Use of feruloylputrescine in the preparation of deoxynivalenol

Abstract

This invention belongs to the field of bioengineering technology and relates to the application of ferulic acid putrescine in the preparation of deoxynivalenol. This invention verifies that ferulic acid putrescine, when used as an additive in the culture medium of *Fusarium graminearum*, can promote the production of deoxynivalenol by *Fusarium graminearum*. This invention provides more pathways for the synthesis and acquisition of deoxynivalenol.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to the application of ferulic acid putrescine in the preparation of deoxynivalenol. Background Technology

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a toxin produced by Fusarium graminearum (…). Fusarium graminearum Trichothecene B toxins produced by pathogenic fungi such as *Fusarium graminearum*. DON is one of the most common mycotoxins in grains such as wheat and corn, posing a serious threat to the health of livestock and humans, causing vomiting, diarrhea, and immunosuppression. It is also a key virulence factor in the infection process of *Fusarium graminearum*. Stable and reliable sources of DON are needed in scientific research, toxicological assessment, and the development of detection methods. Currently, obtaining DON mainly relies on extraction and purification from grains naturally infected or artificially inoculated by *Fusarium graminearum*. However, this method is limited by factors such as crop growth cycle, climatic conditions, and uneven infection levels, resulting in unstable DON yields, high costs, and cumbersome processes, making it difficult to meet the needs of large-scale, standardized applications. Although chemical synthesis of DON is theoretically possible, its synthetic route is complex, involves numerous steps, and is extremely costly, making it currently not an economically feasible preparation method.

[0003] The biosynthesis of DON in Fusarium graminearum is influenced by a process known as THREE The coordinated regulation of multiple genes in a gene cluster, among which THREE5 Genes are key genes that enable toxin synthesis. THREE1 and THREE12 Genes such as DON also play important roles in toxin modification and transport. Studies have shown that DON synthesis occurs in a subcellular structure called the "toxin-producing body," the formation of which is a prerequisite for efficient toxin production. Environmental factors, especially signaling molecules within the host plant, also play a role. THREE Gene expression and toxin-producing body formation play important inductive roles. Therefore, identifying key inducing factors that can effectively activate the toxin-producing mechanism of Fusarium graminearum is of great significance for developing new and efficient methods for DON preparation.

[0004] Currently, there is a lack of culture medium additives or inducers that can clearly and efficiently induce Fusarium graminearum-specific and large-volume DON production. There is an urgent need in this field for a technical solution that can eliminate dependence on naturally infected grains and achieve efficient and stable DON production under controlled conditions. Summary of the Invention

[0005] The purpose of this invention is to provide more methods for preparing deoxynivalenol.

[0006] On one hand, this invention relates to the application of ferulic acid putrescine in the preparation of deoxynivalenol, comprising: ferulic acid putrescine promoting the growth of Fusarium graminearum. Fusarium graminearum It produces deoxynivalenol.

[0007] Furthermore, in the application of ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, ferulic acid putrescine promotes the growth of Fusarium graminearum. Fusarium graminearum of THREE Gene expression.

[0008] Furthermore, in the application of the ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, the... THREE Genes are THREE1 Gene, THREE5 Genes and THREE12 At least one of the genes.

[0009] THREE1 The coding sequence of the gene is shown in SEQ ID NO: 1, as follows:

[0010]

[0011] THREE5 The coding sequence of the gene is shown in SEQ ID NO: 2, as follows:

[0012]

[0013] THREE12 The coding sequence of the gene is shown in SEQ ID NO: 3, as follows:

[0014]

[0015] Furthermore, in the application of the ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, Fusarium graminearum... Fusarium graminearum The cultivation environment includes wheat ears.

[0016] Furthermore, in the application of the ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, Fusarium graminearum... Fusarium graminearum The concentration of ferulic acid putrescine in the culture environment should not be less than 30 μM.

[0017] Furthermore, in the application of the ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, Fusarium graminearum... Fusarium graminearum The concentration of ferulic acid putrescine in the culture environment was 30 μM to 300 μM.

[0018] Furthermore, in the application of ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, ferulic acid putrescine promotes the production of... THREE1 Recombinant bacteria produce toxin-producing bodies.

[0019] Furthermore, in the application of ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, ferulic acid putrescine is *Fusarium graminearum*. Fusarium graminearum A single nitrogen source in the culture environment.

[0020] Furthermore, in the application of the ferulic acid putrescine provided by this invention in the preparation of deoxynivalenol, Fusarium graminearum... Fusarium graminearum Deoxynivalenol is produced in liquid toxin-producing culture medium;

[0021] The 1L liquid toxin-producing culture medium contains 0.3g plant gel, 30g sucrose, 1g KH2PO4, 0.5g MgSO4·7H2O, 0.5g KCl, 10mg FeSO4·7H2O, 264.3mg ferulic acid putrescine, and 20μL trace elements;

[0022] Each 100 mL of the trace elements contains 5 g citric acid, 5 g ZnSO4·7H2O, 0.25 g CuSO4·5H2O, 50 mg MnSO4·H2O, 50 mg H3BO3, and 50 mg Na2MoO4·2H2O.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] This invention is the first to discover and verify that the plant-derived metabolite ferulic acid putrescine can act as a highly efficient inducer, significantly promoting the synthesis of deoxynivalenol (DON) by Fusarium graminearum. Compared to traditional methods that rely on naturally infected grains for extraction, this invention achieves stable and significant increases in DON yield by adding ferulic acid putrescine to a controlled culture medium in the laboratory, eliminating dependence on crop growth cycles and infection environments, and opening up more avenues for the standardized and large-scale preparation of DON. This invention not only verifies the effect of ferulic acid putrescine on increasing the final DON yield, but also further reveals its deeper mechanism of action: namely, by specifically upregulating key components in Fusarium graminearum. THREE Genes (including) THREE1 , THREE5 and THREE12 The expression of feruloyl putrescine is significantly promoted, and the formation of the toxin-producing body structure responsible for DON synthesis is significantly enhanced. This invention optimizes and provides specific liquid toxin-producing culture medium formulations and culture conditions, clarifies the effective concentration range of ferulic acid putrescine (e.g., 30 μM~300 μM), and confirms its effectiveness as a single nitrogen source, providing a reliable guarantee for subsequent scientific research, preparation of detection standards, and other applications. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 To promote the synthesis of deoxynivalenol by ferulic acid putrescine, THREE Diagrams showing gene expression and toxin-producing body formation. In diagram A, DON production is shown in liquid toxin-producing media using ammonium nitrate, putrescine, or ferulic acid putrescine as a single nitrogen source; in diagram B, DON production is shown in liquid toxin-producing media using putrescine and ferulic acid putrescine as single nitrogen sources. THREE The effect of gene expression; the effect of liquid toxin-producing medium with putrescine and ferulic acid putrescine as single nitrogen sources on the formation of toxin-producing bodies (C); DIC indicates observation under field conditions; THREE1 -GFP refers to the fusion protein of green fluorescent protein (GFP), which contains the coding sequence of the TRI1 gene; NH4NO3 is ammonium nitrate, Put is putrescine, and Ferput is ferulic acid putrescine; scale bar = 20 µm. Detailed Implementation

[0027] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0028] Example 1

[0029] This embodiment provides metabolomics analysis showing a significant increase in ferulic acid putrescine in wheat ears infected with Fusarium graminearum.

[0030] Wild-type Fusarium graminearum strain PH-1 was activated in PDA medium (purchased from Beijing Aoboxing Biotechnology Co., Ltd.) and cultured in the dark at 25℃ for 3 days. Mycelial cakes were created using a 60mm punch and inoculated into 50ml CMC liquid medium. 1L of CMC liquid medium contained 1g yeast extract (purchased from OXOID), 1g NH4NO3 (purchased from Shanghai Chemical Reagent Factory), 1g KH2PO4 (purchased from Guangdong Guanghua Technology Co., Ltd.), 0.5g MgSO4·7H2O (purchased from Guangdong Guanghua Technology Co., Ltd.), and 15g carboxymethyl cellulose (purchased from Aladdin). Spores were collected after 3 days, and the spore concentration was adjusted to 1×10⁻⁶. 6 10 μL of spore solution was inoculated into the lemma of Norm wheat at the flowering stage, while the control group was inoculated with water. After bagging and keeping moist for 24 h, the wheat ears were collected and quickly frozen in liquid nitrogen.

[0031] The sample was freeze-dried under vacuum in a Scientz-100F freeze dryer for 63 hours, then ground into powder using a grinder (MM400, Retsch) at 30 Hz for 1.5 minutes. 50 mg of the sample powder was weighed using an electronic balance (MS105DM) and added to 1200 mL of solution at -20°C. Centrifuge in pre-cooled 70% methanol. Vortex every 30 minutes for 30 seconds, for a total of 6 vortexes. After centrifugation (12000 rpm, 3 minutes), collect the supernatant, filter the sample through a 0.22 μm microporous membrane, and store in a vial for UPLC-MS / MS analysis.

[0032] The chromatographic column was an Agilent SB-C18 (1.8µm × 2.1mm × 100mm). Mobile phase A was 0.1% formic acid / water, and mobile phase B was 0.1% formic acid / acetonitrile. The elution gradient was as follows: 0.0 min, B phase ratio 5%, increasing linearly to 95% within 9.0 min and maintaining at 95% for 1 min; 10.00-11.10 min, B phase ratio decreased to 5%, and equilibrated to 5% for 14 min. The flow rate was 0.35 mL / min, the column temperature was 40℃, and the injection volume was 2 μL. The electrospray ionization source temperature was 500℃; the ion spray voltage was 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high. QQQ scan used MRM mode with the collision gas (nitrogen) set to medium. Further optimization of declustering voltage (DP) and collision energy (CE) was performed for each MRM ion pair. A specific set of MRM ion pairs was monitored at each time period based on the eluted metabolites. Compounds were compared using the MWDB (metware database) built by Wuhan Maiwei Metabolic Biotechnology Co., Ltd. The results are shown in Table 1.

[0033] Table 1: Categories of compounds that were significantly elevated in wheat ears infected with Fusarium graminearum

[0034]

[0035] The comparison showed that phenolic amines were the most elevated compounds in wheat ears infected with Fusarium graminearum, and ferulic acid putrescine was enriched in wheat ears infected with Fusarium graminearum.

[0036] Example 2

[0037] This embodiment provides a method for promoting the synthesis of deoxynivalenol (DON) with ferulic acid putrescine.

[0038] Toxin production culture conditions: Add 2 mL of liquid toxin production medium to each 24-well plate. 1 L of liquid toxin production medium contains 0.3 g plant gel, 30 g sucrose, 1 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 0.5 g KCl, 10 mg FeSO₄·7H₂O, 80.043 mg ammonium nitrate (or 88.151 mg putrescine, or 264.3 mg ferulic acid putrescine), and 20 μL of trace elements. Each 100 mL of trace elements contains 5 g citric acid, 5 g ZnSO₄·7H₂O, 0.25 g CuSO₄·5H₂O, 50 mg MnSO₄·H₂O, 50 mg H₃BO₃, and 50 mg Na₂MoO₄·2H₂O. All reagents were purchased from Sigma. Adjust the pH to 5.4–5.6. Add to a final concentration of 1 × 10⁻⁶. 4 The spore suspension was prepared at 1 spore / mL and cultured in a constant temperature incubator at 25℃ in the dark for 7 days. Then, the bacterial film on the surface of the culture medium was quickly placed in liquid nitrogen, freeze-dried for 24 hours, and weighed.

[0039] DON toxin extraction: Take 400 μL of the liquid toxin-producing medium after 7 days of culture, add 1600 μL of methanol and mix well. Pass the resulting extract through a C18 SPE solid-phase extraction column. Take 800 μL of the filtrate, concentrate under vacuum to dryness, add 50 μL of silanizing reagent (trimethylsilylimidazolium to trimethylchlorosilane, volume ratio 100:1, purchased from Sigma), vortex thoroughly to ensure the silanizing reagent and sample are fully mixed, and shake on a shaker for 10 min. Add 800 μL of isooctane, gently invert and mix thoroughly, then add 800 μL of ultrapure water, gently invert and allow to stand for separation, transfer the supernatant to a sample vial.

[0040] GC-MS detection:

[0041] Chromatographic conditions: Rxi-5MS column (30m×0.230-1000μM×0.25μm); split ratio 50:1; injection port temperature 260℃; carrier gas: helium, flow rate 1mL / min; temperature program: initial column temperature 150℃, hold for 1min, increase to 280℃ at 30℃ / min, and hold for 15min.

[0042] Mass spectrometry conditions: mass spectrometer interface temperature 280℃; ion source temperature 250℃; injection volume 1μL; qualitative analysis of DON was performed using m / z 295, 235, and 193 in SIM mode, and quantitative analysis was performed using m / z 235. Mass spectrometry acquisition time was 5–10 min. The effect of ferulic acid putrescine on DON synthesis is as follows: Figure 1 As shown in A, the content of deoxynivalenol was significantly higher than that of ammonium nitrate and putrescine, indicating that ferulic acid putrescine can promote the synthesis of DON.

[0043] Example 3

[0044] This embodiment provides the effect of ferulic acid putrescine on Fusarium graminearum. THREE Effects of gene expression

[0045] exist THREE In genes, THREE5 Catalyzing the cyclization of farnesyl pyrophosphate into trichothecene is the first-step catalytic enzyme in the synthesis of DON toxin. THREE1 The encoded protein catalyzes the formation of C8 trichothecene from lecithin and is localized to the toxin-producing body. THREE12 The encoded transporter protein is involved in the transport of DON toxin.

[0046] Add 50 mL of liquid toxin-producing medium to an Erlenmeyer flask, then add spores of wild-type Fusarium graminearum strain PH-1 to bring the final spore concentration to 1 × 10⁻⁶. 4 Cells / mL. Mycelia were cultured in the dark at 30℃ and 90 rpm for 36 h, then rapidly frozen in liquid nitrogen. RNA was extracted from the mycelia, followed by DNA digestion and reverse transcription. Design THREE Quantitative PCR was performed using primers for the gene and internal reference gene, followed by data analysis and processing. The results are as follows: Figure 1 As shown in B, THREE The quantitative primers for the gene and internal reference gene are shown in Table 2.

[0047] Table 2: Specific primers for the internal reference genes actin and TRI.

[0048]

[0049] Depend on Figure 1 As can be seen from B, ferulic acid putrescine promotes THREE1 , THREE5 and THREE12 The expression levels of these substances increased by more than 6 times.

[0050] Example 4

[0051] This embodiment provides the effect of ferulic acid putrescine on the formation of toxin-producing bodies in Fusarium graminearum.

[0052] Toxin-producing bodies are considered to be a class of round proteins synthesized by DON. Using primers... THREE1 -GFP-F (5'-AGGGAACAAAAGCTGGGGTACCGCTATACTCGGCAGTCCTTTGC-3') and THREE1 PCR amplification was performed using GFP-R (5'-GAACAGCTCCTCGCCCTTGCTCACGGCGTCATTTGGGCTTGAGATAG-3'), which amplified Fusarium graminearum. THREE1 Gene sequence. The pKNTG plasmid was digested with KpnI / HindIII double enzymes to obtain... THREE1 -GFP fragment was introduced into the digested pKNTG plasmid. The pKNTGseq / F (5'-CCCCAGGCTTTACACTTTATGCT-3') and THREE1 PCR verification was performed using GFP-R primers, and the plasmid was sent to a company for sequencing. After confirming the absence of mutations, it was introduced into the protoplasm of PH-1. PCR detection yielded Fusarium graminearum. THREE1 -GFP transformation strain. To THREE1 The GFP-transformed bacterial blocks were incubated in CMC liquid medium with shaking for 3 days (25℃, 200 rpm), filtered through a filter cloth, and centrifuged, discarding the supernatant. 50 mL of liquid toxin-producing medium was added to an Erlenmeyer flask, followed by... THREE1 -GFP strain spore solution, with a final spore concentration of 1×10⁻⁶. 4 Cells / mL. Incubate in the dark with shaking for 36 hours (25℃, 90 rpm). Pick hyphae, rinse with sterile water, and observe the formation of toxin-producing bodies under a fluorescence microscope. Results are as follows: Figure 1 As shown in C in the figure. Compared with the control group, in the toxin-producing medium with ferulic acid putrescine as the single nitrogen source, a large number of toxin-producing bodies were formed in the hyphae of Fusarium graminearum, indicating that ferulic acid putrescine treatment significantly promoted the formation of toxin-producing bodies.

[0053] The embodiments described above are only some embodiments of the present invention, and not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate preferred embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. Use of feruloylputrescine for the preparation of deoxynivalenol, characterized in that, Comprising: feruloyl putrescine promotes fusarium graminearum Fusarium graminearum deoxynivalenol production in liquid toxinogenic medium; 1L of the liquid toxin production medium contains 0.3g plant gel, 30g sucrose, 1g KH2PO4, 0.5g MgSO4·7H2O, 0.5g KCl, 10mg FeSO4·7H2O, 264.3mg feruloyl putrescine, 20μL trace elements; 5g citric acid, 5g ZnSO4·7H2O, 0.25g CuSO4·5H2O, 50mg MnSO4·H2O, 50mg H3BO3, 50mg Na2MoO4·2H2O per 100mL of the trace elements.

2. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, feruloylputrescine promotes fusarium graminearum Fusarium graminearum gene expression. TRI gene expression.

3. Use of feruloylputrescine according to claim 2 for the preparation of deoxynivalenol, characterized in that, The TRI genes are TRI1 gene, TRI5 genes and TRI12 at least one of the genes.

4. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, feruloyl putrescine promotes the production of TRI1 toxophore by recombinant bacteria containing the gene.

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