Aspergillus niger recombinant strain for degrading vomitoxin and application thereof

Abstract

The invention discloses an aspergillus niger recombinant strain for degrading vomitoxin and application thereof, and belongs to the field of gene engineering. In order to solve the problem that a method for efficiently degrading vomitoxin is lacked in the prior art, the invention provides an aspergillus niger recombinant strain for degrading vomitoxin, the aspergillus niger strain for co-expressing FsTRI12 is taken as a recipient bacterium, a vomitoxin acetyltransferase gene segment is selected, an intracellular gene expression vector is constructed, and a homozygous aspergillus niger recombinant strain is screened. The aspergillus niger recombinant strain provided by the invention can be applied to degradation of vomitoxin, provides a new choice for effectively solving the vomitoxin pollution problem in cereal raw materials and feeds, reducing grain loss and guaranteeing food and feed safety, and has potential industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and particularly relates to a recombinant Aspergillus niger strain capable of degrading vomitoxin and an application thereof. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by fungi (such as Fusarium, Alternaria, Aspergillus, and Penicillium) when they infect plant products in the field or during storage. They include aflatoxins, zearalenone, ochratoxins, deoxynivalenol, and patulin. Among them, deoxynivalenol (DON), also known as deoxynivalenol and deoxynaphthotoxin, is widely found in cereals such as wheat, barley, and corn, and their products, making it the most widespread mycotoxin globally.

[0003] Currently, methods for degrading vomitoxin mainly include physical adsorption, chemical degradation, and biodegradation. While physical adsorption (e.g., using adsorbents) is simple to operate, it cannot completely remove the toxin. Chemical degradation (e.g., using oxidants) may produce harmful byproducts. Biodegradation (e.g., using microorganisms) is considered environmentally friendly and highly effective, but still faces the following challenges: Glycosylation: Glycosylation reduces DON's biological activity by transferring sugar molecules (such as glucose) to form glycoside compounds (such as DON-3-glucoside, D3G). Plant UDP-glucosyltransferases (UGTs) typically convert DON to DON-3-glucoside (D3G). While D3G is less toxic than DON, it can be reconverted back into DON under certain conditions (such as intestinal microbial activity), leading to a restoration of toxicity. Existing research on the interaction mechanism, catalytic sites, and reaction kinetics between UGTs and DON is insufficient, and a lack of in-depth understanding of the structure-function relationship of UGTs makes it difficult to improve their catalytic efficiency through genetic engineering.

[0004] De-epoxidation: De-epoxidation destroys the toxic core structure of DON by hydrolyzing the epoxy group (COC). Epoxide hydrolase (EH) may only partially hydrolyze the epoxy group, and the remaining epoxy fragment is still toxic. De-epoxidation may produce a variety of metabolites, the toxicity of which is not yet fully understood. For example, de-epoxidation of DON may produce DOM-1 (de-epoxy-DON), but DOM-1 may be further converted into derivatives of unknown toxicity under the action of intestinal microorganisms. Carbonylation: Carbonylation changes the chemical properties of DON by introducing a carbonyl (C=O) group. Carbonylation may generate more toxic intermediates. For example, during oxidative carbonylation, DON may be converted to DON-8-ketone, which is approximately 20% more toxic to hepatocytes than DON. Furthermore, oxidation reactions may generate free radicals, triggering chain reactions such as lipid peroxidation. Carbonylation reactions typically require precious metal catalysts (such as palladium and platinum). For example, the oxidative carbonylation of amines requires palladium catalysts, which are expensive and easily poisoned by sulfides, leading to catalyst deactivation.

[0005] Therefore, those skilled in the art are eager to develop a method for efficiently degrading vomitoxin to solve the problem of vomitoxin pollution and prevent harm to animal husbandry and human health. Summary of the Invention

[0006] The present invention aims to solve the problem of lack of a method for efficiently degrading vomitoxin in the prior art and provides a recombinant Aspergillus niger strain for degrading vomitoxin and its application.

[0007] One of the purposes of the present invention is to provide a method for degrading vomitoxin. Aspergillus niger ) recombinant strain, the preparation method of the Aspergillus niger recombinant strain is as follows: the vomitoxin acetyltransferase gene fragment is connected 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 transformants; the Agrobacterium transformants are co-cultured with the Aspergillus niger strain Fs, transferred to a membrane, and subcultured for 3-5 days to screen and obtain a genotype homozygous Aspergillus niger recombinant strain; the Aspergillus niger strain Fs is co-expressed FsTRI12 niger strains of genes.

[0008] Preferably, the DON acetyltransferase gene fragment comprises Ff1TRI201、Fcu2TRI101 or Fcr2TRI101 Any one of the Ff1TRI201 The nucleotide sequence of the gene is shown in SEQ ID NO.5. Fcu2TRI101 The nucleotide sequence of the gene is shown in SEQ ID NO.6. Fcr2TRI101 The nucleotide sequence of the gene is shown in SEQ ID NO.7.

[0009] Preferably, the culture medium used for the co-cultivation is PDA+AS culture medium.

[0010] Preferably, the culture medium used in the subculture is PDA liquid culture medium.

[0011] A second object of the present invention is to provide the use of the above-mentioned recombinant Aspergillus niger strain in degrading vomitoxin.

[0012] A third object of the present invention is to provide a method for degrading vomitoxin, comprising the following steps: fermenting the above-mentioned recombinant Aspergillus niger strain to obtain a fermentation bacterial suspension of the recombinant Aspergillus niger strain; and adding the above-mentioned fermentation bacterial suspension to a vomitoxin substrate to be degraded for degradation.

[0013] 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.

[0014] Compared with the prior art, the present invention has the following advantages: the present invention provides a recombinant strain of Aspergillus niger that degrades vomitoxin, by co-expressing FsTRI12 Aspergillus niger strain Fs with the gene was used as the starting strain, and the DON acetyltransferase gene was selected Ff1TRI201 、 Fcu2TRI101 or Fcr2TRI101 , construct an intracellular gene expression vector, and screen homozygous Aspergillus niger recombinant strains. The present invention provides a method for degrading vomitoxin using the above-mentioned Aspergillus niger recombinant strains. The fermentation bacterial suspensions of the three recombinant strains (PFFS, PUFS, PRFS) provided by the present invention are optimized for degradation conditions of vomitoxin, and the optimal fermentation time is determined to be 8 days, the degradation temperature is 25-35°C, the pH value is 5-6, and the time is 24 hours. Under the optimal reaction conditions, the degradation rates of vomitoxin of the three recombinant Aspergillus niger strains (PFFS, PUFS, PRFS) provided by the present invention are 72.37%, 60.93% and 74.51%, respectively; among them, the recombinant strain PFFS is significantly better than that of the strain expressed alone. FsTRI12 The control strain (PFT) increased the degradation rate of vomitoxin by 7.06 times, indicating that the recombinant Aspergillus niger strain provided by the present invention can be used to FsTRI12 It works synergistically with acetyltransferase to significantly improve the acetylation and detoxification efficiency of bacterial suspension.

[0015] The recombinant Aspergillus niger strain provided by the present 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 losses, and ensuring food and feed safety, and has potential industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the construction of the recombinant plasmid in Example 1; a is p-6-g- Fcr-His Schematic diagram of recombinant plasmid construction; b is p-6-g- Fcu-His Schematic diagram of recombinant plasmid construction; c is p-6-g- Ff-His Schematic diagram of recombinant plasmid construction; Figure 2The double enzyme digestion result diagram in Example 1; M is 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 ; Figure 3 The results of PCR identification of bacterial solution in Example 1 are shown; M is DL5000 Marker; Lane 0 is blank control; Lane 01 is blank control; Lane 02 is blank control; Lane 03 is blank control; Lane 04 is blank control; Lane 05 is blank control; Lane 06 is blank control; Lane 07 is blank control; Lane 08 is blank control; Lane 09 is blank control; Lane 01 + 、02 + and 03 + Lanes 1, 2, and 3 are the plasmid controls; lanes 1, 2, and 3 are the Agrobacterium transformant p-6-g -Fcr-His 、p-6-g- Fcu-His and p-6-g- Ff-His ; Figure 4 The results of PCR identification of the genome of the recombinant strain in Example 1 are shown; M is DL5000 Marker; Lane 0 is the blank control; Lane 01 is the blank control; - and 02 - Lanes 01 and 02 represent the negative control of the starting strain; + 、02 + 、03 + and 04 + The lanes are respectively the plasmid positive controls; lane 1 is PFFS; lane 2 is PRFS; lane 3 is PUFS; lane 4 is PFT; Figure 5 The Western-blot detection results of the intracellular proteins of the recombinant strain in Example 1; M is ProteinMarker; 01 - Lanes are Fs; Lane 1 is PFFS; Lane 2 is PRFS; Lane 3 is PUFS; Figure 6 This is a graph showing the effect of fermentation time on the degradation of DON by the recombinant strain in Example 2; Figure 7 This is a graph showing the effect of degradation temperature on the degradation of DON by the recombinant strain in Example 2; Figure 8 This is a graph showing the effect of degradation pH on DON degradation by the recombinant strain in Example 2; Figure 9 This is a graph showing the effect of degradation time on DON degradation by the recombinant strain in Example 2; Figure 10 The figure shows the results of the DON degradation test of the recombinant strain fermentation suspension in Example 2. The data in the figure are expressed as mean ± standard deviation. Different letters indicate significant differences in the degradation rates between the strain samples. p <0.05. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0018] The starting strain for preparing the Aspergillus niger strain Fs of the present invention is purchased from Zhaodong Richeng Enzyme Preparation Company, has a preservation number of Aspergillus niger CICC2462, and is named Aspergillus niger TH-2 in the laboratory.

[0019] Example 1: Preparation and identification of a recombinant strain of Aspergillus niger that degrades vomitoxin 1. Preparation and Identification of Recombinant Plasmids Deoxynivalenol acetyltransferase gene fragment Ff1TRI201 (The nucleotide sequence is shown in SEQ ID NO.5), Fcu2TRI101 (nucleotide sequence is shown in SEQ ID NO.6) and Fcr2TRI101 (nucleotide sequence is shown in SEQ ID NO.7) and connected with p-6-g vector fragment and His tag to obtain recombinant plasmids, which were named p-6-g- Ff-His 、p-6-g- Fcu-Hi s and p-6-g- Fcr-His ,like Figure 1 shown.

[0020] After plasmid extraction from E. coli transformants, the three recombinant plasmids obtained above were identified by double digestion with restriction endonucleases Nhe I and Hind III. The plasmid extraction steps of the E. coli transformants were as follows: (1) Collecting bacteria: Take 1.5 mL of overnight culture solution, centrifuge (12,000 rpm, 1 min), and discard the supernatant; (2) Resuspending: Add 100 μL Solution I (containing RNase A) and resuspend the precipitate by pipetting; (3) Lysing: Add 200 μL Solution II, immediately and gently invert and mix for 5-10 seconds (the solution becomes clear and viscous), and ice bath for 2-5 minutes; (4) Neutralizing: Add 150 μL Solution III, immediately and gently invert and mix for 10 seconds (white flocs appear), and ice bath for 5-10 minutes; (5) Precipitating DNA: Centrifuge (12,000 rpm, 10 min), transfer the supernatant to a new tube; add 0.7 times the volume of room temperature anhydrous ethanol, mix well, and place at room temperature or -20℃ for 10-30 min; (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, turn upside down and dry for 5-10 min (do not dry completely); add 30-50 μL TE buffer or sterile water to dissolve.

[0021] like Figure 2 As shown, the enzyme digestion results showed two DNA bands consistent with the expectations, preliminarily confirming that 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 using vector-specific primers P1 (nucleotide sequence shown in SEQ ID NO. 1) and P2 (nucleotide sequence shown in SEQ ID NO. 2) revealed that the inserted gene sequences were correct, further confirming the successful construction of the three recombinant plasmids.

[0022] 2. Agrobacterium Transformation and Identification of Recombinant Plasmids The above p-6-g- Ff-His 、p-6-g- Fcu-His and p-6-g- Fcr-His The recombinant plasmids were introduced into AGL1 Competent cells were used to obtain Agrobacterium transformants; the Agrobacterium transformants were spread on LB+Rif+Kan plates and cultured at 28°C for 48 h to screen single clones, and p-6-g- Ff-His 、p-6-g- Fcu-His and p-6-g- Fcr- HisAgrobacterium transformants.

[0023] The above three Agrobacterium transformant colonies were used as PCR templates, and PCR verification was performed using glaA homology arm primers P1 and P2 and glaA514 homology arm primers P3 (nucleotide sequence shown in SEQ ID NO. 3) and P2.

[0024] PCR identification results Figure 3 As shown, the transformant bands were 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, successfully obtained p-6-g- Ff-His 、p-6-g- Fcu-His and p-6-g- Fcr-His Agrobacterium transformants.

[0025] 3. Preparation and Identification of Recombinant Aspergillus niger Strains (1) Preparation of Aspergillus niger strain Fs: a) FsTRI12 Preparation of recombinant plasmid 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, and the target gene recovery product was obtained. The recombinant vector was constructed using the T4 DNA ligase catalytic system with the following configuration 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 were added. The reaction system was supplemented to 10 μL and the ligation reaction was carried out at 16°C for 12 h. The ligation product was purified by ethanol precipitation to obtain FsTRI12 Recombinant plasmid.

[0026] b) Preparation of competent Agrobacterium 200 μL of glycerol-stored AGL1 was inoculated into 10 mL of YEB medium containing Rif (50 μg / mL) and cultured with shaking at 28°C and 200 rpm for 16-18 h; the inoculum was transferred to fresh YEB medium containing Rif (50 μg / mL) at a 1:25 (v / v) inoculum ratio and cultured with shaking at 28°C and 200 rpm for 3.5 h; 1.5 mL of the above bacterial solution was transferred to a pre-cooled centrifuge tube, ice-bathed at 4°C for 30 min, centrifuged at 8000 rpm for 8 min, and the supernatant was discarded to retain the bacterial pellet; 1 mL of pre-cooled 50 mM CaCl2 solution (0.278 g CaCl2 to 100 mL) was added to the above bacteria and gently resuspended, and stored at 4°C; the above bacteria were resuspended in 200 μL of pre-cooled 20 mM CaCl2 solution (0.111 g CaCl2 to 100 mL), gently resuspended to obtain competent cells, and stored at 4°C.

[0027] c) Freeze-thaw transformation of Agrobacterium Add 1-5 μL of the culture medium obtained in a) to the competent cells obtained in b). FsTRI12 Recombinant plasmid DNA was mixed by gently tapping the tube wall and placed on ice for 30 min; the mixture was placed on ice at -4°C for 5 min, then immersed in liquid nitrogen and frozen for 8 min; immediately transferred to a 37°C water bath for 5 min, 800 μL of YEB liquid medium was added, and cultured at 28°C and 200 rpm with shaking for 2-4 h; 50 μL of the above bacterial solution was spread on an LB solid plate containing 100 mg / L Kan and 50 mg / mL Rif, and cultured inverted at 28°C for 2-3 d. Single colonies were observed to obtain Agrobacterium transformants.

[0028] d) Co-cultivation of Agrobacterium and Aspergillus niger TH-2 The Agrobacterium transformants obtained in c) were inoculated into YEB+Kan (100 μg / mL) medium and cultured with shaking at 28°C and 200 rpm for 16-18 h. 1 mL of the 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 taken and ground into a sterile PDA homogenate. The mixture was allowed to stand for 10 min to obtain an Aspergillus niger TH-2 mycelial homogenate. 450 μL of the second-activated Agrobacterium culture was centrifuged to collect the precipitate, which was then mixed with 600 μL of the supernatant of the Aspergillus niger TH-2 mycelial homogenate. 50 μL of the supernatant was spread on a PDA plate covered with cellophane (containing 200 μM AS). After inverted co-culture at 28°C for 48 h, the cellophane was transferred to a plate containing cefuroxime sodium (200 μg / mL) and hygromycin (400 μg / mL). μg / mL) screening medium was cultured at 30°C for 24 h, the film was removed, and the culture was continued for 5-8 days until resistant colonies appeared.

[0029] e) Screening of homologous recombination strains Select target colonies from the medium-resistant plate in (d) and inoculate them into PDA liquid culture medium containing dual antibiotics (cefotaxime sodium 200 μg / mL + hygromycin B 400 μg / mL). After incubation on a 30°C constant temperature shaker for 3-5 days, when the mycelial biomass reaches the experimental requirement, collect mature mycelia for genomic DNA extraction.

[0030] Using the extracted genome as a template, ddH2O (blank control), starting strain (Aspergillus niger TH-2) DNA (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 amplified products were analyzed by electrophoresis to screen and obtain the positive Aspergillus niger strain Fs.

[0031] (2) Preparation of recombinant Aspergillus niger strains The p-6-g- Ff-His 、p-6-g- Fcu-His and p-6-g- Fcr-His The Agrobacterium transformants were co-cultured with the above-mentioned Aspergillus niger strain Fs (PDA+AS medium). The resistant colonies grown after the above-mentioned transfer were subcultured in PDA liquid medium for 3-5 days, and the bacteria were collected by centrifugation and genomic DNA was extracted. PCR verification was performed using p-6-g homology arm primers P4 (nucleotide sequence shown in SEQ ID NO.4) and P2, and genotypic homozygous Aspergillus niger recombinant strains were screened and named PFFS, PRFS, and PUFS, respectively.

[0032] The p-6-g- Ff-His The recombinant plasmid was co-cultured with Aspergillus niger TH-2 (PDA+AS medium); the resistant colonies grown after the above transfer were subcultured in PDA liquid medium for 3-5 days, and the bacteria were collected by centrifugation and genomic DNA was extracted. PCR verification was performed using p-6-g homology arm primers P4 (nucleotide sequence shown in SEQ ID NO.4) and P2 to screen for single expression. Ff1TRI201 The control strain (nucleotide sequence is shown in SEQ ID NO.5) was named PFT.

[0033] PCR verification Figure 4 As shown, when the amplified products of the experimental group showed a complete match with the positive control group, it indicated that the genotype homozygous Aspergillus niger recombinant strains (PFFS, PRFS, PUFS) and the control strain (PFT) were successfully screened.

[0034] The target protein levels in the cell lysates of the PFFS, PRFS, and PUFS recombinant strains obtained above were detected. The bacterial cells were disrupted by chemical lysis, and the fermentation supernatants of the three homozygous recombinant strains PFFS, PRFS, and PUFS with His tags and the starting strain (Aspergillus niger strain Fs) after 10 days of fermentation were used for Western-blot analysis.

[0035] 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.

[0036] Example 2: Application of recombinant strains of Aspergillus niger in degrading vomitoxin The PFFS, PRFS, PUFS, and PFT recombinant strains obtained in Example 1 were fermented for 8 days to obtain fermentation suspensions of the recombinant Aspergillus niger strain; the above fermentation suspensions were added to the vomitoxin substrate to be degraded for degradation treatment, and the degradation treatment temperature was 25-35°C, the pH value was 5-6, and the time was 24 hours.

[0037] 1. Optimization of the method for degrading vomitoxin (DON) by recombinant strains (1) Effect of fermentation time on DON degradation by recombinant strains The DON substrate concentration was set at 8 ppm, and the degradation ability of the recombinant strain fermentation suspension was evaluated by reducing the amount of DON substrate in the reaction system.

[0038] The specific experimental steps are: (a) Preparation of bacterial suspension: Take 1 mL of the fermentation broth of the recombinant strain from day 5 to 10, centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash the centrifuged bacteria three times with PBS buffer, and resuspend to the original volume of the bacterial suspension. (b) The total volume of the DON degradation reaction was set to 2 mL, which contained 1 mL of bacterial suspension, 160 μL of 100% μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer; (c) The reaction system was incubated in a constant temperature water bath at 30°C for 24 h and then boiled at 100°C for 10 min to inactivate the enzyme and terminate the reaction. The solid-liquid separation was then performed by centrifugation at 12,000 rpm for 10 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered sample was taken for liquid chromatography-mass spectrometry analysis, and the liquid chromatography method was consistent with that of the fermentation supernatant.

[0039] The results of the optimal fermentation time test for the DON degradation recombinant strain are as follows Figure 6 As shown, during the first eight days of fermentation, the DON degradation efficiency of the recombinant strains PFFS, PRFS, and PUFS showed a significant upward trend. On the eighth day of fermentation, the DON degradation rates of the recombinant strains PFFS, PRFS, and PUFS reached their highest levels, at 65.85%, 56.83%, and 69.30%, respectively. After eight days of fermentation, the DON degradation rates of the recombinant strains PFFS, PRFS, and PUFS decreased in different trends. This indicates that the recombinant strains PFFS, PRFS, and PUFS had the best DON degradation efficiency when obtained on the eighth day of fermentation.

[0040] (2) Effect of degradation temperature on DON degradation by recombinant strains The DON substrate concentration was set at 8 ppm, and the degradation ability of the recombinant strain fermentation suspension was evaluated by reducing the amount of DON substrate in the reaction system.

[0041] The specific experimental steps are: (a) Preparation of bacterial suspension: Take 1 mL of the 8-day fermentation broth of the recombinant strain and centrifuge at 12,000 rpm for 10 min. Discard the supernatant, wash the centrifuged cells three times with PBS buffer, and resuspend them to the original volume of the bacterial suspension. (b) The total volume of the DON degradation reaction was set to 2 mL, which contained 1 mL of bacterial suspension, 160 μL of 100% μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer; (c) The reaction system was set to pH 6.0. After incubation at 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C for 24 h in a water bath, the reaction was terminated by boiling at 100°C for 10 min to inactivate the enzyme. The mixture was then centrifuged at 12,000 rpm for 10 min at room temperature for solid-liquid separation. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered samples were subjected to liquid chromatography-mass spectrometry (LC-MS) analysis, and the results were consistent with those of the fermentation supernatant.

[0042] 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 in the figure, when the degradation temperature was 30℃, the DON degradation efficiency of the recombinant strains PFFS, PRFS and PUFS reached the highest level of 72.86%, 50.82% and 63.2% respectively; when the temperature was 25℃, the DON degradation rates of the recombinant strains PFFS, PRFS and PUFS all decreased to varying degrees, indicating that the optimal degradation temperature of the fermentation suspension of these three strains was in the range of 25-30℃; when the temperature was 50℃, the DON degradation rates of the recombinant strains PFFS, PRFS and PUFS could still reach 44.31%, 30.33% and 11.16% respectively. Among them, the recombinant strain PFFS had the strongest high temperature resistance, while PUFS had the worst high temperature resistance, indicating that the intracellular co-expression FsTRI12 and Ff1TRI201 The Aspergillus niger strain Fs showed a certain degree of high temperature resistance when degrading DON in the form of bacterial suspension. Therefore, the optimal degradation temperature of the recombinant strains PFFS, PRFS, and PUFS fermentation suspension was 25-35℃.

[0043] (3) Effect of degradation pH on DON degradation by recombinant strains The DON substrate concentration was set at 8 ppm, and the degradation ability of the recombinant strain fermentation suspension was evaluated by reducing the amount of DON substrate in the reaction system.

[0044] The specific experimental steps are: (a) Preparation of bacterial suspension: Take 1 mL of the 8-day fermentation broth of the recombinant strain and centrifuge at 12,000 rpm for 10 min. Discard the supernatant, wash the centrifuged cells three times with PBS buffer, and resuspend them to the original volume of the bacterial suspension. (b) The total volume of the DON degradation reaction was set to 2 mL, which contained 1 mL of bacterial suspension, 160 μL of 100% μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer; (c) The reaction system pH was set to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. After incubation in a water bath at 30°C for 24 h, the reaction was terminated by boiling at 100°C for 10 min to inactivate the enzyme. The mixture was then centrifuged at 12,000 rpm for 10 min at room temperature for solid-liquid separation. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered samples were taken for liquid chromatography-mass spectrometry (LC-MS / MS) analysis, and the results were consistent with those of the fermentation supernatant.

[0045] The results of the study on the optimal degradation pH of the fermentation suspension of recombinant strains PFFS, PRFS and PUFS are as follows: Figure 8 As shown, the optimal degradation pH for the recombinant strains PRFS and PUFS was 5.0, and the degradation rates could reach up to 59.82% and 73.20% respectively. The optimal degradation pH for the recombinant strain PFFS was 6.0, with a maximum degradation rate of 71.01%. At pH 3.0, the DON degradation rates of the recombinant strains PFFS, PRFS, and PUFS were 11.19%, 14.87%, and 15.79%, respectively. PUFS had stronger acid resistance than the recombinant strains PFFS and PRFS, but the acidic environment still had a significant impact on the DON degradation efficiency of the recombinant strains. At pH 8.0, the DON degradation rates of the recombinant strains PFFS, PRFS, and PUFS were 21.95%, 17.35%, and 24.51%, respectively. PUFS had stronger alkali resistance than the recombinant strains PFFS and PRFS, and the degradation efficiency of the three strains in alkaline environments was better than that in acidic environments. Compared with the recombinant strain PRFS, the degradation efficiency of the recombinant strains PFFS and PUFS was 21.95% and 24.51%, respectively, indicating a certain degree of alkali resistance. It can be seen that the optimal degradation pH of the fermentation suspension of the recombinant strains PFFS, PRFS and PUFS is 5-6.

[0046] (4) Effect of degradation reaction time on DON degradation by recombinant strains The DON substrate concentration was set at 8 ppm, and the degradation ability of the recombinant strain fermentation suspension was evaluated by reducing the amount of DON substrate in the reaction system.

[0047] The specific experimental steps are: (a) Preparation of bacterial suspension: Take 1 mL of the 8-day fermentation broth of the recombinant strain and centrifuge at 12,000 rpm for 10 min. Discard the supernatant, wash the centrifuged cells three times with PBS buffer, and resuspend them to the original volume of the bacterial suspension. (b) The total volume of the DON degradation reaction was set to 2 mL, which contained 1 mL of bacterial suspension, 160 μL of 100% μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer; (c) The pH of the reaction system was set to 6.0. The reaction was incubated in a water bath at 30°C for 0 h, 12 h, 24 h, and 36 h, and then boiled at 100°C for 10 min to inactivate the enzyme and terminate the reaction. The mixture was then centrifuged at 12,000 rpm for 10 min at room temperature for solid-liquid separation. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered samples were taken for liquid chromatography-mass spectrometry (LC-MS / MS) analysis, and the results were consistent with those for the fermentation supernatant.

[0048] Under the conditions of optimal degradation temperature (30℃) and pH (5-6), the results of the optimal degradation time of recombinant strains PFFS, PRFS and PUFS are as follows: Figure 9 As shown in the figure, within 24 hours of degradation, the DON degradation efficiencies of the recombinant strains PFFS, PRFS, and PUFS all showed an increasing trend. At 24 hours, the DON degradation efficiencies of the recombinant strains PFFS, PRFS, and PUFS reached their highest levels, reaching 72.37%, 60.93%, and 74.51%, respectively, with the recombinant strain PUFS showing the best degradation efficiency. After 24 hours of degradation, the DON degradation efficiencies of the recombinant strains PFFS, PRFS, and PUFS stabilized. Therefore, the optimal degradation time for the fermentation suspensions of the recombinant strains PFFS, PRFS, and PUFS is 24 hours.

[0049] 2. Degradation of DON by the Recombinant Strain The degradation capacity of the recombinant fermentation suspension was assessed by measuring the reduction in DON substrate in the reaction system at a DON substrate concentration of 8 ppm. DON degradation rates were determined under the same conditions using 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).

[0050] The specific experimental steps are: (a) Preparation of bacterial suspension: Take 1 mL of the 8-day fermentation broth of the recombinant strain and centrifuge at 12,000 rpm for 10 min. Discard the supernatant, wash the centrifuged cells three times with PBS buffer, and resuspend them to the original volume of the bacterial suspension. (b) The total volume of the DON degradation reaction was set to 2 mL, which contained 1 mL of bacterial suspension, 160 μL of 100% μg / mL DON stock solution (solvent: methanol) and 840 μL phosphate buffer; (c) The reaction system was set to pH 6.0, incubated in a water bath at 30°C for 24 h, and boiled at 100°C for 10 min to inactivate the enzyme and terminate the reaction. The solid-liquid separation was then performed by centrifugation at 12,000 rpm for 10 min at room temperature. The supernatant was collected and filtered through a 0.22 μm filter membrane. The filtered sample was taken for liquid chromatography-mass spectrometry (LC-MS / MS) analysis, and the liquid chromatography-mass spectrometry method was consistent with that for the fermentation supernatant.

[0051] The results are as follows Figure 10 As shown, the DON degradation rates of the recombinant strain PFT and Aspergillus niger strain Fs were 9.06% and 6.50%, and the DON degradation rates of the recombinant strains PFFS, PRFS, and PUFS were 63.92%, 59.15%, and 70.88%, respectively.

[0052] Compared with non-coexpressed FsTRI12 The DON degradation rate of the recombinant strain PFT and the recombinant strain PFFS increased by 7.06 times, indicating that the DON acetyltransferase and FsTRI12 The degradation efficiency of DON by fermentation bacteria suspension can be effectively improved through synergistic effect.

[0053] Compared with the original strain (Aspergillus niger strain Fs), the DON degradation rate of the recombinant strain PFFS increased by 9.83 times, indicating that the DON acetyltransferase gene Ff1TRI201、Fcu2TRI101 and Fcr2TRI101 The protein was successfully recombinantly expressed in the Aspergillus niger recombinant strain provided by the present invention and had high degradation activity.

[0054] The specific embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The specific embodiments do not describe all details in detail, nor do they limit the invention to the specific embodiments described. Numerous modifications and variations are possible based on the contents of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention.

Claims

1. A method for degrading vomitoxin ( Aspergillus niger ) recombinant strain, characterized in that, The preparation method of the recombinant Aspergillus niger strain comprises: connecting a vomitoxin acetyltransferase gene fragment with a p-6-g vector fragment to obtain a recombinant plasmid; introducing the recombinant plasmid into AGL1 competent cells by a freeze-thaw method to obtain Agrobacterium transformants; co-culturing the Agrobacterium transformants with the Aspergillus niger strain Fs, transferring to a membrane, subculturing for 3-5 days, and screening to obtain a genotype homozygous Aspergillus niger recombinant strain; The Aspergillus niger strain Fs is co-expressed FsTRI12 niger strains of genes.

2. The recombinant Aspergillus niger strain according to claim 1, characterized in that The deoxynivalenol acetyltransferase gene fragment comprises Ff1TRI201、Fcu2TRI101 or Fcr2TRI101 Any one of the Ff1TRI201 The nucleotide sequence of the gene is shown in SEQ ID NO.

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

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

7.

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

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

5. Use of the recombinant Aspergillus niger strain according to any one of claims 1 to 4 in degrading vomitoxin.

6. A method for degrading vomitoxin, characterized in that: The method comprises the following steps: fermenting the recombinant Aspergillus niger strain according to any one of claims 1 to 4 to obtain a fermentation bacterial suspension of the recombinant Aspergillus niger strain; and adding the fermentation bacterial suspension to a vomitoxin substrate to be degraded for degradation.

7. The method according to claim 6, wherein 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.

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