Engineering bacterium capable of efficiently degrading aflatoxin B1 as well as construction method and application of engineering bacterium

Abstract

The invention provides an engineering bacterium capable of efficiently degrading aflatoxin B1 as well as a construction method and application of the engineering bacterium. The engineering bacterium PD630:: lac103sp is obtained by taking rhodococcus turbid PD630 as an original strain and expressing a modified fmb-rL103 protein coding gene, and the nucleotide sequence of the modified fmb-rL103 protein coding gene is as shown in SEQ ID NO: 1. Compared with wild rhodococcus turbid PD630, the engineering bacterium PD630:: lac103sp provided by the invention has the advantages that the degradation efficiency of the engineering bacterium PD630:: lac103sp on aflatoxin B1 is obviously improved, the problems of insufficient degradation efficiency and poor application stability of an existing strain on aflatoxin B1 are solved, and a novel functional strain and a technical support are provided for pollution abatement of aflatoxin B1.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of microbial genetic engineering and food safety, specifically to an engineered bacterium that efficiently degrades aflatoxin B1, its construction method, and its application. Background Technology

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. It is highly carcinogenic, teratogenic, and mutagenic, and has been classified as a Group 1 carcinogen by the World Health Organization (WHO). AFB1 is widely found in grain crops such as corn, wheat, peanuts, and soybeans, as well as various animal feeds. Even at low concentrations, it can cause irreversible damage to the liver and kidneys in humans, while also leading to decreased livestock and poultry production performance and deterioration of product quality, seriously threatening food safety and supply chain security.

[0003] Currently, AFB1 detoxification technologies mainly include three categories: physical adsorption, chemical degradation, and biodegradation. Among them, physical adsorption (such as activated carbon adsorption) easily leads to the loss of nutrients; chemical degradation (such as ozone and alkaline treatment) may produce toxic byproducts and poses a risk of residue; while biodegradation mainly relies on the specific action of microorganisms or their metabolites (such as enzyme preparations), and can degrade AFB1 into low-toxicity or non-toxic products under mild conditions. It has advantages such as being environmentally friendly, efficient, and highly specific, and has become the core direction of current AFB1 detoxification research.

[0004] Existing AFB1-degrading microorganisms still face numerous bottlenecks in practical applications. Many wild-type strains exhibit low degradation efficiency for AFB1, failing to meet the demands for rapid and efficient detoxification in actual production. Some strains may pose biosafety risks, or their genetic background is unclear, making them unsuitable for food-grade applications. Furthermore, while directly using enzymes isolated and purified from microorganisms to degrade AFB1 improves specificity, free enzymes are easily inactivated, difficult to recover, and have high production costs, hindering their large-scale application. Therefore, constructing engineered strains with high efficiency, stable degradation capabilities, and high safety using genetic engineering techniques is crucial to overcoming the current bottlenecks in AFB1 biological detoxification technology. Summary of the Invention

[0005] To address the issues of insufficient degradation efficiency and poor application stability of existing strains for AFB1, this invention provides an engineered strain that efficiently degrades aflatoxin B1, along with its construction method and application.

[0006] According to a first aspect of the present invention, an engineered bacterium PD630::lac103sp that efficiently degrades aflatoxin B1 is provided. The engineered bacterium PD630::lac103sp is obtained by expressing a modified fmb-rL103 protein-coding gene from Rhodococcus turbidus PD630 as the starting strain. The nucleotide sequence of the modified fmb-rL103 protein-coding gene is shown in SEQ ID NO: 1.

[0007] The inventors of this application obtained the lac103sp gene with the nucleotide sequence shown in SEQ ID NO: 1 by codon optimization, promoter fusion, and signal peptide fusion modification of the fmb-rL103 protein-coding gene (i.e., lac103 gene) derived from Bacillus vallismortis fmb-103. The lac103sp gene was then introduced into Rhodococcus opacus PD630 to construct a recombinant strain. After screening the recombinant strain, a genetically engineered strain that efficiently degrades aflatoxin B1 (AFB1) was obtained and named Rhodococcus opacus PD630::lac103sp, which is the highly efficient aflatoxin B1 degrading engineered strain PD630::lac103sp provided by this invention.

[0008] Compared to wild-type Rhodococcus turbidus PD630, the engineered strain PD630::lac103sp provided by this invention significantly improves the degradation efficiency of AFB1, solving the problems of insufficient degradation efficiency and poor application stability of existing strains, and providing new functional strains and technical support for AFB1 pollution control.

[0009] According to a second aspect of the present invention, a method for constructing an engineered bacterium PD630::lac103sp that efficiently degrades aflatoxin B1 is provided, comprising the following steps: S1. The modified fmb-rL103 protein-coding gene was introduced into an expression vector to construct a recombinant plasmid. The nucleotide sequence of the modified fmb-rL103 protein-coding gene is shown in SEQ ID NO: 1. S2. Mix the donor bacteria containing the recombinant plasmid with the recipient bacteria to obtain a bacterial solution; S3. Spread the bacterial solution onto the culture medium, and after culturing, obtain the recombinant strain; S4. The engineered strain PD630::lac103sp was obtained by screening the recombinant strain.

[0010] In the method for constructing the engineered bacterium PD630::lac103sp that efficiently degrades aflatoxin B1 provided in this scheme, the modified fmb-rL103 protein-coding gene (i.e., the lac103sp gene with the nucleotide sequence shown in SEQ ID NO: 1) is cloned into an expression vector. The constructed recombinant plasmid pJXNU-lac103sp is introduced into the donor bacterium and mixed with the recipient bacterium. After being spread on a culture medium for cultivation and screening, the engineered bacterium PD630::lac103sp that can efficiently degrade aflatoxin B1 is obtained.

[0011] The method for constructing the engineered bacterium PD630::lac103sp provided in this scheme includes four core steps in the actual construction process: gene optimization and vector construction, conjugation transfer and host transformation, resistance screening and positive clone identification, and genetic stability verification. This method is highly controllable and can efficiently obtain homozygous and stable recombinant strains, namely the engineered bacterium PD630::lac103sp that efficiently degrades aflatoxin B1 provided by this invention.

[0012] Preferably, in S1, the expression vector is the actinomycete integrative vector pSET152.

[0013] Preferably, in S2, the ratio of the number of donor bacteria to the number of recipient bacteria is (0.5~2):(1~3).

[0014] Preferably, in S2, the donor bacteria is Escherichia coli DH5α and the recipient bacteria is Rhodococcus turbidus PD630.

[0015] Preferably, in S3, the culture medium is M-ISP4 medium containing 5-15 mM MgCl2.

[0016] Preferably, S3 includes the following operation: spreading the bacterial solution onto a culture medium and culturing it at 28-32°C for 18-20 hours to obtain a recombinant strain.

[0017] Preferably, in S3, engineered bacteria PD630::lac103sp are screened from recombinant strains using a mixed antibiotic; the mixed antibiotic includes the following components: 45~55 μg / mL trimethoprim, 45~55 μg / mL apramycin, and 45~55 μg / mL kanamycin.

[0018] According to a third aspect of the present invention, the engineered strain PD630::lac103sp, which is described above as highly efficient at degrading aflatoxin B1, is provided for the application of aflatoxin B1 detoxification in food, feed, and agricultural products.

[0019] The engineered bacterium PD630::lac103sp provided by this invention can efficiently degrade aflatoxin B1. Applying this engineered bacterium PD630::lac103sp to the detoxification treatment of aflatoxin B1 in food, feed, and agricultural products can significantly reduce the aflatoxin B1 content in these products, making them safer. This provides an efficient biological solution to the food safety problems caused by aflatoxin B1 contamination. Furthermore, the engineered bacterium PD630::lac103sp can degrade its products through liquid fermentation, leaving no toxic residues and not affecting the nutritional quality of the substrate, thus showing broad prospects for industrial application.

[0020] According to a fourth aspect of the present invention, the engineered bacterium PD630::lac103sp, which is constructed using the above-described method for constructing an engineered bacterium that efficiently degrades aflatoxin B1, is provided for use in the detoxification of aflatoxin B1 in food, feed, and agricultural products.

[0021] The engineered bacterium PD630::lac103sp, constructed using the method provided in this invention, can be applied to the detoxification treatment of aflatoxin B1 in food, feed, and agricultural products. This significantly reduces the aflatoxin B1 content in these products, making them safer and providing an efficient biological solution to food safety issues caused by aflatoxin B1 contamination. Furthermore, the engineered bacterium PD630::lac103sp can degrade its products through liquid fermentation, leaving no toxic residues and not affecting the nutritional quality of the substrate, thus demonstrating broad prospects for industrial application.

[0022] According to a fifth aspect of the present invention, an aflatoxin B1 detoxifier is provided, the aflatoxin B1 detoxifier comprising the above-mentioned highly efficient aflatoxin B1 degrading engineered bacterium PD630::lac103sp or the highly efficient aflatoxin B1 degrading engineered bacterium PD630::lac103sp constructed using the above-mentioned method for constructing the highly efficient aflatoxin B1 degrading engineered bacterium PD630::lac103sp.

[0023] The engineered bacteria PD630::lac103sp provided by this invention, or the engineered bacteria PD630::lac103sp constructed using the method provided by this invention, can efficiently degrade aflatoxin B1. Therefore, using engineered bacteria PD630::lac103sp as the main active ingredient of aflatoxin B1 detoxifier, the resulting detoxifier can efficiently degrade aflatoxin B1, thereby achieving the detoxification treatment of aflatoxin B1. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the restriction enzyme sites of the modified fmb-rL103 protein-coding gene (lac103sp gene) provided in Example 1.

[0025] Figure 2 The figure shows the effect of the three antibiotics provided in Example 1—trimethoprim (Tmp), apramycin (Apr), and kanamycin (Kan)—on the growth of wild-type Rhodococcus opacus PD630.

[0026] Figure 3 The diagram shows the structure of the actinomycete integrated vector pSET152 provided in Example 1.

[0027] Figure 4 The diagram shows the structure of the recombinant plasmid pJXNU-lac103sp obtained in Example 1.

[0028] Figure 5 The image shows the agarose gel electrophoresis results of the colony PCR amplification products of the recombinant plasmid pJXNU-lac103sp provided in Example 1 after it was introduced into Escherichia coli DH5α.

[0029] Figure 6 The image shows the agarose gel electrophoresis results of the enzyme digestion products obtained after introducing the recombinant plasmid pJXNU-lac103sp provided in Example 1 into Escherichia coli donor bacteria and digesting it with enzymes.

[0030] Figure 7 The diagram shows the colony PCR identification process and agarose gel electrophoresis results of the colony PCR amplification products of the engineered strain PD630::lac103sp provided in Example 2.

[0031] Figure 8 The sequence alignment results are for the 16S rRNA colony PCR amplification products of Rhodococcus turbidus PD630 provided in Example 2.

[0032] Figure 9 The image shows the culture results of the engineered strain PD630::lac103sp provided in Example 2 on M-ISP4 medium containing trimethoprim (Tmp), apramycin (Apr), and kanamycin (Kan).

[0033] Figure 10 The graph shows the degradation rate of AFB1 by the engineered bacteria PD630::lac103sp and wild-type Rhodococcus turbidus provided in Example 3 at 48 h. Detailed Implementation

[0034] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Construction method of genetically engineered strain PD630::lac103sp This embodiment aims to construct a recombinant strain PD630::lac103sp using genetic engineering. The construction method includes the following steps: 1. Optimization of the fmb-rL103 protein-coding gene (i.e., lac103 gene) and construction of recombinant plasmids (1) *Rhodococcus turbidus* PD630 is taxonomically classified as an actinomycete. Based on the codon bias of *Streptomyces ceolicolor*, a cloning sequence was designed. The fmb-rL103 protein-coding gene (denoted as lac103 gene, GenBank accession numbers: KC859464, MK061426) derived from *Bacillus vallismortis* fmb-103 was modified. The modification process included codon optimization, promoter fusion, and signal peptide fusion, resulting in the modified fmb-rL103 protein-coding gene, named lac103sp. Its full length is 1551 bp, and the specific nucleotide sequence is shown in SEQ ID NO: 1. Genscript was commissioned to synthesize the lac103sp gene with the nucleotide sequence shown in SEQ ID NO: 1. The restriction enzyme sites of the synthesized lac103sp gene fragment are as follows: Figure 1 As shown, its upstream contains the NdeI endonuclease recognition site CATATG, and its downstream contains the XbaI endonuclease recognition site TCTAGA. The sequence from the 4th base to the 1545th base is the complete coding region of the recombinant protein Lac103, which has the function of degrading highly active aflatoxin B1 (AFB1).

[0036] SEQ ID NO: 1 2. Host bacterial resistance verification (preliminary experiments) To clarify the resistance characteristics of the wild-type Rhodococcus opacus PD630 strain (the host bacterium, i.e., the recipient bacterium) and verify its resistance, the specific experimental procedures were as follows: Using wild-type Rhodococcus opacus PD630 strain as the experimental object, M-ISP4 medium specifically for actinomycetes was used to test the effects of three antibiotics—trimethoprim (Tmp), apramycin (Apr), and kanamycin (Kan)—on the growth of the strain. The results are as follows: Figure 2 As shown.

[0037] Depend on Figure 2 It is known that Rhodococcus turbidus PD630 is resistant to Tmp, has no resistance to Apr and the "Apr+Kan combination", and only shows weak resistance to Kan. The above resistance characteristics of Rhodococcus turbidus PD630 will serve as the basis for subsequent screening of recombinant strains to determine antibiotic selection.

[0038] 3. Construction and Dual Validation of Recombinant Plasmids (1) Construction of recombinant plasmids Construct recombinant plasmids according to the following steps: [structure as shown in the original text] Figure 3 Based on the actinomycete integrative vector pSET152 shown, the optimized AFB1 degradation gene (lac103sp) was cloned into the expression vector, i.e., the actinomycete integrative vector pSET152, through molecular cloning steps such as XbaⅠ / NdeⅠ restriction enzyme digestion and ligation, resulting in the structure shown below. Figure 4 The recombinant plasmid pJXNU-lac103sp is shown.

[0039] (2) Dual verification of recombinant plasmids First verification (preservation and preliminary identification): The recombinant plasmid pJXNU-lac103sp obtained above was introduced into E. coli DH5α (for plasmid preservation). Verification was performed by colony PCR and sequencing. The colony PCR amplification products were verified using agarose gel electrophoresis. The results are as follows: Figure 5 As shown.

[0040] Depend on Figure 5 It can be seen that the band size (1723 bp) and sequence of the colony PCR amplification product of the constructed recombinant plasmid pJXNU-lac103sp were consistent with the design when it was introduced into Escherichia coli DH5α, confirming that the sequence of the constructed recombinant plasmid pJXNU-lac103sp was correct.

[0041] for Figure 5The above results can be explained as follows: Although the lac103sp gene is 1551 bp in length, while the colony PCR amplification product is 1723 bp, this difference in length is one of the key bases for verifying the successful construction of the recombinant plasmid. The universal primers (M13F / R) used in colony PCR have binding sites on both sides of the cloning site of the recombinant plasmid. Therefore, the obtained colony PCR amplification product contains not only the target gene but also part of the vector sequence, resulting in a total length greater than the gene itself. If the lac103sp gene is not successfully inserted into the plasmid, the region between the universal primers in colony PCR will only be a part of the recombinant plasmid backbone, and the expected colony PCR amplification product will be much smaller than 1723 bp (e.g., only about 172 bp), which is clearly inconsistent with the current results. In addition, the colony PCR amplification product is longer than the target gene, which helps to ensure complete coverage of the target insertion fragment and the connecting regions at both ends in subsequent sequencing, thereby accurately verifying whether the gene is correctly integrated, whether the reading frame is complete, and whether there are any mutations in the sequence.

[0042] Therefore, the agarose gel electrophoresis results showed that the size of the colony PCR amplification product was consistent with the expectation. Combined with the sequencing results from Sangon Biotech (Shanghai) Co., Ltd., the sequencing results were compared with those obtained by SnapGene and the peak shape was judged. The bases in the reliable regions were selected and spliced ​​upstream and downstream on SeqMan to obtain a reliable sequencing base sequence. Combined with the sequencing results, it can be confirmed that the recombinant plasmid construction was correct.

[0043] The second verification (donor bacteria introduction verification): The correctly verified recombinant plasmid pJXNU-lac103sp was introduced into the E. coli donor bacteria (ET12567 / pUZ8002). After enzyme digestion, the digestion products were verified by agarose gel electrophoresis. The results are as follows: Figure 6 As shown, lane 1's original plasmid refers to the undigested recombinant plasmid pJXNU-lac103sp, while lanes 2 and 3's digested plasmids refer to the digested recombinant plasmid pJXNU-lac103sp.

[0044] Depend on Figure 6 The band size of the recombinant plasmid pJXNU-lac103sp after enzyme digestion was 1544 bp, which met the expected design. This confirms the integrity and correct construction of the recombinant plasmid, preparing it for subsequent conjugation and transfer.

[0045] for Figure 6The above results are explained as follows: The size of the band obtained after enzyme digestion is 1544 bp, which is slightly smaller than the 1551 bp of the lac103sp gene itself. This difference is normal because the insert fragment of the lac103sp gene is designed with specific enzyme digestion sites at both ends. During the enzyme digestion process, some enzyme digestion site sequences will be removed. Therefore, the enzyme digestion product often does not contain complete enzyme digestion site sequences, resulting in its length being slightly smaller than the original gene fragment. Enzyme digestion verification can only reflect whether the fragment size and enzyme digestion pattern meet expectations. Further sequencing of the enzyme digestion product by Sangon Biotech (Shanghai) Co., Ltd., combined with the obtained sequencing results, confirms that the constructed recombinant plasmid is correct.

[0046] 4. Conjugation transfer and screening of recombinant strains (1) Based on the Escherichia coli-Rhodococcus indirect co-transfer technology, the recombinant plasmid pJXNU-lac103sp was introduced into Escherichia coli DH5α to obtain donor bacteria containing pJXNU-lac103sp. Rhodococcus turbidus PD630 was used as recipient bacteria. The donor bacteria and recipient bacteria were mixed at a ratio of 1:2 to obtain bacterial solution. (2) The bacterial culture was spread on M-ISP4 medium containing MgCl2 and cultured at 30°C for 19 hours to promote the transfer of recombinant plasmid pJXNU-lac103sp from the donor bacteria to the recipient bacteria, and the recombinant strain was obtained. (3) Prepare a mixed antibiotic containing 50 μg / mL trimethoprim (Tmp), 50 μg / mL apramycin (Apr), and 50 μg / mL kanamycin (Kan); (4) The recombinant strain obtained by the above-prepared mixed antibiotics is screened to kill the donor bacteria and retain the conjugate containing the recombinant plasmid to obtain the potential recombinant strain and name it PD630::lac103sp, which is the engineered strain PD630::lac103sp that provides the present invention for the efficient degradation of aflatoxin B1.

[0047] Example 2: Molecular and phenotypic verification of engineered bacterium PD630::lac103sp This implementation aims to perform a final identification of the engineered bacteria PD630::lac103sp obtained in Example 1. The specific identification experiments and operation steps are as follows: 1. Molecular identification (1) Validation of target gene integration The engineered bacteria PD630::lac103sp constructed in Example 1 were cultured in M-ISP4 medium containing trimethoprim (Tmp), apramycin (Apr), and kanamycin (Kan). Single colonies were picked and colony PCR was performed using the first verification primer pair (used to amplify the lac103sp gene, including the first upstream primer M13F and the first downstream primer M13R). The colony PCR amplification products were then identified by agarose gel electrophoresis, and the colony PCR amplification products and the target sequence (i.e., the recombinant plasmid pJXNU-lac103sp constructed in Example 1) were sequenced. The agarose gel electrophoresis results of the colony PCR amplification products are shown below. Figure 7 As shown, the sequencing results are as follows: the length of the colony PCR amplification product is 1636 bp, and the nucleotide sequence is shown in SEQ ID NO: 2; the length of the recombinant plasmid pJXNU-lac103sp is 1635 bp, and the nucleotide sequence is shown in SEQ ID NO: 3.

[0048] SEQ ID NO: 2 SEQ ID NO:3 The sequencing results show that both SEQ ID NO: 2 and SEQ ID NO: 3 are correct. The two nucleotide sequences are complementary sequences with opposite directions. The sequencing results of the product obtained from colony PCR amplification of the engineered bacterium PD630::lac103sp are basically consistent with the target sequence (i.e., the recombinant plasmid pJXNU-lac103sp constructed in Example 1) (only one base error, which is an accidental sequencing error). Figure 7 The agarose gel electrophoresis results showed that the lac103sp specific band (1723 bp) could be successfully obtained from the engineered bacteria PD630::lac103sp constructed in Example 1 using colony PCR.

[0049] Based on the sequencing results and agarose gel electrophoresis results, it can be confirmed that the lac103sp gene has been successfully integrated into the genome of Rhodococcus turbidus PD630.

[0050] (2) Host background purity verification The engineered bacteria PD630::lac103sp constructed in Example 1 were cultured in M-ISP4 medium containing trimethoprim (Tmp), apramycin (Apr), and kanamycin (Kan). Single colonies were picked and colony PCR was performed using the second verification primer pair (used to amplify the 16S rDNA of Rhodococcus turbidus PD630, which includes the second upstream primer 27F and the second downstream primer 1492R). Simultaneously, the 16S rDNA gene obtained by PCR was sequenced. The sequencing results are shown in SEQ ID NO: 4. The sequencing results were compared with sequences in the NCBI database. Figure 8 As shown.

[0051] SEQ ID NO: 4 SEQ ID NO:5 Depend on Figure 8 It can be seen that the product of colony PCR amplification of the 16S rDNA of engineered bacterium PD630::lac103sp using the second verification primer pair (nucleotide sequence as shown in SEQ ID NO: 4) has 100% homology with the 16S rDNA of wild-type Rhodococcusopacus PD630 (nucleotide sequence as shown in SEQ ID NO: 5).

[0052] The above results demonstrate that the sequencing results of the product obtained by colony PCR amplification of engineered bacteria PD630::lac103sp are basically consistent with the target sequence (with only one base error, which is an accidental sequencing error). This confirms that the lac103sp gene has been successfully integrated into the genome of Rhodococcus turbidus PD630, and the host background is still PD630, with no contamination from other bacteria or strain variation.

[0053] 2. Phenotypic validation (resistance consistency validation) To test the growth of the engineered bacterium PD630::lac103sp constructed in Example 1 on M-ISP4 medium containing "Tmp+Apr+Kan", the engineered bacterium PD630::lac103sp constructed in Example 1 was cultured in M-ISP4 medium containing trimethoprim (Tmp), apramycin (Apr), and kanamycin (Kan). The results are as follows. Figure 9 As shown.

[0054] Depend on Figure 9 It can be seen that the engineered strain PD630::lac103sp can grow normally on M-ISP4 medium containing Tmp+Apr+Kan, and its resistance marker is completely matched with that of the recombinant plasmid pJXNU-lac103sp, confirming that the genetically engineered strain PD630::lac103sp was successfully constructed.

[0055] 3. Verification of genetic stability The engineered bacterium PD630::lac103sp constructed in Example 1 was passaged for 10 generations on antibiotic-free M-ISP4 medium. The results of resistance plate retrospection and PCR detection showed that the lac103sp gene has genetic stability. This indicates that the engineered bacterium PD630::lac103sp constructed by introducing the lac103sp gene into Rhodococcus turbidus PD630 has good application stability.

[0056] Example 3: Validation of AFB1 degradation performance of genetically engineered strain PD630::lac103sp This embodiment aims to verify the degradation performance of the engineered strain PD630::lac103sp obtained in Example 1 on aflatoxin B1 (AFB1). The specific experimental methods and operation steps are as follows: 1. Experimental Materials and Instruments (1) Strain The engineered bacteria PD630::lac103sp and wild-type Rhodococcus turbidus PD630 were constructed in Example 1; (2) Culture medium NB liquid medium (nutrient broth medium, used for liquid degradation experiments), M-ISP4 solid / liquid medium (used for bacterial culture). (3) AFB1 standard Aflatoxin B1 (AFB1) with a purity ≥98% was used as a standard. It was dissolved in methanol to prepare a stock solution with a concentration of 100 μg / mL and stored at -20℃ in the dark. (4) Instruments High-performance liquid chromatograph (HPLC, equipped with UV detector), high-speed centrifuge, nitrogen evaporator, constant temperature shaker, pH meter.

[0057] 2. Degradation Activity Assay Method (1) HPLC detection conditions A Waters C18 column (5 μm, 4.6 × 150 mm) was used as the chromatographic column; (2) Mobile phase Acetonitrile and ultrapure water were mixed evenly at a volume ratio of 40:60 (v / v) to form the mobile phase; (3) Flow velocity The flow rate of the mobile phase was 1.0 mL / min; (4) Column temperature The column temperature chamber is controlled at 30℃; (5) Detection wavelength The detection wavelength of the ultraviolet spectrophotometer is 365 nm (the characteristic absorption wavelength of AFB1). (6) Injection volume The injection volume for each test is 20 μL; (7) Quantitative methods Quantification was performed using the external standard method (a standard curve of AFB1 was plotted, with a concentration range of 0.1–1.0 μg / mL, R...). 2 ≥0.999); (8) Sample pretreatment Take the bacterial culture or crude enzyme solution containing AFB1, centrifuge at 8000 rpm for 10 min, and collect the supernatant; add an equal volume of dichloromethane, vortex for 5 min, allow to stand for layering, and collect the organic phase; repeat the extraction 3 times, combine the organic phases, and blow dry with nitrogen at 40℃; redissolve the residue with 1 mL of acetonitrile, filter through a 0.22 μm organic phase filter membrane, and use for HPLC analysis; (9) Degradation rate calculation AFB1 degradation rate (%) = (1 - AFB1 concentration in the treatment group / AFB1 concentration in the control group) × 100%; where the control group is a blank culture medium without the strain (containing the same concentration of AFB1), which is tested after the same pretreatment.

[0058] 3. Degradation effect verification results The degradation performance of AFB1 under liquid culture conditions was tested according to the following steps: The engineered strain PD630::lac103sp obtained in Example 1 and wild-type Rhodococcus turbidus PD630 were inoculated into NB medium containing 0.4 μg / mL AFB1, respectively, and cultured at 30℃ and 160 rpm for 48 h with shaking. Samples were taken to detect the residual AFB1 in the system. The results are shown in Table 1 and... Figure 10 As shown, where, Figure 10 The graph shows the degradation rate of AFB1 by engineered strain PD630::lac103sp and wild-type Rhodococcus turbidus PD630 at 48h. The horizontal axis represents strain type, and the vertical axis represents AFB1 degradation rate (%).

[0059] Table 1. Degradation rate of AFB1 by engineered strain PD630::lac103sp and wild-type Rhodococcus turbidus PD630

[0060] Depend on Figure 10 As shown in Table 1, the degradation rate of AFB1 by the engineered bacterium PD630::lac103sp constructed in Example 1 was as high as 85.2%, while the degradation rate of AFB1 by the wild-type Rhodococcus turbidus was 60.4%, showing a significant difference between the two. P <0.05).

[0061] The above results indicate that the introduction of the lac103sp gene significantly improved the degradation efficiency of AFB1 by Rhodococcus turbidus PD630.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. An engineered bacterium, PD630::lac103sp, that efficiently degrades aflatoxin B1, characterized in that: The engineered bacterium PD630::lac103sp was obtained by expressing the modified fmb-rL103 protein-coding gene using Rhodococcus turbidus PD630 as the starting strain. The nucleotide sequence of the modified fmb-rL103 protein-coding gene is shown in SEQ ID NO:

1.

2. A method for constructing an engineered bacterium, PD630::lac103sp, that efficiently degrades aflatoxin B1, characterized in that, Includes the following steps: S1. The modified fmb-rL103 protein-coding gene was introduced into an expression vector to construct a recombinant plasmid. The nucleotide sequence of the modified fmb-rL103 protein-coding gene is shown in SEQ ID NO:

1. S2. Mix the donor bacteria containing the recombinant plasmid with the recipient bacteria to obtain a bacterial solution; S3. Spread the bacterial solution onto a culture medium, and after culturing, obtain a recombinant strain; S4. The engineered strain PD630::lac103sp was obtained by screening the recombinant strain.

3. The method for constructing the engineered strain PD630::lac103sp, which efficiently degrades aflatoxin B1 as described in claim 2, is characterized in that: In S1, the expression vector is the actinomycete integrative vector pSET152.

4. The method for constructing the engineered strain PD630::lac103sp, which efficiently degrades aflatoxin B1 as described in claim 2, is characterized in that: In S2, the ratio of the number of donor bacteria to the number of recipient bacteria is (0.5~2):(1~3). And / or, In S2, the donor bacteria is Escherichia coli DH5α, and the recipient bacteria is Rhodococcus turbidus PD630.

5. The method for constructing the engineered strain PD630::lac103sp, which efficiently degrades aflatoxin B1 as described in claim 2, is characterized in that: In S3, the culture medium is M-ISP4 medium containing 5-15 mM MgCl2.

6. The method for constructing the engineered strain PD630::lac103sp, which efficiently degrades aflatoxin B1 as described in claim 2, is characterized in that, S3 includes the following operation: spreading the bacterial solution onto a culture medium and culturing it at 28-32°C for 18-20 hours to obtain a recombinant strain.

7. The method for constructing the engineered strain PD630::lac103sp, which efficiently degrades aflatoxin B1 as described in claim 2, is characterized in that: In step S3, the engineered strain PD630::lac103sp was screened from the recombinant strain using a mixture of antibiotics. The mixed antibiotic comprises the following components: 45-55 μg / mL trimethoprim, 45-55 μg / mL apramycin, and 45-55 μg / mL kanamycin.

8. The application of the engineered strain PD630::lac103sp, which is described in claim 1, for the detoxification of aflatoxin B1 in food, feed, and agricultural products.

9. The application of the engineered bacterium PD630::lac103sp, which is constructed using the method described in any one of claims 2 to 7 for the efficient degradation of aflatoxin B1, in the detoxification of aflatoxin B1 in food, feed, and agricultural products.

10. An aflatoxin B1 detoxifying agent, characterized in that: The aflatoxin B1 detoxifier includes the engineered bacterium PD630::lac103sp that efficiently degrades aflatoxin B1 as described in claim 1, or the engineered bacterium PD630::lac103sp that efficiently degrades aflatoxin B1 as described in any one of claims 2 to 7.

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