A method for constructing a yeast surface display of a two-enzyme system and its application in cascade detoxification of vomitoxin

Abstract

The present application relates to the field of bioengineering and microbial technology, and relates to a yeast surface display construction method of a double-enzyme system. AGA1 The gene promoter is replaced by a strong constitutive promoter TEF1 and a glucose-responsive promoter HXT7 ; a double-enzyme free expression vector containing a galactose-inducible promoter GAL1 driven detoxification enzyme gene is constructed, and is transformed into a modified chassis cell to obtain a double-enzyme co-display engineering bacterium; the chassis protein AGA1 is expressed in a culture medium, and YtdepA and YtdepB are induced in a galactose-induced culture medium, so that the YtdepA and YtdepB are co-displayed on the yeast surface through the coupling mechanism of AGA1-AGA2. The strong constitutive promoter TEF1 and the glucose-repressed promoter HXT7 are coupled with the galactose-inducible promoter GAL1 respectively, so that the chassis protein and the detoxification enzyme are matched in the expression timing and intensity, and the detoxification enzyme is displayed on the yeast surface.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and microbial technology, and in particular to a method for constructing a yeast surface display of a dual-enzyme system and its application in the detoxification of vomitoxin cascade. Background Technology

[0002] Deoxynivalenol (DON), also known as deoxynivalenol, is a class of toxins widely found in Fusarium wilt bacteria. Fusarium Trichothecene toxins (DON) are fungal toxins found in contaminated grains (such as wheat, corn, barley, and oats). These toxins are chemically stable and difficult to completely remove during grain growth, harvesting, storage, and processing, thus posing a persistent threat to food and feed safety globally. DON has various toxicological effects on human and animal health, primarily including inhibition of protein synthesis, induction of cellular stress responses, disruption of the intestinal barrier function, and immune dysregulation. Long-term intake can lead to clinical symptoms such as growth retardation, loss of appetite, vomiting, and diarrhea; in severe cases, it can even cause acute poisoning, making it a highly concerning contaminant in the food and feed industries.

[0003] Traditional control measures for DON mainly fall into two categories: physical adsorption and chemical degradation. Physical methods primarily rely on adsorbents such as activated carbon and aluminosilicates, reducing the bioavailability of toxins through non-specific adsorption. However, these methods generally suffer from unstable removal efficiency, non-selective adsorption of nutrients (such as vitamins and amino acids), and the adsorbed toxins remain in the system, failing to truly eliminate them. Chemical degradation methods utilize chemical reagents such as ozone, hydrogen peroxide, and alkali treatment to destroy the molecular structure of DON. While this can reduce toxicity to some extent, the reaction conditions are often quite harsh, easily introducing harmful byproducts and posing a risk of secondary pollution. Furthermore, it may adversely affect the original nutritional quality and sensory characteristics of grains. In contrast, biodegradation typically offers advantages such as mild reaction conditions, high substrate specificity, minimal damage to nutrients, and environmental friendliness, and is therefore widely considered a more promising strategy for DON detoxification and control.

[0004] Isomerization detoxification of vomitoxin typically relies on a multi-step enzymatic cascade reaction, where two or more enzymes work synergistically to convert DON into a less toxic or non-toxic metabolite. Specifically, the dehydrogenase DepA first oxidizes the C3 hydroxyl group of the DON molecule to a ketone group, generating the intermediate 3-keto-deoxynivalenol (3-keto-DON); then, the reductase DepB performs a stereospecific reduction, ultimately converting it into the less toxic 3-epi-DON. This isomerization pathway, due to its significantly reduced end product toxicity, is considered a highly promising detoxification approach. However, free multi-enzyme cascade catalytic systems face multiple challenges in practical applications. First, free enzymes exhibit poor stability and tolerance under complex processing environments (such as temperature, pH, metal ions, and proteases), making it difficult to maintain long-term catalytic activity. Second, the poor reusability of enzymes leads to high application costs. More importantly, in the three-dimensional liquid phase, the intermediate metabolite 3-keto-DON generated by the first-step enzyme catalysis is prone to random diffusion and escape, failing to efficiently enter the active pocket of the second-step enzyme. This results in low cascade reaction efficiency, and the accumulation of intermediate products may even pose potential toxicity risks, thus severely limiting the overall conversion throughput.

[0005] Yeast surface display technology constructs whole-cell catalysts by fusing exogenous proteins with the AGA2 carrier protein and anchoring them to the cell wall via AGA1. Existing Saccharomyces cerevisiae display systems typically utilize the natural endogenous base protein AGA1 and the galactose-induced fusion protein AGA2, which anchor the target protein to the cell wall via disulfide bonds. However, in conventional systems, there is often a quantitative imbalance between the transcription and expression of AGA1 and AGA2: AGA1 expression is relatively insufficient, while the expression level of the AGA2 fusion protein is high, resulting in a large number of fusion proteins failing to be effectively anchored, thus limiting display efficiency. Furthermore, existing systems lack systematic expression regulation design for the co-display of multiple detoxification enzymes, making it difficult to achieve the spatially coordinated distribution of two or more enzymes on the cell surface. Therefore, there is an urgent need to develop an engineered bacteria construction method that can achieve co-display of detoxification enzymes, shorten the physical spatial distance of cascade enzymes, and induce spatial proximity effects, so as to effectively inhibit the liquid-phase diffusion escape of intermediate products, improve the degradation efficiency of vomitoxin, and meet the comprehensive requirements of industrial applications for conversion efficiency, stability and economy. Summary of the Invention

[0006] The biodegradation of fungal toxins often relies on multi-step enzymatic cascade reactions. In existing enzymatic detoxification methods for vomitoxin (DON), intermediate metabolites (such as 3-keto-DON) easily diffuse and escape from the environment, limiting the conversion efficiency of the final product 3-epi-DON and making it difficult to meet industrial requirements. The purpose of this invention is to provide a yeast surface display construction method for a dual-enzyme system and its application in vomitoxin cascade detoxification. By synergistically modifying the promoter elements of *Saccharomyces cerevisiae* and constructing a surface display expression vector, the co-display of dehydrogenase (YtdepA) and reductase (YtdepB) on the cell surface is achieved, realizing the structural transformation of DON into the low-toxicity final product 3-epi-DON, thus achieving DON detoxification.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for displaying and expressing vomitoxin detoxifying enzymes on the surface of yeast, comprising the following steps: (1) Targeted modification of the chassis cells of Saccharomyces cerevisiae, replacing the promoter of its endogenous anchoring protein AGA1 gene with a constitutive promoter. TEF1 and glucose-responsive promoters HXT7 Two modified chassis cells were obtained. (2) Construct a dual-detoxification enzyme yeast free expression vector, wherein the expression vector contains a galactose-inducible promoter. GAL1 Driven detoxification enzyme A gene YtdepA and detoxification enzyme B gene YtdepB , wherein YtdepA The nucleotide sequence is shown in SEQ ID NO: 5. YtdepB The nucleotide sequence is shown in SEQ ID NO: 7; (3) The expression vector constructed in step (2) was co-transformed into the chassis cells modified in step (1) to obtain two strains of double enzyme co-display engineered bacteria; (4) The engineered bacteria obtained in step (3) were induced to express the anchoring protein AGA1. First, they were cultured in a glucose-containing medium to express the anchoring protein AGA1. Then, they were induced to be cultured in an induction medium with galactose as the carbon source so that the detoxification enzymes YtdepA and YtdepB were co-displayed on the surface of the yeast cells.

[0008] Preferably, in step (1), the TEF1 The nucleotide sequence of the promoter is shown in SEQ ID NO: 1. HXT7 The nucleotide sequence of the promoter is shown in SEQ ID NO: 2.

[0009] Preferably, the brewer's yeast chassis cells in step (1) are brewer's yeast CEN.PK2.

[0010] Preferably, the detoxification enzyme A gene described in step (2) YtdepA The detoxification enzyme B gene carries a Strep-Tag II tag coding sequence. YtdepB Carrying a 6xHis tag encoded sequence.

[0011] Preferably, the expression vector in step (2) uses the pYD1 plasmid as a backbone. YtdepA and YtdepB The fusion protein AGA2 is expressed by fusing it with the encoding sequence of the carrier protein AGA2, and the nucleotide sequence of the fusion protein AGA2 is shown in SEQ ID NO: 4.

[0012] Preferably, the glucose concentration in the glucose-containing culture medium in step (4) is 18-22%, and the induction culture medium contains 18-22% galactose.

[0013] This invention provides a dual-enzyme co-display engineered bacterium constructed by the method described above, wherein the genome of the engineered bacterium contains... AGA1 Genes are subject to constitutive promoters TEF1 and glucose-responsive promoters HXT7 Drive, the TEF1 The nucleotide sequence of the promoter is shown in SEQ ID NO: 1. HXT7 The nucleotide sequence of the promoter is shown in SEQ ID NO: 2; this genetically engineered bacterium carries the nucleotide sequence of the promoter. GAL1 Promoter-driven detoxification enzyme genes YtdepA and YtdepB The expression carrier, the YtdepA The nucleotide sequence is shown in SEQ ID NO: 5. YtdepB The nucleotide sequence is shown in SEQ ID NO: 7.

[0014] Preferably, the detoxification enzymes YtdepA and YtdepB are co-displayed on the surface of yeast cells via an AGA2 fusion protein and an AGA1 anchoring protein; wherein the amino acid sequence of YtdepA is shown in SEQ ID NO: 6, and the amino acid sequence of YtdepB is shown in SEQ ID NO: 8.

[0015] This invention provides the application of the aforementioned dual-enzyme co-display engineered bacteria in the preparation of biocatalysts for degrading vomitoxin.

[0016] The present invention provides a fusion protein comprising a carrier protein AGA2 and a detoxification enzyme YtdepA or YtdepB; the amino acid sequence of the detoxification enzyme YtdepA is shown in SEQ ID NO: 6, the amino acid sequence of the detoxification enzyme YtdepB is shown in SEQ ID NO: 8, and the nucleotide sequence encoding the carrier protein AGA2 is shown in SEQ ID NO: 4.

[0017] The technical solution provided by this invention has the following innovative features: Based on the synergistic regulation mechanism of carbon source response elements, this invention is the first to incorporate a constitutive promoter. TEF1 and glucose-responsive HXT7 Promoter and galactose-induced GAL1 Promoter coupling is applied to a yeast surface display system. Among these, TEF1 and HXT7 The expression of the basal protein AGA1 is driven. GAL1 This drives the expression of the fusion enzymes AGA2-YtdepA and AGA2-YtdepB. For strongly constitutive... TEF1 Promoters, regardless of the carbon source conditions, TEF1 The promoters can all achieve high-level expression of the anchoring protein AGA1, under galactose induction. GAL1 The promoter induces the expression of the fusion enzyme AGA2-YtdepA / B, enabling yeast surface display of this two-enzyme system; for HXT7 The promoter, under glucose-free conditions, HXT7 High expression of the promoter leads to high-level expression of the anchoring protein AGA1, while simultaneously... GAL1 The promoter induces the expression of fusion enzymes AGA2-YtdepA and AGA2-YtdepB, thereby enabling the yeast surface display of this two-enzyme system.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention, through a spatiotemporal coordinated regulation mechanism, for the first time combines constitutive promoters, glucose-responsive promoters, and galactose-inducible promoters in a yeast surface display system, achieving stoichiometric matching between the anchoring protein and the detoxification enzyme fusion protein. Specifically, the strong constitutive promoter can drive efficient expression of the anchoring protein under various carbon source conditions; while the glucose-responsive promoter, under glucose-free induction conditions, removes carbon repression, also achieving efficient expression of the anchoring protein. These two strategies together ensure an ample supply of anchoring proteins, providing abundant anchoring sites for the fusion enzyme driven by the galactose-inducible promoter, thus overcoming the display efficiency bottleneck caused by expression imbalance in traditional systems.

[0019] Functional validation results showed that, compared with engineered bacteria using constitutive promoters to regulate anchored protein expression, the engineered bacteria based on glucose-responsive promoters exhibited superior catalytic performance: their degradation rate of vomitoxin was significantly improved, the accumulation of the final product 3-epi-DON increased, while the residual amount of the intermediate product 3-keto-DON was significantly reduced. These results confirm that this surface-displaying genetically engineered bacterium effectively opened up the cascade degradation pathway of DON, achieving highly efficient biological detoxification.

[0020] In summary, this invention provides a feasible cell catalyst solution for the industrial-scale biological detoxification of fungal toxins, with promising application prospects and industrialization value. Attached Figure Description

[0021] Figure 1 Genetically engineered bacteria Sc -TG construction diagram.

[0022] Figure 2 Genetically engineered bacteria Sc -HG construction diagram.

[0023] Figure 3 The image shows the results of ELISA detection of the relative fluorescence intensity (RFU) of detoxification enzymes displayed on the surface of recombinant engineered strains. In the image, NC represents the empty vector control, and Exp1 represents... Sc -Detection of the detoxification enzyme YtdepA in TG strain, Exp2 is Sc -Detection of the detoxification enzyme YtdepB in TG strain, Exp3 is Sc -Detection of the detoxification enzyme YtdepA in strain HG, Exp4 is Sc Detection of the detoxification enzyme YtdepB in strain HG.

[0024] Figure 4 Sc - A graph showing the changes in the content of each component over time in the TG strain detoxification system.

[0025] Figure 5 Sc - Curves showing the changes in the content of each component over time in the HG strain detoxification system.

[0026] Figure 6 Sc -TG and Sc -HG strain DON degradation rate curve over time.

[0027] Figure 7 This is a schematic diagram of the pESC-URA vector.

[0028] Figure 8 A schematic diagram for constructing pESC-URA3-AGA1-step1.

[0029] Figure 9 A schematic diagram for constructing pESC-URA3-TEF1-AGA1-step1.

[0030] Figure 10 A schematic diagram is constructed for pUC57-left-AGA1-URA3-right-step2.

[0031] Figure 11 A schematic diagram for constructing pESCtp01-TEF-a-YTdepA-PGK1-a-YTdepB-step2.

[0032] Figure 12 A schematic diagram for constructing pESCtp01.

[0033] Figure 13 This is a schematic diagram of the pESC-LEU vector. Detailed Implementation

[0034] The sequences used in the following examples: SEQ ID NO: 1 (TEF1 promoter nucleotide sequence): AGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAA AAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCATTGATATTTAAGTTAATAAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA SEQ ID NO: 2 (HXT7 promoter nucleotide sequence): ACTACTTCTCGTAGGAACAATTTCGGGCCCCTGCGTGTTCTTCTGAGGTTCATCTTTTACATTTGCTTCTGCTGGATAATTTTCAGAGGCAACAAGGAAAAATTAGATGGCAAAAAGTCGTCTTTCAAGGAAAAATCCCCACCATCTTTCGAGATCCCCTGTAACTTATTGGCAACTGAAAGAATGAAAAGGAGGAAAATACAAAATATACTAGAACTGAAAAAAAAAAAGTATAAATAGAGACGATATATGCCAATACTTCACAATGTTCGAATCTATTCTTCATTTGCAGCTATTGTAAAATAATAAAACATCAAGAACAAACAAGCTCAACTTGTCTTTTCTAAGAACAAAGAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAA SEQ ID NO: 3 (AGA1 nucleotide sequence): SEQ ID NO: 4 (AGA2 nucleotide sequence): ATGCAGTTACTTCGCTGTTTTTCAATATTTCTGTTATTGCTTCAGTTTTAGCACAGGAACTGACAACTATATGCGAGCAAATCCCCTCACCAACTTTAGAATCGACGCCGTACTCTTTGTCAACGACTA CTATTTTGGCCAACGGGAAGGCAATGCAAGGAGTTTTTGAATATTACAAATCAGTAACGTTTGTCAGTAATTGCGGTTCTCACCCCTCAACAACTAGCAAAGGCAGCCCCATAAACACACAGTATGTTTTT SEQ ID NO: 5 ( YtdepA (Carrying a Strep-Tag II nucleotide sequence) SEQ ID NO: 6 ( YtdepA carrying the amino acid sequence of Strep-Tag II): QHADGAATEAASAQSAIENFKPVTADDLAGANSANWPILRGNYQGWGYTPLDQINKDNVGQLQLAWSRTMEPGSNEGAAIAYNGVVYLGNANDVIQAIDGKTGDLIWEYRRKLPPASKFINSLGQAKRSIALFGDKVYFVSWDNFVVALDAKTGKLAWETNRGQGVEEGVSNSSGPIVVDGVVIAGSTCQFSGFGCYVTGTDAESGEELWRNTFIPRPGEEGDDTWGGAPYENRWMTGAWGQITYDPELDLVYYGSTGAGPASEVQRGTEGGTLAGTNTRFAVKPKTGEVVWKHQTLPRDNWDQECTFEMMVVSTTVNPNADADGMFSVGATLPRGETRKVLTGVPCKTGVAWQFDAETGDYFWSKATVAQNAVASIDDKGLVTVNEDMILKEPGKNYDFCPTFLGGRDWPSAGYLPASNLYVIPLSNACYDLTARTTEATPADVYNTDATVKLAPGKTNMGRVDAIDLATGETKWSFETTAALYDPVMTTGGDLVFVGSTDRVFRALDAATGKEVWSTRLPGAISGYTTSYSIDGRQYVAVVAGGSLGTSFFKAAVPNVDAVQGGNGIYVFALPEAKSAWSHPQFEK SEQ ID NO: 7 ( YtdepB carrying the nucleotide sequence of 6xHis): GACGAAACTACCTCTCACTTGATCTTGGATGATTATGTTGAAGCTGGTGGTAACTTCATTGATACCGCTAATGTTTACTCCTTGGGTGTCTCTGAAGAAATAGTTGGTAGATGGTTGAAAGCTAGACCAGAAGCTGCTTCTCAAGTTGTTTTGGCTACAAAAGGTAGATTTCCAATGGGTGCTGGTCCAAATGATATTGGTTTGTCCAGAAAGCACTTGAACAGAGCTTTGGAAGATTCCTTGAGAAGATTGGGTGTTGAACAAATCGACTTGTACCAAATGCATGCTTGGGATGCTTTGACTCCAATTGAAGAAACTTTGAGATTCTTGGACGATGCTGTTTCTGCTGGTAAAATTGCTTACTACGGTTTCTCCAATTACCTAGGTTGGCAAGTTACAAAGGCTGTTCATTGCTAAAGCTAATCATTGGTCTGCTCCAGTTACTTTACAACCACAGTACAACTTGTTGGTCAGAGATATT GAACACGAAATCGTTCCAGCTTGTTTGGATGCTGGTATGGGTTTGTTGCCATGGTCACCATTAGGTGGTGGTTGGTTGGCTGGTAAATATCAAAGGGATGTTATGCCATCTGGTGCTACTAGATTAGGTGAAAATCCAAACAGAGGCATGGAATCTTTTGGTCCAAGAAATGCTCAAGAAAGAACCTGGCAAATTATTGATGCTGTTGCTGAAATTGCCAAGGATAGAGGTGCTTCAGCTG CTCAAGTTGCTTTAGCTTGGGTTGAAGCAAGACCTGCTGTTACTTCTGTTATTTTGGGTGCTAGAACCAGAGAACAATTGGCTGATAATTTGGGTGCCTCTAAGGTTAAGTTATCTGCTGAAGAAACCGACAAGTTGACCAGAATTTCTATGCCACAAATGTCTGATTACCCATACGGTGAAAGAGGTGTTTCTCAAAGATTCAGAAAAATGGAAGGTGGTAGACATCATCATCACCATCAC SEQ ID NO: 8) YtdepB Carrying a 6xHis-tag amino acid sequence): DETTSHLILDDYVEAGGNFIDTANVYSLGVSEEIVGRWLKARPEAASQVVLATKGRFPMGAGPNDIGLSRKHLNRALEDSLRRLGVEQIDLYQMHAWDALTPIEETLRFLDDAVSAGKIAYYGFSNYLGWQVTKAVHVAKANHWSAPVTLQPQYNLLVRDIEHEIVPACLDAGMGLLPWSPLGGGWLAGKYQRDVMPSGATRLGENPNRGMESFGPRNAQERTWQIIDAVAEIAKDRGASAAQVALAWVEARPAVTSVILGARTREQLADNLGASKVKLSAEETDKLTRISMPQMSDYPYGERGVSQRFRKMEGGRHHHHHH As shown in SEQ ID NO: 1-8.

[0035] The primer sequences used in the following examples: TEF1-F: GTCAAGGAGAAAAAACCCCGAGTGATCCCCCACACACCATAG TEF1-R: AGATAATGTCATTGTTTTGTAATTAAAACTTAGATTAGATTGCTATGC AGA1-F1: TTTAATTACAAAacaATGACATTATCTTTCGCTCATTTTAC AGA1-R: ATCTTAGCTAGCCGCGGTACTTAACTGAAAATTACATTGCAAGCAAC AGA1-LA-F: TTGTAAAACGACGGCCAGTGGCAAAAGGATGTTGCCAGAG AGA1-LA-R1: GGGGATCACTGCGCTTATATACGTTTTAATTGCTTG TEF1-AGA1-F: ATATAAGCGCAGTGATCCCCCACACACCATAG HXT7-AGA1-R: CCGCATCAGGCTTCGAGCGTCCCAAAAC URA3-F: ACGCTCGAAGCCTGATGCGGTATTTTCTCC URA3-R:AATTGTTCTGCCATACCACAGCTTTTCAATTC AGA1-RA-F:TGTGGTATGGCAGAACAATTTAATTCATTTATAAAACATATACAATCAATCG AGA1-RA-R:CTATGACCATGATTACGCCAGTTTCGCCAGGCTCACCTGTG HXT7-Fnew:GTCAAGGAGAAAAAACCCCGACTACTTCTCGTAGGAACAATTTC HXT7-R:AGATAATGTCATTGTTTTTGATTAAAATTAAAAAAACTTTTTGTTTTTGTG AGA1-F:TTTAATCAAAAACAATGACATTATCTTTCGCTCATTTTAC AGA1-LA-Rnew:ACGAGAAGTAGTGCGCTTATATACGTTTTAATTGCTTG HXT7-AGA1-Fnew:ATAAGCGCACTACTTCTCGTAGGAACAATTTC YTdepA-II-F:TGTACGACGATGACGATAAGCAACATGCTGATGGTGCTGC YTdepA-II-R:GTTATCAGATCAGCGGGTTTTCACTTTTCGAATTGTGGATGTGACC YTdepB-His-F:TGTACGACGATGACGATAAGGACGAAACTACCTCTCACTTG YTdepB-His-R:TAGAGCGGATTTAGTGATGGTGATGATGATGTC YTdepB-CYC1-F:CCATCACTAAATCCGCTCTAACCGAAAAGG YTdepB-CYC1-R:TCCCACAGTTCTTCGAGCGTCCCAAAACC CYC1t-Leu-F:ACGCTCGAAGAACTGTGGGAATACTCAGGTATC CYC1t-Leu-R:GTGCCCAATAGAAAGAGAACGGAACTTTCACCATTATGGGAAATG depA-seq:GCTACCGGTAAAGAAGTTTG A-TRP-seq:GGCAAGAATACCAAGAGTTC depB-seq:GCAAGACCTGCTGTTACTTC B-Leu-step3-seq2:GGAACTTTCACCATTATGGG As shown in SEQ ID NO: 9~37 The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1: Targeted modification of the genome of Saccharomyces cerevisiae chassis cells (1) Construction of a genome targeting vector for Saccharomyces cerevisiae chassis cells For the construction of TEF1-AGA1-CENPK2 chassis cells, pESC-URA (preserved in the laboratory) was used. Figure 7 Based on the carrier as the framework, utilizing BamH I and Kpn Linearization was achieved by double digestion with restriction endonucleases. Using genomic DNA from *Saccharomyces cerevisiae* CEN.PK2 as a template, a 465 bp amplification was performed using NEB Q5 high-fidelity DNA polymerase and specific primer pairs TEF1-F and TEF1-R. TEF1 Promoter fragment; simultaneously using AGA1-F1 and AGA1-R primers, from pESC-URA3-AGA1-step1 stored in the laboratory ( Figure 8 A 2213 bp amplification was obtained from the plasmid template. AGA1 Gene sequence. Amplified. TEF1 promoter fragments and AGA1 The gene sequence was seamlessly assembled with the linearized pESC-URA vector using Gibson to obtain the intermediate vector pESC-URA3-TEF1-AGA1-step1'. Figure 9 Subsequently, using EcoR I and Hind III. The pUC57 cloning vector was linearized by double enzyme digestion. The pUC57-left-AGA1-URA3-right-step2 vector, stored in the laboratory, was used. Figure 10 Using the plasmid as a template, primers AGA1-LA-F and AGA1-LA-R1 were used to amplify a 635 bp sample. AGA1Left homologous arm; simultaneously, using the pESC-URA3-TEF1-AGA1-step1 plasmid constructed in the first step as a template, amplification of 2804 bp was performed using primers TEF1-AGA1-F and HXT7-AGA1-R. TEF1-AGA1 Expression cassette; simultaneously, using the laboratory-preserved pUC57-left-AGA1-URA3-right-step2 plasmid as a template, primer pairs URA3-F and URA3-R were used to amplify 1189 bp of [the expression cassette]. URA3 Nutritional Deficiency Screening Tags; Simultaneously, primer pairs AGA1-RA-F and AGA1-RA-R were used to amplify 635 bp of [a specific gene / molecule]. AGA1 Right homologous arm. The four purified nucleic acid fragments were mixed with the linearized pUC57 backbone to complete Gibson seamless splicing. Transformants were amplified and verified by PCR using primers AGA1-LA-F and AGA1-RA-R, with the target band at 4623 bp. Positive transformants were sequenced and confirmed to contain... TEF1 promoter AGA1 Gene promoter targeting plasmid pUC57-left-TEF1-AGA1-URA3-right-step2.

[0037] For the construction of HXT7-AGA1-CENPK2 chassis cells, vector linearization was the same as that for TEF1-AGA1-CENPK2 chassis cells. Using genomic DNA from *Saccharomyces cerevisiae* CEN.PK2 as a template, a three-step method with NEB Q5 high-fidelity DNA polymerase and specific primer pairs HXT7-F and HXT7-R was used to amplify 675 bp of DNA. HXT7 Promoter fragment; AGA1 The gene sequence was amplified in conjunction with the construction of TEF1-AGA1-CENPK2 chassis cells. The amplified... HXT7 promoter fragments and AGA1 Gene fragments and linearized pESC-URA vector were seamlessly assembled using Gibson to obtain the intermediate vector pESC-URA3-HXT7-AGA1-step1new. Linearization of the cloning vector pUC57 was performed in accordance with the construction of TEF1-AGA1-CENPK2 chassis cells. Using the laboratory-preserved pUC57-left-AGA1-URA3-right-step2 plasmid as a template, a 635 bp amplification was performed using primers AGA1-LA-F and AGA1-LA-Rnew. AGA1 Left homologous arm; simultaneously, using the pESC-URA3-HXT7-AGA1-step1new plasmid constructed in the previous step as a template, and with the help of primers HXT7-AGA1-Fnew and HXT7-AGA1-R, amplification of 2804 bp was performed. HXT7-AGA1Expression cassette; simultaneously, using the laboratory-preserved pUC57-left-AGA1-URA3-right-step2 plasmid as a template, primers URA3-F and URA3-R were used to amplify 1189 bp. URA3 Nutritional auxotroph selection markers; simultaneously using primers AGA1-RA-F and AGA1-RA-R, a 635 bp amplification was performed. AGA1 Right homologous arm. The four purified nucleic acid fragments were mixed with the linearized pUC57 backbone to complete Gibson seamless splicing. Transformants were amplified and verified by PCR using primers AGA1-LA-F and AGA1-RA-R, with the target band at 5205 bp. Positive transformants were verified by sequencing. Finally, the desired band was obtained. HXT7 promoter AGA1 Gene promoter targeting plasmid pUC57-left-HXT7-AGA1-URA3-right-step2new.

[0038] (2) Transformation screening of engineered chassis cells The above-mentioned targeting vector was amplified by PCR using M13 universal sequencing primers to obtain linearized targeting fragments. Electroporation was used to transform the targeting fragments into competent *Saccharomyces cerevisiae* CEN.PK2 cells (preserved in the laboratory) in logarithmic growth phase. Screening was performed on uracil-free plates (SC-Ura), and genomic DNA was extracted. PCR verification was performed using primers AGA1-LA-F and AGA1-RA-R. The target band sizes were consistent with the verification results of the TEF1-AGA1-CENPK2 and HXT7-AGA1-CENPK2 targeting plasmids, namely 4623 bp and 5205 bp, respectively. Successful integration of the targeting fragment into the chromosome was confirmed, completing the assay. AGA1 By changing the gene promoter, modified chassis cells TEF1-AGA1-CENPK2 and HXT7-AGA1-CENPK2 were successfully obtained.

[0039] Example 2: Construction and co-transformation of a double-detoxified enzyme free expression vector (1) Construction of free carriers is demonstrated The pYD1 plasmid, which possesses surface-displaying anchoring elements, was used as a framework. Acc65 I and Pme pYD1 was linearized using an I-endonuclease combination. Specific primers YTdepA-II-F and YTdepA-II-R were used, with pESCtp01-TEF-a-YTdepA-PGK1-a-YTdepB-step2 (preserved in the laboratory) as the linearization method. Figure 11 Using a plasmid template, amplify the detoxification enzyme A gene fragment carrying the Strep-Tag II affinity tag. YtdepA1807 bp. It was seamlessly cloned into the linearized pYD1. GAL1 Downstream of the promoter, a single expression vector pYD1-YTdepA-II-step1-1 was constructed. This expression vector carries... TRP1 Filtering tags.

[0040] To construct the expression vector for the detoxification enzyme YtdepB, the following was used: Acc65 I and Mfe I linearized the pYD1 backbone by double enzyme digestion. Primers YTdepB-His-F, YTdepB-His-R and YTdepB-CYC1-F, YTdepB-CYC1-R and CYC1t-Leu-F, CYC1t-Leu-R were used to extract plasmid templates pESCtp01-TEF-a-YTdepA-PGK1-a-YTdepB-step2, pESCtp01 (…) from the laboratory-preserved plasmid templates pESCtp01-TEF-a-YTdepA-PGK1-a-YTdepB-step2, pESCtp01 (…) from the laboratory-preserved plasmid templates pESCtp01-TEF-a-YTdepA-PGK1-a-YTdepB-step2, pESCtp01 (…) Figure 12 ), pESC-LEU ( Figure 13 The detoxification enzyme B sequence with a 6xHis tag was amplified on the amplified image. YtdepB ,999 bp), CYC1 Termination subregion (210 bp) and from pESC-LEU LEU2 Defective screening tags (1973 bp) were used to seamlessly assemble these fragments with linearized pYD1 via Gibson, resulting in the recombinant expression vector pYD1-YTdepB-His-Leu-step1-2, which carries... LEU2 Filtering tags.

[0041] (2) Co-transformation of two plasmids The two purified recombinant plasmids were mixed in equal proportions and co-transfected into the two types of chassis cells obtained in Example 1 by electroporation. The mixtures were then plated on triple-deficient selective medium (SC-Ura-Trp-Leu) plates lacking uracil, tryptophan, and leucine. After incubation at 29°C, positive transformants were picked and named... Sc -TG (TEF1-AGA1-GAL1-AGA2-YtdepA-YtdepB-CENPK2) and Sc -HG(HXT7-AGA1-GAL1-AGA2-YtdepA-YtdepB-CENPK2), Sc -TG and Sc -HG's construction diagram is as follows Figure 1 , Figure 2Transformants were validated by colony PCR. Primer pairs depA-seq and A-TRP-seq were used to verify the correctness of the expression vector pYD1-YTdepA-II-step1-1 (963 bp) transformed into chassis cells; depB-seq and B-LEU-step3-seq2 were used to verify the correctness of the expression vector pYD1-YTdepB-His-Leu-step1-2 (2352 bp) transformed into chassis cells.

[0042] Example 3: Induction of expression of detoxification enzyme on surface and ELISA identification The genetically engineered bacteria prepared in Example 2 Sc -TG、 Sc HG cells were inoculated overnight in SC-Ura-Trp-Leu liquid medium containing 20% ​​glucose for activation, followed by transfer at a 2% inoculum for scale-up culture for 12-24 h to fully express the base protein AGA1. Cells were collected by centrifugation, washed three times with glucose-free medium to remove glucose, and then starved for 5 h. The cells were resuspended in medium containing 20% ​​galactose and induced at 29°C for 12-24 h to drive cell expression. GAL1 Promoter expression.

[0043] After induction, add 100 μL of cell suspension to each well of a clear 96-well microplate. Centrifuge the plate (1000g, 5 min) to ensure the cells settle firmly and evenly at the bottom of the wells, and carefully aspirate the supernatant. Add 100 μL of 4% paraformaldehyde (PFA) in PBS to each well and fix for 15 min at room temperature. Discard the fixative and wash three times with PBST (PBS + 0.05% Tween-20) (shaking the plate), soaking for 2 minutes each time. Pat dry on absorbent paper after each wash. Add 200 μL of blocking buffer (5% skim milk in PBST) to each well and block for 2 h at room temperature. The blocking buffer should be prepared in advance and filtered to clarify. Discard the blocking buffer, wash three times with PBST, and pat dry. Seal the plate and store at 4°C. Dilute the specific antibodies Anti-Strep and Anti-His (1:3000) with the blocking buffer. Add 100 μL of the corresponding diluted primary antibody solution to each well. Seal the ELISA plate and incubate overnight at 4°C. Discard the primary antibody solution, wash thoroughly 5 times with PBST, and blot dry. Dilute the HRP (horseradish peroxidase)-labeled secondary antibody (HRP-anti-mouse IgG) with blocking buffer (1:2000). Add 100 μL of the diluted secondary antibody solution to each well and incubate at room temperature in the dark for 1 h. Discard the secondary antibody solution, wash thoroughly 6 times with PBST to ensure complete removal of unbound secondary antibody, and blot dry. According to the Beyotime SignalUp™ ELISA kit (HRP fluorescence method) instructions, mix thoroughly and in the dark to obtain 100 μL of ADHP working solution. Add 100 μL of ADHP working solution to each well and incubate strictly in the dark at room temperature for 10 min. Add 20 μL of Stop Solution to each well to terminate the reaction. Allow the signal to stabilize for at least 1 hour after termination. Detect using a fluorescence microplate reader. Set the parameters: excitation wavelength (Ex) = 530 nm, emission wavelength (Em) = 590 nm. Control group: empty vector yeast cells; Experimental group: Exp1 ( Sc -TG detection YtdepA), Exp2 ( Sc -TG detection YtdepB), Exp3 ( Sc -HG detection YtdepA), Exp4 ( Sc -HG detection of YtdepB). Detection results are as follows: Figure 3 The results showed that the display level of YtdepA (dehydrogenase) in Sc-HG was lower than that in Sc-TG, but the display level of YtdepB (reductase) was higher than that in Sc-TG.

[0044] Example 4: Determination efficiency and metabolic kinetics of vomitoxin (1) Establishment of detoxification reaction system The galactose-induced bacterial culture was centrifuged, eluted, resuspended, and the cell concentration was adjusted to OD0.05. 600=5.0. DON (final concentration 2 μg / mL), cofactor PQQ (20 μM) and CaCl2 (2 mM) were added to a 10 mL reaction system. The system was placed in a constant temperature shaker at 29℃ for detoxification transformation. Samples were taken at time points of 0, 24, 48, 72 and 96 h, and the supernatant was collected by centrifugation.

[0045] (2) HPLC-Q-TOF MS detection and analysis To quantify the efficiency of the in vitro cascade catalytic conversion of DON to 3-epi-DON by engineered bacteria, high-performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (HPLC-Q-TOF MS) was used to monitor the substrate consumption and metabolite formation in the fermentation broth throughout the entire process. Specific characteristic ion peaks (DON and 3-epi-DON plus Na) were extracted. + Mass-to-charge ratio: m / z is 319.1152; 3-keto-DON plus H + The mass-to-charge ratio (m / z = 295.1176) was precisely quantified, and its specific dynamic changes were analyzed as follows: In the degradation kinetics analysis of the substrate DON, two engineered bacteria co-displaying the dual enzymes were used. Sc -TG and Sc -HG was placed in a galactose-containing induced fermentation system and co-incubated with DON. Quantitative data from liquid chromatography and the DON degradation rate over time confirmed that both genetically engineered bacteria exhibited significant and stable time-dependent degradation ability of DON, but the chassis cells... AGA1 Differences in gene promoter background determined the differences in DON transformation performance between the two strains.

[0046] for Sc -TG strain, the initial average concentration of DON in the system was approximately 1973.36 μg / L. As the reaction proceeded, the DON concentration showed a steady decreasing trend, such as... Figure 4 After 96 h of reaction, the absolute residual concentration of DON in the system decreased to 1467.31 μg / L, corresponding to a DON degradation rate of 25.64%. In comparison, Sc -HG strains exhibited superior catalytic performance, such as Figure 5 In this system, the initial average concentration of DON was approximately 2051.78 μg / L. At the same time point, the absolute residual concentration of DON rapidly decreased to 1427.88 μg / L, corresponding to a degradation rate of 30.41%. A line graph comparing the degradation rates of the two strains is shown below. Figure 6 This intuitively reflects Sc -HG consistently leads the substrate consumption rate throughout the entire reaction cycle. Sc -TG, which fully confirms the sugar-free environment HXT7The derepression effect of the promoter is more advantageous in driving the synthesis of the anchored protein AGA2, which increases the enzyme loading density on the cell surface and improves substrate conversion efficiency.

[0047] exist Sc -TG system ( Figure 4 ), intermediate metabolite 3 - The accumulation of keto-DON was quite significant. At 24 h of reaction, the concentration of 3-keto-DON reached 38.13 μg / L, and steadily increased at subsequent time points, accumulating to 148.89 μg / L by 96 h. Simultaneously, the formation of the target low-toxicity end product, 3-epi-DON, showed a relatively delayed and slow increase. No significant formation was detected in the early stage of the reaction (0-24 h), but it began to appear at 48 h (90.63 μg / L), reaching 354.23 μg / L by 96 h. Based on this metabolite change trend, it can be inferred that due to… Sc The limited density of dual-enzyme display on the TG surface leads to insufficient substrate supply for the second-step reduction reaction, thus limiting conversion capacity. Sc -HG system throughout the entire 96 h reaction cycle ( Figure 5 The concentration of the intermediate 3-keto-DON was drastically suppressed to extremely low levels: 37.60 μg / L at 24 h, 40.61 μg / L at 48 h, 50.76 μg / L at 72 h, and only slightly increased to 55.41 μg / L at 96 h. Corresponding to the strong suppression of the intermediate was the continuous formation of the final product 3-epi-DON. It was also undetectable in the 0-24 h stage, but at 48 h, the concentration of 3-epi-DON rose to 201.48 μg / L, and accumulated to 360.73 μg / L and 565.09 μg / L at 72 h and 96 h, respectively. However, from... Figure 3 It can be seen from this that YtdepA is in Sc -HG expression is lower in HG bacteria than in Sc In -TG bacteria, it may be due to... Sc YtdepB occupies more cell surface sites in HG bacteria. However, YtdepB in Sc The expression level in HG bacteria was significantly higher than that in HG bacteria. Sc -TG, which also explains 3-epi-DON in Sc The concentration of HG bacteria was much higher than that in... Sc -TG bacteria. The above results confirm that it is a genetically engineered bacterium. Sc -HG plays a significant role in the degradation of DON.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for displaying and expressing vomitoxin detoxifying enzymes on the surface of yeast, characterized in that, Includes the following steps: (1) Targeted modification of the chassis cells of Saccharomyces cerevisiae, replacing the endogenous promoter of its endogenous anchoring protein AGA1 gene with a strong constitutive promoter. TEF1 and glucose-responsive promoters HXT7 Two modified chassis cells were obtained. (2) Construct a dual-detoxification enzyme yeast free expression vector, wherein the expression vector contains a galactose-inducible promoter. GAL1 Driven AGA2 With detoxification enzyme A gene YtdepA and detoxification enzyme B gene YtdepB , wherein YtdepA The nucleotide sequence is shown in SEQ ID NO:

5. YtdepB The nucleotide sequence is shown in SEQ ID NO: 7; (3) The expression vector constructed in step (2) was co-transformed into the chassis cells modified in step (1) to obtain two strains of double-enzyme co-display engineered bacteria. The engineered bacteria TEF1-AGA1-GAL1-AGA2-YtdepA-YtdepB-CENPK2 was named Sc -TG,HXT7-AGA1-GAL1-AGA2-YtdepA-YtdepB-CENPK2 is named Sc -HG; (4) The engineered bacteria obtained in step (3) were induced to express the base protein AGA1. First, they were cultured in a glucose-containing medium to express the base protein AGA1. Then, they were induced to be cultured in an induction medium with galactose as the carbon source so that the detoxification enzymes YtdepA and YtdepB, which were respectively coupled with AGA2, were co-displayed on the surface of the yeast cells.

2. The method according to claim 1, characterized in that, In step (1), the TEF1 promoters and HXT7 The nucleotide sequences of the promoter are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

3. The method according to claim 1, characterized in that, The brewer's yeast chassis cells mentioned in step (1) are brewer's yeast CEN.PK2.

4. The method according to claim 1, characterized in that, The detoxification enzyme YtdepA in step (2) carries a Strep-Tag II tag, and the detoxification enzyme YtdepB carries a 6xHis tag.

5. The method according to claim 1, characterized in that, The expression vector described in step (2) uses the pYD1 plasmid as a backbone. YtdepA Genes and YtdepB Genes and anchoring proteins AGA2 Gene fusion expression, the nucleotide coding sequence of the anchoring protein AGA2 is shown in SEQ ID NO:

4.

6. The method according to claim 1, characterized in that, The glucose concentration in the glucose-containing culture medium in step (4) is 18-22%, and the induction culture medium contains 18-22% galactose.

7. A dual-enzyme co-display engineered bacterium constructed by the method according to any one of claims 1 to 6, characterized in that, The engineered bacteria Sc -TG genome AGA1 Genes are subject to constitutive promoters TEF1 Drive, the TEF1 The nucleotide sequence of the promoter is shown in SEQ ID NO: 1, and the engineered bacteria Sc -HG genome AGA1 Glucose-responsive promoters HXT7 Drive, the HXT7 The nucleotide sequence of the promoter is shown in SEQ ID NO:

2. Sc -TG and Sc -HG all carry by GAL1 Promoter-driven detoxification enzyme genes YtdepA and YtdepB The expression carrier, the YtdepA The nucleotide sequence is shown in SEQ ID NO:

5. YtdepB The nucleotide sequence is shown in SEQ ID NO:

7.

8. The engineered bacteria for dual-enzyme co-display according to claim 7, characterized in that, The detoxification enzymes YtdepA and YtdepB are bound to the AGA1 anchoring protein via disulfide bonds through the AGA2 carrier protein, achieving co-extension on the surface of yeast cells and forming a cascade catalytic system with spatial proximity effect; wherein the amino acid sequence of YtdepA is shown in SEQ ID NO: 6, and the amino acid sequence of YtdepB is shown in SEQ ID NO:

8.

9. The use of the dual-enzyme co-display genetically engineered bacteria according to claim 7 or 8 in the preparation of a biocatalyst for degrading vomitoxin.

10. A fusion protein, characterized in that, The fusion protein comprises a carrier protein AGA2 and a detoxification enzyme YtdepA or YtdepB; the amino acid sequence of the detoxification enzyme YtdepA is shown in SEQ ID NO: 6, the amino acid sequence of the detoxification enzyme YtdepB is shown in SEQ ID NO: 8, and the nucleotide sequence encoding the anchoring protein AGA2 is shown in SEQ ID NO: 4.

Images