A method of degrading zearalenone toxin

Abstract

The present disclosure provides a nucleic acid molecule encoding RmZHD enzyme, and also discloses a composition comprising the nucleic acid molecule, a vector, a host cell, and the use of the above-mentioned nucleic acid molecule, composition, vector and host cell in the preparation of a product for degrading zearalenone. In another aspect, the present disclosure also provides a method for degrading zearalenone toxin.

Description

Technical Field

[0001] This disclosure pertains to the field of bioengineering, and in particular relates to a method for degrading zearalenone toxin. Background Technology

[0002] A survey report on mycotoxin contamination in feed and raw materials in China reveals that 99.49% of feed and raw materials in the country are contaminated with mycotoxins. Among these, zearalenone (ZEN), a major and highly hazardous mycotoxin found in corn processing byproducts, is the most prevalent. ZEN, also known as F-2 toxin, is a non-steroidal mycotoxin produced by Fusarium fungi. It has estrogenic effects and can cause chronic poisoning in humans and animals, leading to developmental loss, severe reproductive impairment, and in severe cases, death.

[0003] ZEN detoxification strategies mainly include three types: physical, chemical, and biological methods. Physical methods use aluminosilicates, activated carbon, and organic matter to adsorb toxins, but these are costly and cannot completely remove toxins. Chemical methods mainly include oxidation methods such as ozone and hydrogen peroxide to degrade toxins, but these introduce exogenous compounds, which is not conducive to green farming. Biological methods include microbial degradation and enzymatic hydrolysis, which are highly efficient, specific, environmentally friendly, and low-pollution. They also have mild treatment conditions, are safe and effective, and are the most likely methods to remove ZEN from corn by-products.

[0004] The pH of both grain processing byproducts and the animal stomach environment is acidic, while the reported ZEN-degrading bacteria and enzymes react under neutral and alkaline conditions. Only a few reports have screened out acid-resistant ZEN-degrading bacteria, but their degradation effects are poor. Therefore, the degradation of ZEN toxins under acidic conditions is an urgent problem that needs to be solved. Summary of the Invention

[0005] This disclosure provides a nucleic acid molecule encoding an RmZHD enzyme, comprising a nucleic acid sequence as shown in SEQ ID NO: 6 or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with the sequence of SEQ ID NO: 6.

[0006] In one embodiment, the nucleic acid molecule encoding the RmZHD enzyme is as shown in the nucleic acid sequence of SEQ ID NO: 6 or a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with the sequence of SEQ ID NO: 6.

[0007] This disclosure provides a composition comprising an RmZHD enzyme encoded by the aforementioned nucleic acid molecule.

[0008] In one specific embodiment, the composition further comprises a hydrolase having the ability to degrade zearalenone.

[0009] In one specific embodiment, the hydrolase having the ability to degrade zearalenone is selected from one or more of Laccase 1, ZENG, and zlhy-6.

[0010] This disclosure provides a carrier comprising the above-mentioned nucleic acid molecule, preferably further comprising a nucleic acid molecule encoding a hydrolase that degrades zearalenone, more preferably, wherein the hydrolase that degrades zearalenone is selected from one or more of Laccase 1, ZENG and zlhy-6.

[0011] In one specific embodiment, the vector is an expression vector, preferably a plasmid.

[0012] In one specific implementation, it further includes a promoter, preferably selected from the PGK strong promoter.

[0013] On the other hand, this disclosure provides a host cell comprising the above-described nucleic acid molecules or compositions, wherein the host cell is a fungal cell or a prokaryotic cell.

[0014] In one specific embodiment, the fungal cells are selected from yeast cells, preferably Pichia pastoris cells or Saccharomyces cerevisiae cells.

[0015] In one specific embodiment, the prokaryotic cell is a bacterial cell, preferably selected from Escherichia coli and Bacillus subtilis.

[0016] On the other hand, this disclosure provides the use of the aforementioned nucleic acid molecules, compositions, vectors or host cells in the preparation of products that degrade zearalenone.

[0017] This disclosure provides a method for degrading zearalenone toxin, the method comprising: contacting the aforementioned host cell or a host cell expressing ZEN degrading enzyme with zearalenone under acidic solution conditions to carry out a reaction.

[0018] In one specific implementation, the method further includes: 1) Introduce the above-mentioned nucleic acid molecules into the host cell; 2) The host cells are contacted with a substrate solution containing zearalenone under acidic conditions to carry out the reaction.

[0019] In one specific embodiment, the pH range of the acidic solution is less than 5, preferably pH 3-4.

[0020] This disclosure also provides a method for degrading zearalenone, comprising: contacting a host cell expressing a ZEN-degrading enzyme with zearalenone under acidic solution conditions to react. The ZEN-degrading enzyme includes one or more of RmZHD enzyme, Laccase1, ZENG, and zlhy-6. In one specific embodiment, the host cell expressing the ZEN-degrading enzyme is selected from Pichia pastoris cells, Saccharomyces cerevisiae cells, Escherichia coli, or Bacillus subtilis; preferably, the ZEN-degrading enzyme is expressed in the cell or on the cell membrane. The pH range of the acidic solution is less than 5, preferably pH 3-4.

[0021] Beneficial effects This disclosure provides a method for degrading ZEN toxin under acidic conditions, with higher degradation efficiency. Attached Figure Description

[0022] This disclosure can be more fully understood with reference to the following figures.

[0023] Figure 1 The degradation effects of different recombinant Escherichia coli on ZEN toxin in concentrated gluten were demonstrated.

[0024] Figure 2 The recombinant Pichia pastoris GS115- was shown. RmZHD Degradation effect on ZEN toxin.

[0025] Figure 3 The recombinant brewer's yeast BY4741- is shown. RmZHD Degradation effect on ZEN toxin.

[0026] Figure 4 The degradation effects of RmZHD degradative enzymes expressed at different locations in Saccharomyces cerevisiae cells on ZEN toxin were demonstrated. Among them, α-Rm: extracellular expression strain of RmZHD degradative enzyme; CWP1: cell wall display strain of RmZHD degradative enzyme; DCW1: cell membrane display strain of RmZHD degradative enzyme; Rm: intracellular expression strain of RmZHD degradative enzyme.

[0027] Figure 5 The degradation effect of recombinant Bacillus subtilis SCK6-RmZHD on ZEN toxin was demonstrated.

[0028] Figure 6 The degradation effect of constitutive recombinant yeast on ZEN toxin before and after codon optimization was shown. Detailed Implementation

[0029] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0030] Experimental materials Table 1. Strains and Plasmids

[0031] Table 2. Sequence Information

[0032]

[0033]

[0034]

[0035] Table 3. Primer Information

[0036] Culture media and reagents (1) LB: 0.5% yeast extract, 1% tryptone, 1% sodium chloride, dissolved in deionized water, sterilized at 121 °C for 20 min, solid culture medium with 1.5~2 g agar powder, dispensed and sterilized at 121 °C for 20 min.

[0037] (2) YPD: 2% glucose, 1% yeast extract, 2% tryptone, dissolved in deionized water, 1.5~2 g agar powder added to solid culture medium, autoclave at 115 °C for 20 min.

[0038] (3) SC-Ura: 2% glucose, dissolved in deionized water, sterilized at 115 °C for 20 min, and 1.5~2g agar powder added to solid culture medium. 0.67% YNB (ammonia-free yeast nitrogen source, added separately), DO Supplement-Ura (sterilized by membrane, added separately when pouring solid culture medium, concentration 1.29 g / L) purchased from Shanghai Maokang (Jiqi) Biotechnology Co., Ltd. (4) MD: 2% glucose, 2% agar powder, add 90 mL of deionized water, sterilize at 115 °C for 20 min. When pouring the plate, add 10 mL of 10×YNB and 200 μL of biotin.

[0039] (5) YPG medium: 2% glycerol, 2% tryptone, 1% yeast extract, add deionized water and stir to accelerate dissolution, autoclave at 121 °C for 20 min.

[0040] (6) BMGY medium: 1% glycerol, 2% tryptone, 1% yeast extract, add deionized water and stir to accelerate dissolution, autoclave at 121 °C for 20 min.

[0041] (7) BMMY medium containing 2% tryptone and 1% yeast extract, add deionized water and stir to accelerate dissolution, then autoclave at 121 °C for 20 min.

[0042] (8) 10×TE: 0.4712 g Na2EDTA·2H2O, 1.21 g Tris, add 90 mL of ultrapure water to dissolve, adjust the pH to 7.5 with HCl, make up to 100 mL with ddH2O, filter with a 0.22 μm sterile microporous membrane, pour into a sterile glass bottle and store in a 4 °C refrigerator for later use.

[0043] (9) 10×LiAc: Weigh 10.2 g LiAc, dissolve it in 90 mL of ultrapure water, adjust the pH to about 7.5 with glacial acetic acid, make up to 100 mL with ddH2O, filter it with a 0.22 μm sterile microporous filter membrane to sterilize it, and place it in a pre-sterilized glass bottle at 4 °C.

[0044] (10) 1×LiAc / 1×TE: Take 10 mL of 10×LiAc and 10×TE prepared in advance, add 80 mL of deionized water, and store in a sterile glass bottle at 4 °C.

[0045] (11) 1×LiAc / 40%PEG-3350 / 1×TE: Weigh 40 g PEG3350 and dissolve it in 80 mL of deionized water. Add 10 mL each of 10×LiAc and 10×TE, bring the volume up to 100 mL, sterilize at 121 °C for 20 min, cool to room temperature and store at 4 °C.

[0046] (12) Biotin (500×Biotin): 0.6 g of biotin solid powder, dissolved in 10 mL of ultrapure water, filtered through a 0.22 μm sterile microporous membrane for sterilization, dispensed into 1.5 mL EP tubes, and stored at -20 °C.

[0047] (13) 10×YNB solution: Weigh 13.4 g and dissolve in 100 mL of ultrapure water. Sterilize by high-pressure steam at 107 °C for 10 min. After cooling to room temperature, store at 4 °C.

[0048] (14) Potassium phosphate buffer: 132 mL of 1 mol / L K2HPO4 solution and 868 mL of 1 mol / L KH2PO4 solution, adjust the pH to 6.0, and sterilize at 121 °C for 20 min.

[0049] (15) Galactose: Dissolve 10 g of galactose in 36 mL of deionized water, seal in a glass bottle, and sterilize at 115 °C for 20 min.

[0050] (16) Henzimidox resistance: Weigh 10 g of henzimidox powder, dissolve it in ddH2O and bring the volume to 100 mL. Filter the solution through a 0.22 μm sterile microporous membrane to remove bacteria, dispense it into 1.5 mL EP tubes, and store at -20 °C.

[0051] (17) Ampicillin: Weigh 10 g of ampicillin, dissolve it in ddH2O and bring the volume to 100 mL. Filter the solution through a 0.22 μm sterile microporous membrane to remove bacteria, dispense it into 1.5 mL EP tubes, and store at -20 °C.

[0052] (18) 0.01M PBST dilution: Take 8 g sodium chloride, 0.2 g potassium chloride, 0.2 g KH2PO4, and 1.16 g Na2HPO4·12H2O, add 800 mL of ultrapure water to dissolve, add 2 mL of Tween-20, and make up to 1 L.

[0053] (19) 1 M sorbitol: Weigh 182.17 g of sorbitol, add 800 mL of pure water to dissolve, make up to 1 L, and sterilize at 121 °C for 20 min.

[0054] Example To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0055] Example 1: The degradation effect of different ZEN-degrading enzymes on ZEN toxin in cells Through literature review, hydrolase genes from different sources that exhibit zearalenone degradation effects were selected, and respectively... Laccase 1, ZENG, RmZHD, zlhy-6 Four genes were sequenced at a sequencing company and constructed into the pET22b expression vector, yielding the recombinant plasmid pET22b- Laccase 1 pET22b- ZENG pET22b- RmZHD pET22b-zlhy6 strains.

[0056] The recombinant plasmids were transformed into [various methods]. Escherichia℃oli Recombinant strains were constructed from BL21 (Shanghai Weidi Biotechnology) competent cells. The construction steps are as follows: (1) Activate the strain containing the recombinant plasmid, transfer it to LB (Amp) resistant test tubes for culture, and extract the plasmid for later use.

[0057] (2) Preparation Escherichia℃oli BL21 competent cells.

[0058] (3) Transformation of recombinant plasmids into Escherichia℃oli BL21 and verification.

[0059] (4) Pick transformants and transfer them to LB tubes, and incubate overnight at 37 °C. The next day, transfer 1 mL of bacterial culture to a 100 mL LB shake flask and incubate until OD500. 600 When the concentration reaches approximately 0.6, IPTG (prepared to a final concentration of 0.1M) is added (Sangon Biotech (Shanghai) Co., Ltd.) and the target proteins (Laccase 1, ZENG, RmZHD, zlhy-6) are induced to express overnight at 16 °C. The crude enzyme solution is then used for toxin degradation.

[0060] Reaction system: 100 μL of 1 mg / mL ZEN toxin was added to 9 mL of pH 7.0 PBS solution to prepare the substrate solution. The experimental group was prepared by adding 1 mL of crude enzyme solution to the substrate solution, the control group (CK) was prepared by adding 1 mL of water to the substrate solution, and the negative control group was prepared by adding 1 mL of inactivated crude enzyme solution to the substrate solution. The reaction was carried out at 30°C and 200 r / min for 1 h. The reaction was terminated by adding 10 mL of methanol, and the ZEN content was detected.

[0061] The results are shown in Table 3. All four degrading enzymes can degrade zearalenone toxin. Among them, ZENG and RmZHD enzymes showed better degradation effects, with degradation rates of over 80%. The spatial structure of RmZHD enzyme has been resolved, making it easy to modify and design. Therefore, RmZHD enzyme was selected for subsequent experiments.

[0062] Table 3. Degradation effects of different zearalenone-degrading enzymes

[0063] Example 2: The degradation effect of ZEN-degrading enzymes on ZEN toxin in different strains Different host bacteria, such as Bacillus subtilis, Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae, were selected to investigate the degradation effect of ZEN degrading enzymes on ZEN toxin in different strains.

[0064] 2.1 The effect of ZEN-degrading enzyme expression on ZEN toxin degradation in Escherichia coli Contains recombinant plasmid pET22b- Laccase 1 pET22b- ZENG pET22b- RmZHD pET22b- zlhy6 The specific steps for constructing the Escherichia coli strain are detailed in Example 1.

[0065] Using genes containing four different enzymes zlhy6 , Laccase 1 , ZENG , RmZHD The recombinant *E. coli* strain was used to degrade ZEN toxin in concentrated gluten. The recombinant bacteria were transferred to LB tubes and incubated overnight at 37 °C. The next day, 1 mL of the bacterial culture was transferred to a 100 mL LB shake flask and incubated until OD500 was reached. 600 When the pH reaches approximately 0.6, 0.1 M IPTG (Sangon Biotech (Shanghai) Co., Ltd.) is added and the target protein is induced at 16 °C. Bacterial cells expressing the target protein are added at a concentration of 20% to 80 mL of concentrated corn by-product gluten (Tongliao Meihua Biotechnology Co., Ltd.). The reaction is carried out at 37 °C with shaking at 200 rpm for 24 h. After the reaction, the detoxified concentrated gluten sample is poured into a glass dish and dried at 70 °C. After drying, the sample is plated and ground into powder. 100 mL of extraction buffer (80% acetonitrile) is added and mixed well. The mixture is homogenized at high speed for 1 min or shaken on a shaker for 20 min. The sample is then rapidly filtered through qualitative filter paper, and the filtrate is collected. 10 mL of the filtrate is diluted with 40 mL of diluent and filtered again through microfiber filter paper. The collected filtrate is used as the loading solution. 25 mL of the solution is then passed through an immunoaffinity purification column. Remove the immunoaffinity column, place it on the rack and secure it. Open the stopper and bottom plug of the column to drain the liquid inside, allowing it to flow out at a rate of 1-2 drops / second. Add the sample loading solution, drain all the sample loading solution, and wash twice with 10 mL of water at a flow rate of 2-3 drops / second. After draining the liquid, plug the bottom plug, add 1 mL of methanol, let it stand for a period of time, and then collect the eluent. Detect the remaining ZEN toxin using liquid chromatography. The results are as follows: Figure 1 All four strains achieved good degradation effects, especially RmZHD The recombinant bacteria achieved a 100% degradation rate, completely degrading ZEN toxins in concentrated gluten. Since concentrated gluten has an acidic pH of 3-4, this experiment demonstrates that the cell membrane can act as a barrier, allowing ZEN-degrading enzymes to remain active intracellularly, thus detoxifying acidic concentrated gluten.

[0066] 2.2 The effect of ZEN-degrading enzymes expressed in Saccharomyces cerevisiae on the degradation of ZEN toxin 2.2.1 Recombinant plasmid pYES2- RmZHD Build design Bam H І / Eco R-I double enzyme digestion and ligation of primers P1 / P2 to recombinant plasmid pET22b- RmZHD Using pYES2 as a template, the target gene fragment was cloned and purified using a standard DNA product purification kit (Tiangen Biotech). The purified product was then double-digested with pYES2, and the digested product was subsequently recovered using a Tiangen standard agarose gel DNA recovery kit to retrieve the target band. pYES2 and the target gene fragment were ligated using T4 ligase and transformed into [a specific DNA product]. Escherichia℃oli T1 (Shanghai Weidi Biotechnology) competent cells were used to select transformants for verification, and plasmids were extracted and sent for testing.

[0067] 2.2.2 Brewing yeast BY4741-pYES2- RmZHD Construction of recombinant bacteria (1) Activation: Take the BY4741 strain (Baosai Biotechnology) frozen at -80 °C and streak it in the three zones of YPD plate.

[0068] (2) Enrichment: Select a single colony and transfer it to a YPD test tube, and incubate overnight at 30 °C.

[0069] (3) Shake flask transfer: Add the bacterial culture to a 50 mL shake flask and initially allow the OD to rise. 600 Reach 0.4, incubate at 30 °C for 3-4 h until OD. 600 Once the pH reaches 1.2-1.5, pre-cool and centrifuge at 5000 rpm for 10 min, then discard the supernatant.

[0070] (4) Add 20-25 mL of 1×TE buffer to resuspend the bacterial cells, centrifuge and discard the supernatant. Add 1 mL of 1×LiAc / 1×TE and aliquot evenly by pipetting.

[0071] (5) Add 20 μL salmon sperm DNA, 5-10 μL recombinant plasmid and 700 μL 1×LiAc / 40%PEG3350 / 1×TE to the competent cells and mix well by pipetting. Let stand at 30 °C for 30 min.

[0072] (6) Add 88 μL of DMSO and mix well. Incubate in a 42°C water bath for 7 min.

[0073] (7) Centrifuge at 14000 rpm for 10 s, discard the supernatant, add 1×TE and blow, centrifuge at 14000 rpm for 10 s, discard the supernatant, and retain the sample to be plated on an SC-Ura plate.

[0074] 2.2.3 Validation of Saccharomyces cerevisiae transformants Pick a single colony and transfer it to a 5 mL YPD tube, incubate overnight at 30 °C. Centrifuge to collect the cells, extract plasmids using a yeast plasmid extraction kit (Omega), and verify the target fragment using the universal validation primers GAL1 / CYC1 for Saccharomyces cerevisiae.

[0075] 2.2.4 Shake-flask fermentation of brewer's yeast (1) Select a transformant with good growth and transfer it to 5 mL SC-Ura liquid culture medium in a test tube and incubate overnight at 30 °C.

[0076] (2) Transfer to 50 mL YPD liquid medium and incubate at 30 °C in a shaker for 16-20 h.

[0077] (3) Add 4.5 mL of 20×galactose to induce expression.

[0078] 2.2.5 The effect of ZEN-degrading enzymes expressed in Saccharomyces cerevisiae on the degradation of ZEN toxin Using genes containing ZEN degradase RmZHD The degradation of ZEN toxin by recombinant Saccharomyces cerevisiae was investigated. The yeast cells were added to 90 mL of corn-based by-product gluten concentrate at a dosage of 10%, and the reaction was carried out at 30°C with shaking at 200 r / min for 12 h. ZEN toxin was then extracted and detected. The results are as follows: Figure 3 As shown: Saccharomyces cerevisiae BY4741 and pYES2 are the blank host strain control and empty plasmid control, respectively. Similar to Pichia pastoris, the adsorption of ZEN toxin by the yeast cell wall reduces it by 15%-20%; recombinant strain BY4741-pYES2- RmZHD (RmZHD) can degrade nearly 60% of toxins, indicating that the ZEN degrading enzyme gene... RmZHD It was successfully expressed in Saccharomyces cerevisiae and was able to degrade ZEN toxin under acidic conditions.

[0079] 2.3 The effect of ZEN-degrading enzymes expressed at different locations in Saccharomyces cerevisiae cells on the degradation of ZEN toxin The ZEN-degrading enzyme RmZHD was expressed in the intracellular, cell membrane, cell wall, and extracellular regions of Saccharomyces cerevisiae to investigate the degradation effect of RmZHD enzyme on ZEN toxin in different cellular locations under acidic conditions, thereby verifying the protective effect of microbial cell walls / membranes on enzymes.

[0080] 2.3.1 Construction and transformation of different expression vectors By using Saccharomyces cerevisiae BY4741 as the host cell, PKG-T cyc The plasmid pYES2-PGK-T was obtained by introducing it into the pYES2 expression vector. cyc As an expression vector, the gene is... RmZHD pYES2-PGK is constructed-RmZHD Expressive vehicle, thereby achieving RmZHD Intracellular expression; to enable RmZHD and cell membrane proteins DCW1 Fusion, constructing pYES2-PGK -DCW1 - RmZHD Expression vectors were used to achieve cell membrane display and expression of RmZHD enzyme; to fuse RmZHD enzyme with cell wall protein CWP1, pYES2-PGK- was constructed. CWP1 - RmZHD Expression vectors were used to achieve cell wall display expression of RmZHD enzyme; in order to... RmZHD The enzyme and α signal peptide were fused to construct pYES2-PGK-α- RmZHD Expression vectors were used to achieve extracellular secretory expression of the RmZHD enzyme. The four recombinant plasmids were transformed into competent cells of Saccharomyces cerevisiae BY4741, and screened using Ura-deficient medium. The corresponding transformants were obtained after verification and named Rm, DCW1, CWP1, and α-Rm, respectively.

[0081] 2.3.2 Expression and activity study of RmZHD enzyme The four transformants Rm, DCW1, CWP1, and α-Rm were inoculated into test tubes containing 5 mL of SC-Ura medium and cultured at 30°C with shaking at 200 r / min for 24 h. 1 mL of each transformant was then transferred to YPD liquid medium and cultured at 30°C with shaking at 200 r / min for another 24 h. The cells were collected by centrifugation and washed 2-3 times with pH 7.0 buffer. The reaction system is shown in the table below. The reaction was carried out at 30°C with 200 r / min for 30 min, and the reaction was terminated by adding 10 mL of methanol. The ZEN content was then measured.

[0082] Table 4. ZEN toxin degradation reaction system

[0083] The ZEN-degrading enzyme RmZHD was expressed in the intracellular (Rm), cell membrane (DCW1), cell wall (CWP1), and extracellular (α-Rm) regions of *Saccharomyces cerevisiae*. Degradation experiments using these four strains of ZEN toxin were conducted. Results are as follows: Figure 4As shown, the degradation activity of RmZHD degrading enzyme increases from the outer to the inner part of the cell. The intracellular Rm strain exhibits the highest degradation activity against ZEN toxin, reaching 78%, while the extracellular α-Rm strain shows the lowest activity. The DCW1 strain expressed on the cell membrane shows higher degradation activity than the CWP1 strain expressed on the cell wall. This indicates that through the layered protection of the yeast cell wall and cell membrane, the RmZHD degrading enzyme is gradually less affected by the external acidic environment, allowing it to fully exert its degradation activity intracellularly and achieve detoxification of acidic maize byproducts.

[0084] 2.4 The effect of ZEN-degrading enzymes expressed in Pichia pastoris on the degradation of ZEN toxin 2.4.1 Recombinant plasmid pPIC9k- RmZHD Build design Eco R І / Not Double digestion of primers P3 / P4 with recombinant plasmid pET22b- RmZHD Using a template, the target gene fragment was cloned and purified using the Tiangen purification kit; the purified product was then processed together with pPIC9k. Eco R І / Not The DNA fragments were digested with two enzymes (I and B), and the digestion products were then recovered using a standard agarose gel DNA recovery kit (Tiangen Biotech). The pPIC9k and target gene fragments were ligated using T4 ligase and transformed into [a specific gene / technology]. Escherichia℃oli Transformants of DH5α (Shanghai Weidi Biotechnology) were selected for verification, and plasmids were extracted and sent for testing.

[0085] 2.4.2 Construction of recombinant Pichia pastoris (1) Activation: Take out Pichia pastoris GS115 strain (Baosai Biotechnology) from -80 °C and streak it on YPD plate in three zones.

[0086] (2) Enrichment: Select a single colony and transfer it to a YPD test tube, and incubate overnight at 30 °C.

[0087] (3) Transfer in shake flask: Take 2 mL of bacterial culture and transfer it to a 50 mL shake flask. Incubate at 30°C for 3-4 h and measure OD. 600 Centrifuge at 4 °C for 5000 rpm for 10 min until the pH reaches 1.3-1.5, then discard the supernatant.

[0088] (4) Add 20-25 mL of sterile water to resuspend the bacterial cells, centrifuge and discard the supernatant. Add 20 mL of sterile 1 M sorbitol (Sangon Biotech (Shanghai) Co., Ltd.) and wash twice by blowing and aspiration.

[0089] (5) Add 500 μL of sorbitol and dispense evenly by blowing and suction.

[0090] (6) Take 30-50 μL of linearized plasmid and add it to competent cells, transfer it to an electroporation cup, and let it stand for 5 min.

[0091] (7) Set parameters to 1.5 KV, 5 ms; immediately after the electric shock, add 1 mL of sorbitol for recovery. Centrifuge at 6000 rpm for 5 min and plate onto MD plates.

[0092] 2.4.3 High-copy transformant screening (1) The transformants grown on MD medium were spotted onto YPD medium with 0.5 mg / mL G418 resistance and incubated at 30°C for 2-3 days.

[0093] (2) Select single colonies with good growth from the colonies grown on the YPD medium with 0.5 mg / mL G418 resistance, and then replicate them on YPD plates with 2.0 mg / mL and 4.0 mg / mL G418 resistance in sequence. Incubate at 30°C for 2-3 days.

[0094] (3) Select bacteria with good growth and large colonies from the 4.0 mg / mL G418 resistant YPD plate, spread them densely on the YPD medium plate for verification.

[0095] 2.4.4 Validation of Pichia pastoris transformants Pick a single colony from an MD plate and transfer it to 40 μL of a solution containing 1% SDS. Mix well by pipetting and boiling for 5 min, then incubate on ice for 5 min (increase the number of repetitions to increase the extraction rate, 2-3 times). Centrifuge at 12000 rpm for 1 min and use the supernatant as a PCR template. Verify with the universal primers 5AOX / 3AOX for Pichia pastoris.

[0096] 2.4.5 Pichia pastoris shake-flask fermentation (1) Select a transformant with good growth and transfer it to 30 mL of YPG liquid medium and ferment it in a shaker at 30 °C for 24 h.

[0097] (2) Transfer 500 μL of bacterial culture to 24 mL of BMGY medium (prepare 3 mL of phosphate buffer, 3 mL of 10×YNB, and 100 μL of biotin before transfer), and ferment in a shaker at 30°C for about 20 h. Take about 15 mL of bacterial culture (measure OD). 600 (To ensure consistent bacterial count) Add to a 50 mL centrifuge tube, centrifuge at 4°C for 5000 r / min for 5 min, and discard the supernatant.

[0098] (3) Transfer to BMMY medium. Before transfer, add 3 mL of phosphate buffer, 3 mL of 10×YNB, 200 μL of biotin, and 300 μL of methanol to induce the expression of the target protein for 3-4 days. Add 600 μL of methanol daily during the fermentation period.

[0099] 2.4.6 The effect of ZEN-degrading enzyme expression on ZEN toxin degradation in Pichia pastoris Using genes containing ZEN degradase RmZHD The degradation of ZEN toxin by recombinant Pichia pastoris was investigated. 20% of the bacterial cells were added to 80 mL of a buffer solution containing corn by-products, concentrated gluten, and pH 4.0. The reaction was carried out at 30°C with shaking at 200 r / min for 24 h. ZEN toxin was then extracted and detected. The results are as follows: Figure 2 As shown: Pichia pastoris GS115 and 9K are the blank host strain control and empty plasmid control, respectively, which reduce ZEN toxin by 15%-20%. This is because the yeast cell wall has a certain adsorption effect on ZEN toxin, which is consistent with relevant research results. The recombinant Pichia pastoris strain R1 can almost completely degrade the toxin, indicating that the ZEN degrading enzyme gene... RmZHD Successfully expressed in Pichia pastoris cells, expression RmZHD Pichia pastoris can degrade ZEN toxin under acidic conditions.

[0100] 2.5 The effect of ZEN-degrading enzyme expression on ZEN toxin degradation in Bacillus subtilis 2.5.1 Recombinant plasmid pMA5- RmZHD Build Amplification RmZHD The target gene and pMA5 vector backbone fragment were tested using gel electrophoresis to verify the band size. Once confirmed, the correct band size was added. Dpn The template was digested, and the DNA was purified and recovered using the Tiangen Common DNA Product Purification Kit. The purified and recovered DNA was then used as the final product. RmZHD The target gene and the pMA5 vector backbone fragment were used as templates and added in an equimolar ratio, with the pMA5 vector backbone concentration at 4 ng / μL. The two fragments were ligated by PCR to complete the recombinant plasmid pMA5- RmZHD Construction. After amplification, agarose gel electrophoresis was performed to verify the band size.

[0101] 2.5.2 Construction of Recombinant Bacillus subtilis Take 100 μL of prepared SCK6 competent cells, thaw them on ice, add 2 μL of PCR product, mix well, and incubate at 37°C in a shaker for 2 h. Spread the entire bacterial culture onto LB agar plates containing kanamycin and incubate overnight at 37°C. Pick single-clone transformants for verification and sequencing to obtain recombinant Bacillus subtilis.

[0102] 2.5.3 The effect of ZEN-degrading enzyme expression on ZEN toxin degradation in recombinant Bacillus subtilis The recombinant Bacillus subtilis was used to degrade ZEN toxin. The bacterial cells were added to 90 mL of corn and by-product gluten concentrate at a dosage of 10%, and the reaction was carried out at 30°C with shaking at 200 r / min for 12 h. ZEN toxin was then extracted and detected. The results are as follows: Figure 4 As shown: Group CK consisted of wild-type Bacillus subtilis. Recombinant Bacillus subtilis SCK6-RmZHD showed poor toxin degradation under acidic conditions, with a degradation rate of only 5.58%. Under the same conditions, the degradation effect of recombinant Bacillus subtilis SCK6-RmZHD on toxins under acidic conditions was far lower than that of recombinant Saccharomyces cerevisiae Rm (…). Figure 4 Therefore, Saccharomyces cerevisiae was chosen as the expression host in the future.

[0103] 2.6 The effect of ZEN-degrading enzymes on the degradation of ZEN toxin in different Saccharomyces cerevisiae In the synthesis of P by Genewiz pgk1 -T cyc (SEQ ID NO: 7), P pgk1 -T cyc The recombinant expression vector pYES2-P was constructed into the Saccharomyces cerevisiae expression vector pYES2-P. pgk1 -T cyc pET22b- RmZHD Design as a template RmZHD Amplification primers ( Hin d Ш / Kpn PCR products were purified using a purification kit; the purified products and pYES2-P pgk1 -T cyc The plasmids were double-digested together, and the digestion products were purified and recovered. The digested fragments were ligated overnight at 16°C using T4 ligase; the ligation product was transformed into a product purchased from Weidi Biotechnology. Escherichia℃oli DH5α was used to select transformants for verification and testing, resulting in pYES2-P expressed by the PGK strong promoter. pgk1 -T cyc -RmZHD recombinant plasmid.

[0104] 2.6.1 pYES2-P pgk1 -RmZHD Build to different brewer's yeast hosts The successfully constructed recombinant plasmid pYES2-P pgk1 -RmZHD Converted to Saccharomyces℃ erevisiae BY4742 Saccharomyces℃erevisiae For the W303 host and yeast conversion method, please refer to Example 2.2.2.

[0105] 2.6.2 Degradation of ZEN toxin by recombinant strains of different Saccharomyces cerevisiae hosts Using genes containing ZEN degradase RmZHD Different brewing yeasts were used to degrade ZEN toxin. The yeast cells were added at a concentration of 20% to 80 mL of a buffer solution containing corn by-products, concentrated gluten, and pH 4.0. The mixture was reacted at 30°C with shaking at 200 r / min for 24 h. ZEN toxin was then extracted and detected. The results are as follows: Figure 5 As shown, the toxin degradation rates of recombinant expression strains BY4741 and BY4742 were 78.63% and 70.52%, respectively, with similar toxin degradation effects; W303 showed a slightly lower degradation effect. Further optimization was performed. RmZHD Degrading gene codons, and in Saccharomyces℃erevisiae BY4741 is expressed in the host.

[0106] Example 3: The effect of RmZHD gene optimization on the degradation of pure ZEN toxin To increase the expression level of degradation genes in Saccharomyces cerevisiae, we will RmZHD The gene codons were optimized according to yeast preferences (SEQ ID NO: 6), and the optimized codons were processed using the same method described above. RmZHD Build to pYES2-P pgk1 -T cyc Expression was performed to obtain the optimized recombinant plasmid pYES2-P, which expresses the PGK strong promoter. pgk1 -T cyc -RmZHD(pYES2-P pgk1 -RmZHD optimization). The degradation rate of the toxin by recombinant bacteria with different bacterial loads before and after optimization was compared under acidic conditions. The required volume of bacterial solution was calculated for different bacterial loads, and bacterial cells were collected. The cells were resuspended in pH 4 buffer, and 1 μg of toxin was added. The reaction was carried out at 37 ℃ with shaking at 200 r / min for 30 min. After the reaction, an equal volume of methanol was added to terminate the reaction. Samples were tested using the Huaan Maike Zearalenone Rapid Detection Kit. The results are as follows: Figure 6 As shown: Before and after optimization, the degradation rate increased with the increase of bacterial count; compared with before optimization, the toxin degradation rate increased significantly after optimization, and the degradation rate of bacterial counts at 8 OD levels could reach more than 90%; in the bacterial counts at 4 OD levels, the degradation rate after gene optimization was about twice that before optimization.

[0107] By incorporating references The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0108] Equivalence This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A nucleic acid molecule encoding an RmZHD enzyme, comprising a nucleic acid sequence as shown in SEQ ID NO: 6 or a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with the sequence of SEQ ID NO:

6.

2. A composition comprising an RmZHD enzyme encoded by the nucleic acid molecule of claim 1.

3. The composition of claim 2, further comprising an enzyme that degrades zearalenone.

4. The composition of claim 3, wherein the hydrolase having zearalenone-degrading enzyme is selected from one or more of Laccase 1, ZENG and zlhy-6.

5. A vector comprising the nucleic acid molecule of claim 1, preferably further comprising a nucleic acid molecule encoding a hydrolase that degrades zearalenone, more preferably, wherein the hydrolase that degrades zearalenone is selected from one or more of Laccase 1, ZENG and zlhy-6.

6. The vector as claimed in claim 5, wherein the vector is an expression vector, preferably, the expression vector is a plasmid.

7. The carrier as claimed in claim 5 or 6, further comprising a promoter, preferably, the promoter being selected from the PGK strong promoter.

8. A host cell comprising a nucleic acid molecule as claimed in claim 1 or a composition as claimed in any one of claims 2 to 4, wherein the host cell is a fungal cell or a prokaryotic cell.

9. The host cell of claim 8, wherein the fungal cell is selected from yeast cells, preferably, the yeast cell is Pichia pastoris cells or Saccharomyces cerevisiae cells.

10. The host cell of claim 8, wherein the prokaryotic cell is a bacterial cell, preferably, the bacterial cell is selected from Escherichia coli and Bacillus subtilis.

11. Use of the nucleic acid molecule of claim 1, the composition of any one of claims 2 to 4, the vector of any one of claims 5 to 7, or the host cell of claims 8 to 10 in the preparation of products degrading zearalenone.

12. A method for degrading zearalenone, comprising: The host cell or host cell expressing ZEN degrading enzyme as described in any one of claims 8 to 10 is contacted with a substrate solution containing zearalenone under acidic solution conditions to carry out the reaction.

13. The method of claim 12, wherein the host cell expressing the ZEN degrading enzyme is selected from Pichia pastoris cells, Saccharomyces cerevisiae cells, Escherichia coli or Bacillus subtilis, preferably, the ZEN degrading enzyme is expressed in the cell or on the cell membrane.

14. The method of claim 12 or 13, wherein the pH range of the acidic solution is less than 5, preferably pH 3-4.

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