μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer;

[0075] (c) The pH of the reaction system was set to 6.0. After being bathed in water at 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃ for 24 h, the enzyme was inactivated and the reaction was terminated by boiling at 100℃ for 10 min. Then, the solid and liquid were separated by centrifugation at 12000 rpm for 10 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered sample was then subjected to liquid chromatography analysis using the same method as that used for the fermentation supernatant.

[0076] The results of the study on the optimal degradation temperature of the fermentation suspension of recombinant strains PFFS, PRFS and PUFS are as follows: Figure 7 As shown, when the degradation temperature is 30℃, the DON degradation efficiencies of recombinant strains PFFS, PRFS, and PUFS reach their highest levels of 72.86%, 50.82%, and 63.2%, respectively. When the temperature is 25℃, the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS all show varying degrees of decrease, indicating that the optimal degradation temperature for the fermentation suspension of these three strains is within the range of 25-30℃. When the temperature is 50℃, the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS can still reach 44.31%, 30.33%, and 11.16%, respectively. Among them, recombinant strain PFFS exhibits the strongest thermostability, while PUFS exhibits the weakest thermostability, indicating intracellular co-expression. FsTRI12 and Ff1TRI201 The Aspergillus niger strain Fs exhibits a certain degree of heat resistance when degrading DON in the form of bacterial suspension. It is evident that the optimal degradation temperature for the fermentation suspensions of recombinant strains PFFS, PRFS, and PUFS is 25-35℃.

[0077] (3) Effect of degradation pH on the degradation of DON by recombinant strains

[0078] The DON substrate concentration was set at 8 ppm, and the degradation capacity of the recombinant strain fermentation suspension was evaluated by the reduction of DON substrate in the reaction system.

[0079] The specific experimental steps are as follows:

[0080] (a) Preparation of bacterial suspension: Take 1 mL of the fermentation broth of the recombinant strain on day 8, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the centrifuged bacterial cells 3 times with PBS buffer, and resuspend to the original bacterial volume to obtain the bacterial suspension.

[0081] (b) Set the total volume of the DON degradation reaction to 2 mL, which includes 1 mL of bacterial suspension and 160 μL of 100% DON.

[0082] μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer;

[0083] (c) The pH of the reaction system was set to 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. After being in a water bath at 30°C for 24 h, the enzyme was inactivated and the reaction was terminated by boiling at 100°C for 10 min. Then, the solid and liquid were separated by centrifugation at 12000 rpm for 10 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered sample was then subjected to liquid chromatography analysis, and the liquid chromatography method was the same as that for the fermentation supernatant.

[0084] The optimal degradation pH of the fermentation suspensions of recombinant strains PFFS, PRFS, and PUFS is as follows: Figure 8 As shown, the optimal degradation pH for recombinant strains PRFS and PUFS is 5.0, with degradation rates reaching up to 59.82% and 73.20%, respectively. The optimal degradation pH for recombinant strain PFFS was 6.0, with a maximum degradation rate of 71.01%. At pH 3.0, the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS were 11.19%, 14.87%, and 15.79%, respectively. Among them, PUFS showed stronger acid resistance than recombinant strains PFFS and PRFS, but acidic environments still significantly affected the DON degradation efficiency of the recombinant strains. At pH 8.0, the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS were 21.95%, 17.35%, and 24.51%, respectively. Among them, PUFS showed stronger alkali resistance than recombinant strains PFFS and PRFS, and all three strains exhibited better degradation efficiency in alkaline environments than in acidic environments. Compared to recombinant strain PRFS, recombinant strains PFFS and PUFS showed degradation efficiency of 21.95% and 24.51%, respectively, indicating some alkali resistance. It is evident that the optimal degradation pH for the recombinant strains PFFS, PRFS, and PUFS fermentation suspension is 5-6.

[0085] (4) Effect of degradation reaction time on the degradation of DON by recombinant strains

[0086] The DON substrate concentration was set at 8 ppm, and the degradation capacity of the recombinant strain fermentation suspension was evaluated by the reduction of DON substrate in the reaction system.

[0087] The specific experimental steps are as follows:

[0088] (a) Preparation of bacterial suspension: Take 1 mL of the fermentation broth of the recombinant strain on day 8, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the centrifuged bacterial cells 3 times with PBS buffer, and resuspend to the original bacterial volume to obtain the bacterial suspension.

[0089] (b) Set the total volume of the DON degradation reaction to 2 mL, which includes 1 mL of bacterial suspension and 160 μL of 100% DON.

[0090] μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer;

[0091] (c) The pH of the reaction system was set to 6.0. The reaction was terminated by water bath at 30°C for 0 h, 12 h, 24 h and 36 h, followed by boiling at 100°C for 10 min to inactivate the enzyme. Then, the solid-liquid separation was performed by centrifugation at 12000 rpm for 10 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered sample was then subjected to liquid chromatography analysis using the same method as that used for the fermentation supernatant.

[0092] The optimal degradation times for recombinant strains PFFS, PRFS, and PUFS were determined under the optimal degradation temperature (30℃) and pH (5-6) conditions. Figure 9 As shown in the figure, the DON degradation efficiency of recombinant strains PFFS, PRFS, and PUFS all showed an increasing trend within 24 h of the degradation reaction. At 24 h, the highest DON degradation efficiencies of recombinant strains PFFS, PRFS, and PUFS were 72.37%, 60.93%, and 74.51%, respectively, with recombinant strain PUFS showing the best degradation effect. After 24 h, the DON degradation efficiency of recombinant strains PFFS, PRFS, and PUFS tended to stabilize. Therefore, the optimal degradation time for the fermentation suspensions of recombinant strains PFFS, PRFS, and PUFS was 24 h.

[0093] 2. Detection of the degradation ability of recombinant strains against devotoxin (DON)

[0094] The DON substrate concentration was set at 8 ppm, and the degradation capacity of the recombinant strain fermentation suspension was evaluated by detecting the reduction of DON substrate in the reaction system. Fermentation suspensions of the PFFS, PRFS, and PUFS recombinant strains obtained in Example 1, as well as the starting strain (Aspergillus niger strain Fs) and the PFT recombinant strain (control group), were used to determine the DON degradation rate under the same conditions.

[0095] The specific experimental steps are as follows:

[0096] (a) Preparation of bacterial suspension: Take 1 mL of the fermentation broth of the recombinant strain on day 8, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the centrifuged bacterial cells 3 times with PBS buffer, and resuspend to the original bacterial volume to obtain the bacterial suspension.

[0097] (b) Set the total volume of the DON degradation reaction to 2 mL, which includes 1 mL of bacterial suspension and 160 μL of 100% DON.

[0098] μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer;

[0099] (c) The pH of the reaction system was set to 6.0, and the reaction was terminated by water bath at 30°C for 24 h and boiling at 100°C for 10 min to inactivate the enzyme. Then, the solid-liquid separation was performed by centrifugation at 12000 rpm for 10 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered sample was then subjected to liquid chromatography analysis, and the liquid chromatography method was the same as that for the fermentation supernatant.

[0100] The results are as follows Figure 10 As shown, the DON degradation rates of recombinant strain PFT and Aspergillus niger strain Fs were 9.06% and 6.50%, respectively, while the DON degradation rates of recombinant strains PFFS, PRFS, and PUFS were 63.92%, 59.15%, and 70.88%, respectively.

[0101] Compared to non-co-expression FsTRI12 The recombinant strain PFT showed a 7.06-fold increase in DON degradation rate, indicating that the DON acetyltransferase and DON degradation rate were significantly improved in the recombinant Aspergillus niger strain provided by this invention. FsTRI12 The synergistic effect can effectively improve the degradation efficiency of DON by the fermentation suspension.

[0102] Compared to the original strain (Aspergillus niger strain Fs), the recombinant strain PFFS showed a 9.83-fold increase in DON degradation rate, indicating that the DON acetyltransferase gene... Ff1TRI201、Fcu2TRI101 and Fcr2TRI101 The recombinant strain of Aspergillus niger provided in this invention was successfully recombinantly expressed and exhibited high degradation activity.

[0103] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A type of Aspergillus niger that degrades vomitoxin ( Aspergillus niger The recombinant strain is characterized by, The preparation method of the recombinant Aspergillus niger strain is as follows: the vomitoxin acetyltransferase gene fragment is ligated with the p-6-g vector fragment to obtain a recombinant plasmid; the recombinant plasmid is introduced into AGL1 competent cells by freeze-thaw method to obtain Agrobacterium tumefaciens transformants; the Agrobacterium tumefaciens transformants are co-cultured with Aspergillus niger strain Fs, transferred to a membrane, passaged for 3-5 days, and screened to obtain genotype homozygous Aspergillus niger recombinant strains; The Aspergillus niger strain Fs is co-expressed FsTRI12 Genetically modified Aspergillus niger strains; The FsTRI12 The gene nucleotide sequence is shown in SEQ ID NO. 8; The vomitoxin acetyltransferase gene fragment is Ff1TRI201、Fcu2TRI101 or Fcr2TRI101 Any one of them; the Ff1TRI201 The gene nucleotide sequence is shown in SEQ ID NO.

5. Fcu2TRI101 The gene nucleotide sequence is shown in SEQ ID NO.

6. Fcr2TRI101 The gene nucleotide sequence is shown in SEQ ID NO.

7.

2. The recombinant Aspergillus niger strain according to claim 1, characterized in that, The culture medium used for co-culture is PDA+AS medium.

3. The recombinant Aspergillus niger strain according to claim 1, characterized in that, The culture medium used for the subculture was PDA liquid medium.

4. The use of the recombinant Aspergillus niger strain according to any one of claims 1 to 3 in the degradation of vomitoxin.

5. A method for degrading vomitoxin, characterized in that, The method includes the following steps: fermenting the recombinant Aspergillus niger strain according to any one of claims 1 to 3 to obtain a fermentation suspension of the recombinant Aspergillus niger strain; adding the fermentation suspension to a vomitoxin substrate to be degraded for degradation treatment.

6. The method according to claim 5, characterized in that, The fermentation treatment lasted for 8 days, and the degradation treatment was carried out at a temperature of 25-35℃, a pH of 5-6, and a time of 24 hours.

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