A recombinant expression vector, a recombinant bacillus subtilis and application thereof

Abstract

The application belongs to the technical field of gene editing, and particularly relates to a recombinant expression vector, a recombinant bacillus subtilis and application thereof. The recombinant expression vector comprises an upstream homologous arm sequence, a downstream homologous arm sequence and a gene sequence coding high-sulfur zein for knocking out an endo-1, 4-beta-D-glucanase gene coding region. The application uses a homologous recombination method to induce expression of the Zein gene under control of an endo-1, 4-beta-D-glucanase promoter and a signal peptide sequence, and obtains a homologous recombination bacillus subtilis for efficiently secreting Zein protein. The Met content in fermentation liquor of the obtained homologous recombination bacillus subtilis can be increased by 94.18% compared with that of wild bacteria.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a recombinant expression vector, recombinant Bacillus subtilis, and their applications. Background Technology

[0002] Methionine (Met) is one of the limiting amino acids that animals can only obtain from external sources. Furthermore, Met is a substrate for protein synthesis and an important exogenous signaling stimulant for promoting protein and lipid synthesis. The Met content in animal feed has a significant impact on feed protein conversion rate. Therefore, methionine and methionine-rich proteins are often used as feed additives in livestock production.

[0003] Zeatin is an important source of methionine, and high-sulfur zeatin is a novel methionine-rich corn seed storage protein with a methionine content as high as 37%. Previously, zeatin was obtained by using free plasmids to carry exogenous genes for heterologous protein expression in bacteria. This method has drawbacks such as plasmid instability, dependence on antibiotics, and toxicity of inducers.

[0004] In recent years, domestic and international studies have found that using integrative plasmids for homologous recombination expression of heterologous proteins has the advantages of being more efficient and stable. Summary of the Invention

[0005] This invention utilizes homologous recombination to replace the coding region of the endo-1,4-β-D-glucanase gene in Bacillus subtilis with the zein gene while retaining the signal peptide sequence of the coding region, thereby obtaining a genetically engineered Bacillus subtilis strain that secretes zein protein.

[0006] To achieve the above objectives, the present invention may adopt the following technical solutions;

[0007] This invention provides a recombinant expression vector comprising an upstream homologous arm sequence for knocking out the coding region of an endonuclease-1,4-β-D-glucanase gene, a downstream homologous arm sequence for knocking out the coding region of an endonuclease-1,4-β-D-glucanase gene, and a gene sequence encoding high-sulfur zein; wherein the upstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene, the gene sequence encoding high-sulfur zein, and the downstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene are tandemly linked; wherein the gene sequence encoding high-sulfur zein is shown in SEQ ID NO:3.

[0008] Preferably, the upstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene is shown in SEQ ID NO:1; and / or the downstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene is shown in SEQ ID NO:2.

[0009] Another aspect of the present invention provides a biomaterial, including the recombinant expression vector of the present invention, wherein the biomaterial includes a gene expression kit or engineered cells.

[0010] In another aspect, the present invention provides a recombinant Bacillus subtilis, wherein the gene encoding endonuclease-1,4-β-D-glucanase in Bacillus subtilis is knocked out, and then the recombinant expression vector of the present invention is transferred into Bacillus subtilis to obtain recombinant Bacillus subtilis.

[0011] Preferably, the Bacillus subtilis has the accession number ATCC 23857.

[0012] In another aspect, the present invention provides a fermentation broth or culture medium obtained by fermentation or culture of the recombinant Bacillus subtilis of the present invention.

[0013] In another aspect, the present invention provides a method for preparing high-sulfur zein, comprising: culturing the recombinant Bacillus subtilis of the present invention in a culture medium to obtain high-sulfur zein, wherein the culture medium includes cellulose.

[0014] Preferably, the above preparation method may include one or more of the following conditions: (a) the cellulose includes one or more combinations of sodium carboxymethyl cellulose, microcrystalline cellulose or hay powder; (b) the mass fraction of cellulose is 1%; and (c) the culture time is 12h.

[0015] The application of the recombinant expression vector, biomaterial, recombinant Bacillus subtilis, fermentation broth or culture medium in this invention in increasing the methionine content in animal feed.

[0016] The beneficial effects of this invention include at least the following: This invention utilizes homologous recombination to induce the expression of the Zein gene under the control of the endonuclease promoter and signal peptide sequence, thereby obtaining homologous recombinant Bacillus subtilis that efficiently secretes Zein protein. The Met content in the fermentation broth of the obtained homologous recombinant Bacillus subtilis can be increased by 94.18% compared with that of the wild-type strain. Attached Figure Description

[0017] Figure 1 The results of identification of Bacillus subtilis strain 168 are shown; where M: DL1000 DNA Marker; 1: negative control (Escherichia coli); 2-3: Bacillus subtilis.

[0018] Figure 2 PCR validation of pHY300PLK-Z; where M: DL10000 DNA Marker; 1: pHY300PLK; 2-3: pHY300PLK-Z (parallel experiments);

[0019] Figure 3 Screening of Bacillus subtilis clones homologous recombinant with antibiotic-free and antibiotic-containing agar plates was conducted.

[0020] Figure 4 To detect the integration effect of homologous recombination expression cassettes using PCR amplification and agarose gel electrophoresis; where M: DL5000 DNA Marker; 1: blank control: H2O; 2: negative control (Bacillus subtilis); 3: homologous recombination positive clones;

[0021] Figure 5 To determine the growth performance of recombinant Bacillus subtilis using growth curve analysis;

[0022] Figure 6 To detect the stability of recombinant Bacillus subtilis through sequence analysis; where 1: recombinant bacterial PCR product; 2: target replacement fragment sequence;

[0023] Figure 7 To analyze the expression level of Zein under different induction times and cellulose concentrations using qRT-PCR;

[0024] Figure 8 The expression of the target protein in recombinant Bacillus subtilis was detected by SDS-PAGE; where M: 180kDa Marker; 1: fermentation broth of wild-type Bacillus subtilis; 2: fermentation broth of Bacillus subtilis after cellulose induction; 3: fermentation broth of recombinant Bacillus subtilis; 4: fermentation broth of recombinant Bacillus subtilis after cellulose induction; 5: fermentation broth of recombinant Bacillus subtilis after cellulose + Met induction.

[0025] Figure 9 To determine the Met content in the fermentation broth of wild-type and recombinant strains using HPLC-MS; (a) supernatant of fermentation broth from the trypsin + streptomycin E digestion group; (b) cell precipitate from the trypsin + streptomycin digestion group; (c) supernatant of fermentation broth from the trypsin digestion group; (d) cell precipitate from the trypsin digestion group.

[0026] Figure 10 Comparison of Met content in fermentation broths of wild-type and recombinant strains. Detailed Implementation

[0027] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0028] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0029] This invention provides a recombinant expression vector comprising an upstream homologous arm sequence for knocking out the coding region of an endonuclease-1,4-β-D-glucanase gene, a downstream homologous arm sequence for knocking out the coding region of an endonuclease-1,4-β-D-glucanase gene, and a gene sequence encoding high-sulfur zein; wherein the upstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene, the gene sequence encoding high-sulfur zein, and the downstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene are tandemly linked; wherein the gene sequence encoding high-sulfur zein is shown in SEQ ID NO:3.

[0030] It should be noted that this invention uses the integrative plasmid pHY300PLK for homologous recombination to express the Zein gene in Bacillus subtilis, aiming to provide Met-rich Bacillus subtilis for livestock and poultry production. Furthermore, Bacillus subtilis is a probiotic strain recognized by the US FDA as "Generally Regarded as Safe" (GRAS) and has also been approved in China as a probiotic strain for use as a feed additive; Bacillus subtilis does not exhibit significant codon bias, making it an excellent choice for expressing exogenous proteins.

[0031] It should also be noted that endo-1,4-β-D-glucanase (EC3-2-1-4) (gene bank ID: 938607) is a cellulase that specifically hydrolyzes and cleaves the β-1,4-glycosidic bonds in cellulose molecules in Bacillus subtilis, thereby breaking down cellulose into reducing oligosaccharides.

[0032] It should also be noted that the present invention can use overlap extension PCR to link the 500bp sequences of each of the upstream and downstream homologous arms of the endonuclease-1,4-β-D-glucanase gene with the Zein sequence of the high-sulfur zein gene to form a tandem relationship. Overlap extension PCR is a method known in the art.

[0033] In some specific embodiments, the upstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene is shown in SEQ ID NO:1; and / or the downstream homologous arm sequence for knocking out the coding region of the endonuclease-1,4-β-D-glucanase gene is shown in SEQ ID NO:2.

[0034] This invention also provides a biomaterial, including the recombinant expression vector of this invention, wherein the biomaterial includes a gene expression kit or engineered cells.

[0035] It should be noted that the recombinant expression vector in this invention can be made into a gene expression kit or engineered cells, which is more conducive to the expression of the zein gene.

[0036] This invention also provides a recombinant Bacillus subtilis, which involves knocking out the gene encoding endonuclease-1,4-β-D-glucanase in Bacillus subtilis, and then transferring the recombinant expression vector of this invention into Bacillus subtilis to obtain recombinant Bacillus subtilis.

[0037] It should be noted that in this invention, the coding region sequence of the endonuclease-1,4-β-D-glucanase gene in Bacillus subtilis is replaced with the high-sulfur zein gene sequence, and the coding region signal peptide sequence of the endonuclease-1,4-β-D-glucanase gene is retained.

[0038] In some specific embodiments, the Bacillus subtilis described above has the accession number ATCC 23857.

[0039] It should be noted that the Bacillus subtilis in this invention can be any Bacillus subtilis known in the art, preferably Bacillus subtilis 168, whose accession number is ATCC 23857.

[0040] This invention also provides a fermentation broth or culture medium obtained by fermentation or culture of the recombinant Bacillus subtilis of this invention.

[0041] This invention also provides a method for preparing high-sulfur zein, comprising: culturing the recombinant Bacillus subtilis of this invention in a culture medium to obtain high-sulfur zein, wherein the culture medium includes cellulose.

[0042] It should be noted that the expression of the endonuclease-1,4-β-D-glucanase gene is induced by the metabolic enzyme substrate cellulose. Cellulose activates the promoter of the endonuclease-1,4-β-D-glucanase gene through signal transduction, resulting in the large-scale expression of the enzyme gene. Under artificial culture conditions, Bacillus subtilis can grow normally without cellulose, indicating that this gene is a non-essential metabolic enzyme gene that can be induced by the cellulose substrate.

[0043] In some specific embodiments, the above preparation method may include one or more of the following conditions: (a) the cellulose includes one or more combinations of sodium carboxymethyl cellulose, microcrystalline cellulose or hay powder; (b) the mass fraction of cellulose is 1%; and (c) the culture time is 12 h.

[0044] This invention also provides an application of the recombinant expression vector, biomaterial, recombinant Bacillus subtilis, fermentation broth or culture medium of this invention in increasing the methionine content in animal feed.

[0045] It should be noted that the recombinant expression vector, biological material, recombinant Bacillus subtilis, fermentation broth or culture medium in this invention can be used as feed additives to supplement Met and improve feed protein conversion rate in animal (such as livestock and aquatic animals) production.

[0046] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0047] The various identification primers used in the following examples are shown in Table 1 below.

[0048] Table 1. Various identification primers used for PCR amplification

[0049]

[0050] In the following examples, Bacillus subtilis 168 (ATCC 23857) was purchased from the Shanghai Center for Microbiological Preservation (SHBCC), strain number SHBCC D25186, abbreviated as Bacillus subtilis 168 strain.

[0051] In the following example, the method for obtaining the Up arm-Zein-Down arm tandem sequence by overlap extension PCR is as follows:

[0052] (1) Search for the sites of endo-1,4-β-D-glucanase (1940625-1942124) on Bacillus subtilis (NC_000964) in NCBI. Based on the sequences 1000bp upstream and 1000bp downstream of the insertion site, use SnapGene software to design corresponding upstream and downstream primers egls Up and egls Down. Design the gene identification primer Zein based on the target gene sequence Zein. Design fusion PCR primers Zein O1 and Zein O2 based on the upstream and downstream homologous arm sequences of wild-type Bacillus subtilis, the Zein gene sequence, and the Bam HI single enzyme restriction site sequence on plasmid pHY300-PLK. The primers used were designed by SanpGene software and synthesized by BGI. The primer sequences are shown in Table 2.

[0053] Table 2 Primers used for overlap extension PCR

[0054]

[0055]

[0056] (2) Extraction of bacterial DNA from Bacillus subtilis. Using DNA as a template, PCR was performed with corresponding primers egls Up and egls Down to amplify the upstream and downstream homologous arms of the substitution site (endo-1,4-β-D-glucanase). The reaction system and steps are shown in Tables 3 and 4. After electrophoresis, the bands of the corresponding lengths were recovered by gel extraction.

[0057] Table 3. PCR reaction system for upstream and downstream homologous arm amplification.

[0058] Components volume 2×Taq PCR StarMix(Dye) 10μL Forward Primer 0.5μL Reverse Primer 0.5μL DNA sample 1μL Sterile Water 8μL Total 20μL

[0059] Table 4. PCR reaction procedures for upstream and downstream homologous arm amplification.

[0060]

[0061] (3) Using the downstream homologous arm and the target gene Zein as templates, fusion PCR was performed with primers Zein O2. The reaction system and steps are shown in Tables 5 and 6. After electrophoresis, the band with a size of about 1109 bp was recovered by gel extraction. The product obtained is the target gene-downstream homologous arm tandem sequence.

[0062] Table 5. PCR reaction system for Zein-downstream homologous arm fusion

[0063]

[0064]

[0065] Table 6. PCR reaction procedure for Zein-downstream homologous arm fusion.

[0066]

[0067] (4) Then, using the upstream homologous arm as a template, fusion PCR was performed using primers F of Zein O1 and R of Zein O2. The reaction system and steps are shown in Tables 7 and 8. After electrophoresis, the bands with a size of about 1632 bp were recovered by gel and sequenced.

[0068] Table 7 PCR reaction system for upstream homologous arm-Zein-downstream homologous arm fusion

[0069] Components volume <![CDATA[ddH2O]]> 22μL <![CDATA[KODOne TM PCRMaster Mix]]> 25μL Forward Primer 1μL Reverse Primer 1μL Zein-downstream homologous arm 0.5μL Upstream homology arm 0.5μL Total 50μL

[0070] Table 8. PCR reaction procedure for upstream homologous arm-Zein-downstream homologous arm fusion.

[0071]

[0072] In the following examples, seamless cloning was performed using the One Step Cloning Kit (Vazyme, Nanjing). The seamless cloning system is shown in Table 9, and the specific steps are as follows:

[0073] (1) Measure the concentration of the vector and the insert fragment respectively, and calculate the optimal dosage according to the concentration and number of base pairs in the instructions;

[0074] (2) On ice, add the linearization vector, insert, buffer, Exnase II and ddH2O sequentially to a 1.5 mL EP tube according to the system size;

[0075] (3) Gently blow and mix well, then briefly centrifuge and collect to the bottom of the tube;

[0076] (4) After bathing in a 37°C water bath for 30 minutes, immediately place it on ice for subsequent operations, or store it in a -20°C refrigerator.

[0077] Table 9 Seamless Cloning System

[0078] Components 50 μL reaction system Linearized carrier ≥1μL Insert fragment ≥1μL 5×CE II Buffer 4μL Exnase II 2μL <![CDATA[ddH2O]]> up to 20μL

[0079] In the following example, the specific method for PCR identification of bacterial strains is as follows: Several Bacillus subtilis colonies were picked and cultured in LB liquid medium at 37°C and 180 rpm for 12 h. Using the bacterial culture as a template, PCR was performed using universal rpoA primers. The reaction system and steps are shown in Tables 10 and 11. Positive bands were recovered from the gel and sent for testing. Bacillus subtilis culture with matching sequencing results was frozen in glycerol.

[0080] Table 10 PCR reaction system for Bacillus subtilis species identification

[0081] Components volume 2×Taq PCR StarMix(Dye) 10μL Forward Primer 0.5μL Reverse Primer 0.5μL DNA sample 1μL Sterile Water 8μL Total 20μL

[0082] Table 11 PCR reaction procedure for Bacillus subtilis species identification

[0083]

[0084] I. Construction of integration expression vectors for homologous recombination double exchange

[0085] (1) The upstream homologous arm sequence Up arm 500bp for replacing the coding region of the Bacillus subtilis 168 endonuclease gene (gene bank ID: 938607) was designed and synthesized, as shown in SEQ ID No. 1; the downstream homologous arm sequence Down arm 500bp for replacing the coding region of the Bacillus subtilis 168 endonuclease gene (gene bank ID: 938607) was designed and synthesized, as shown in SEQ ID No. 2; the high-sulfur zein gene Zein sequence (gene bank ID: KT948686.1) was designed and synthesized, as shown in SEQ ID No. 3;

[0086] (2) The Up arm-Zein-Down arm tandem sequence was obtained by overlapping extension PCR.

[0087] (3) The linearized plasmid pHY300PLK (4872bp) and the upstream homologous arm-Zein-down homologous arm tandem sequence (Up arm-Zein-Down arm, 1632bp) were seamlessly cloned. Both contained BamHI restriction sites and their upstream and downstream homologous sequences. Homologous recombination expression vector was constructed and named pHY300PLK-Zein (also known as pHY300PLK-Z).

[0088] II. Vector transformation and positive clone screening

[0089] (I) Identification of Bacillus subtilis strain 168

[0090] Bacillus subtilis strain 168 was cultured in LB liquid medium (LB medium formula as follows: NaCl 10g, tryptone 10g, yeast extract 5g, agar powder (solid) 20g, deionized water to 1L) to obtain bacterial suspension. Using the bacterial suspension as a template, PCR identification of Bacillus subtilis strain was performed using primer rpoA. The results are as follows. Figure 1As shown, Bacillus subtilis can amplify positive bands (lanes 2 and 3), while Escherichia coli cannot amplify positive bands (lane 1).

[0091] (II) Preparation of competent cells of Bacillus subtilis

[0092] (1) Take out the above-mentioned cultured Bacillus subtilis 168 bacterial culture from the -80℃ freezer, thaw it on ice, and then use an inoculation loop to streak the bacterial culture onto LB solid medium (LB medium formula is as follows: NaCl 10g, tryptone 10g, yeast extract powder 5g, agar powder (solid) 20g, deionized water to 1L) and incubate overnight at 37℃;

[0093] (2) Pick a single colony and inoculate it into 20 mL of LB liquid medium, and incubate at 37°C and 180 rpm for 16 h;

[0094] (1)(3) Take 2.5 mL of bacterial solution into 40 mL of growth medium (the growth medium formula is as follows: NaCl 1 g, yeast extract powder 0.5 g, tryptone 1 g, sorbitol 18.2 g, deionized water to 100 mL) (the volume of bacterial solution is 1 / 16 of the volume of growth medium), and incubate at 37℃ and 180 rpm for 5 h.

[0095] (4) After culturing the bacterial culture medium on ice for 10 min, 20 mL of the culture medium was dispensed into two 50 mL centrifuge tubes; centrifuged at 4℃ and 5000 rpm for 7 min.

[0096] (5) Discard the supernatant and resuspend the bacterial pellet in 50 mL of pre-cooled electrostatic washing buffer (electrostatic washing buffer: 18.2 g sorbitol, 18.2 g mannitol, 34.2 g trehalose, and deionized water to 200 mL). Mix well by pipetting and centrifugation at 4 °C for 5000 rpm for 15 min. Repeat 4 times.

[0097] (6) Discard the supernatant, resuspend the precipitate in 1 mL of electroporation washing solution in each centrifuge tube, and dispense 70 μL into 1.5 mL EP tubes. It can be used immediately or frozen in a -80℃ freezer. The conversion effect will not be significantly affected within 1 month.

[0098] (III) Transformation of Escherichia coli with recombinant vector pHY300PLK-Z

[0099] (1) Take out Escherichia coli DH5α competent cells from the -80℃ freezer and place them on an ice box to thaw slowly;

[0100] (2) In a clean bench, use a pipette to add 5 μL of the ligation product (pHY300PLK-Zein) to 50 μL of competent cells, gently tap the tube wall with your finger, and let it stand on ice for 30 min.

[0101] (3) After heat shock in a 42℃ water bath for 45 seconds, immediately place the ice bath for 2 minutes.

[0102] (4) Add 900 μL of antibiotic-free LB liquid medium and incubate at 37°C and 180 rpm for 60 min with shaking.

[0103] (5) Centrifuge at 5000 rpm for 5 min, and discard 900 μL of supernatant;

[0104] (6) After mixing the remaining 100 μL by pipetting and precipitating, spread it onto LB solid medium containing 100 μg / mL of ampicillin.

[0105] (7) Incubate the plate upright for 1 hour, then invert it and incubate for 16 hours;

[0106] (8) Pick a single colony and inoculate it into 20 ml of LB liquid medium for culture;

[0107] Meanwhile, pHY300PLK was used as a control and transformed into E. coli DH5α competent cells using the above method.

[0108] (iv) Extraction and validation of recombinant vector pHY300PLK-Zein from Escherichia coli

[0109] Plasmids were extracted from the cultured *E. coli* DH5α competent cells using a microbial plasmid extraction kit (Beyotime, Beijing). After extraction, the plasmid concentration was measured and verified by electrophoresis. The results are as follows: Figure 2 As shown, the successfully ligated recombinant vector pHY300PLK-Z (lanes 2 and 3, two parallel lanes) is the same size as expected (4872+1632=6504bp). It has three bands with different separation rates than the original plasmid pHY300PLK (lane 1): open circular, linear and supercoiled structures. The results show that the recombinant vector pHY300PLK-Zein was successfully transformed in E. coli.

[0110] (V) Conditions for electroporation of Bacillus subtilis using pHY300PLK-Zein

[0111] (1) Take out the cultured Bacillus subtilis competent cells prepared in (II) above. If they are taken out from the -80℃ freezer, they need to be placed on ice for slow freezing.

[0112] (2) Add 1 μL of the recombinant vector pHY300PLK-Z to the clean bench;

[0113] (3) Gently blow the carrier and competent cells, mix well, and then transfer them into a sterile electric transfer cup (1mm electrode spacing) that has been pre-cooled at -20℃ for more than 30 minutes. The range of motion should be kept as small as possible throughout the process.

[0114] (4) After the electroporation cup is placed in an ice bath for 5 minutes, wipe the water off the electrode surface with paper, and then place the electroporation cup into the electroporator for electroporation. Electroporation conditions: 2.2 kV, 25 μF, 200 Ω, 4 ms;

[0115] (5) Immediately after the electroporation is completed, remove the electroporation cup, use a pipette to draw 1 mL of recovery solution into the cup, mix it by blowing and then transfer it into a 1.5 mL EP tube;

[0116] (6) Incubate at 37℃ and 90rpm for 6 hours;

[0117] (7) Centrifuge at 7000 rpm for 2 min, resuspend the precipitate in 200 μL of supernatant, mix well by pipetting, and spread on LB solid medium containing 15 μg / mL tetracycline. Incubate overnight at 37℃ for 16 h.

[0118] (8) Pick several single colonies from the plate every other day and inoculate them into LB liquid medium for subsequent testing.

[0119] (vi) Screening of positive clones of transformants

[0120] The bacteria were streaked on LB agar containing tetracycline. Using the electroporated bacterial culture as a template, the target fragment (upstream homologous arm-Zein-down arm) was identified by PCR and sequenced for screening transformant positive clones using primers Uparm-Zein-Down arm. The identification PCR and reaction system and steps are shown in Tables 12 and 13.

[0121] Table 12 PCR reaction system for gene identification of electroporation-positive bacteria

[0122]

[0123]

[0124] Table 13 PCR reaction procedure for gene identification of electroporation-positive bacteria

[0125]

[0126] (vii) Screening of positive clones for homologous recombination

[0127] (1) Streak the positive clones of the above-screened transformants for activation and incubate overnight;

[0128] (2) Pick a single colony and place it in a 10 mL EP tube containing 3 mL of LB liquid medium with a tetracycline concentration of 10 μg / mL, and incubate at 37°C with shaking for 12 h;

[0129] (3) Subculture every 12 hours, using LB liquid medium containing tetracycline throughout the process;

[0130] (4) After continuous subculturing to the 7th generation, take a portion of the bacterial culture every 4 generations to perform upstream homologous arm exchange identification PCR and downstream homologous arm exchange identification PCR. After the product is subjected to agarose gel electrophoresis, if both PCR identification results are negative, continue subculturing and identify once every 4 generations. If one PCR identification result is positive, record it and focus on subculturing and identify once every 2 generations until both PCR results are positive.

[0131] (viii) Screening of Bacillus subtilis clones based on homologous recombinant culture without antibiotic resistance vector

[0132] (1) Take the above-screened homologous recombination positive clones, streak them on LB solid medium containing tetracycline, and incubate overnight at 37°C for 16 hours;

[0133] (2) Pick several single colonies every other day, label them and inoculate them into antibiotic-free LB liquid medium. Subculture at 37°C, subculture once every 12 hours. When the 10th generation is reached, streak them on LB solid medium and incubate upside down at 37°C for 16 hours.

[0134] (3) In the clean bench, use a toothpick to pick up the homologous recombination positive clone, first lightly place it on the antibiotic-free LB solid medium, and then place it on the corresponding position on the tetracycline-containing LB solid medium. When placing the bacteria with the toothpick, control the force so that it can leave an imprint without puncturing the medium. Incubate at 37°C upside down for 12-16 hours.

[0135] (4) Observe the culture dishes every other day, select colonies that grow only on the antibiotic-free plate and do not survive on the resistant plate, label and record them, and then transfer them to both antibiotic-free LB liquid medium and tetracycline-containing LB liquid medium. Incubate at 37°C and 180 rpm for 14 hours. If there are no colonies that meet the conditions on the plate, continue to subculture according to step (2). Repeat the selection operation of step (3) every night until antibiotic-free colonies appear.

[0136] (5) Observe the bacterial culture every other day. If the LB liquid medium without antibiotics becomes turbid while the LB liquid medium containing tetracycline becomes clear, then the screening of Bacillus subtilis without antibiotics is successful. In addition, in order to rule out the possibility of bacterial culture contamination, a portion of the bacterial culture is taken for PCR identification of bacterial species and replacement. The accuracy of the recombinant sequence is determined by PCR amplification and sequencing. The obtained strain is the target strain that stably undergoes homologous recombination.

[0137] Similarly, blank control H2O and negative control Bacillus subtilis were set up;

[0138] Screening results of homologous recombinant Bacillus subtilis clones without resistance vectors, as follows: Figure 3As shown, Bacillus subtilis colonies that only grow on LB plates and do not survive on TET plates (colonies shown in red circles) are selected. Colonies meeting these criteria are recombinant Bacillus subtilis without antibiotic resistance vector and containing the plasmid pHY300PLK-Z, indicating successful homologous recombination. PCR amplification and agarose gel electrophoresis results are shown below. Figure 4 As shown in the figure, after several further purifications of the corresponding colonies, a replacement identification PCR was performed using the bacterial culture as a template and primers Full Re. A single band appeared only at the positive position (2047 bp). This indicates that Bacillus subtilis with successful homologous recombination was successfully screened after multiple passages and purifications.

[0139] (ix) Detection of growth performance of recombinant Bacillus subtilis

[0140] (1) Simultaneously, recombinant bacteria and wild-type Bacillus subtilis were streaked and activated, and then continuously passaged for 20 generations in LB liquid medium.

[0141] (2) Take the bacterial culture of the 20th generation and inoculate it into LB liquid medium at a ratio of 1%. Incubate at 37°C and 180 rpm with shaking. Measure the absorbance at a wavelength of 570 nm every 2 hours using an ELISA reader and plot the growth curve.

[0142] The results are as follows Figure 5 As shown, the growth performance of recombinant Bacillus subtilis was not significantly different from that of wild-type Bacillus subtilis, meaning that the growth performance of recombinant Bacillus subtilis was unaffected.

[0143] (x) Stability test of recombinant Bacillus subtilis

[0144] The recombinant bacterial culture was streaked and activated, then passaged 20 times continuously in antibiotic-free LB liquid medium, and the growth curve was determined. The results are as follows: Figure 5 As shown, the growth curves of the 20th generation recombinant and wild-type Bacillus subtilis are basically the same, indicating that the replacement of the target gene has no effect on the growth performance of the strain.

[0145] In addition, the 20th generation bacterial culture was used for simultaneous PCR identification of the bacterial species and genes to rule out bacterial contamination and verify the presence of the replaced gene. The PCR products were sent for analysis, and sequence alignment was performed using DNAMAN software. The homologous recombination sequence was 100% identical to that of the first generation homologous recombination strain. The results indicate that there was no gene loss during passage, and the replaced target gene can stably exist in the strain. Figure 6 ).

[0146] II. Substrate Induction Methods for Zein Expression in Homologous Recombinant Bacillus subtilis

[0147] (I) qRT-PCR detection of differences in Zein expression induced by cellulose from different sources

[0148] To investigate the effects of cellulose from different sources on Zein gene expression, three induction media were prepared: three cellulose induction groups from different sources and an LB control group. The three cellulose sources included sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose (MCC), and hay powder. After inducing recombinant Bacillus subtilis according to the experimental groups, bacterial RNA was extracted from each group. After reverse transcription, the effects of cellulose from different sources on Zein gene expression were detected by qRT-PCR. The results were then analyzed using 2... -ΔΔCt The method is used for relative quantitative analysis.

[0149] The formulations of the three induction media (1% MCC induction medium, 1% CMC-Na induction medium and 1% sphagnum moss powder induction medium) are shown in Tables 14 to 16 below.

[0150] Table 14 1% MCC induction medium

[0151] NaCl 0.2g trypsin 0.2g Yeast extract powder 0.1g MCC 0.2g <![CDATA[ddH2O]]> up to 20mL

[0152] Table 15 1% CMC-Na Induction Medium

[0153] NaCl 0.2g trypsin 0.2g Yeast extract powder 0.1g CMC-Na 0.2g <![CDATA[ddH2O]]> up to 20mL

[0154] Table 16. Induction Culture Medium with 1% Bacillus subtilis Powder

[0155] NaCl 0.2g trypsin 0.2g Yeast extract powder 0.1g dried grass powder 0.2g <![CDATA[ddH2O]]> up to 20mL

[0156] The results are as follows Figure 7 As shown, the results indicated that, at the same concentration (1% cellulose) and induction time (12 h), the mRNA expression level of the Zein gene in the MCC-induced group was significantly higher than that in the LB control group, slightly higher in the chlorophyll powder-induced group, and lower in the CMC-Na-induced group than in the control group.

[0157] (II) SDS-PAGE detection of differences in Zein protein expression induced by cellulose from different sources

[0158] Wild-type and recombinant Bacillus subtilis were induced with microcrystalline cellulose (MCC) for 12 h, and the fermentation broth was ultrasonically disrupted to prepare protein samples; after SDS-PAGE electrophoresis, Coomassie brilliant blue staining was performed.

[0159] The results are as follows Figure 8 As shown, the results indicate that the recombinant bacteria exhibited a target band near the expected location (21.7 kDa) after cellulose induction, demonstrating that the induced recombinant bacteria can express the target protein. Quantitative analysis of the gel electrophoresis bands using ImageJ software revealed that the intensity of the target band in recombinant Bacillus subtilis increased after cellulose induction.

[0160] (III) Detection of methionine content in recombinant Bacillus subtilis fermentation broth

[0161] L-Met standard detection: The L-Met standard was qualitatively and quantitatively detected using HPLC-MS (TripleTOF 5600, SCIEX, USA) in positive ion mode. The molecular formula is C5H. 11 NO2S; The total ion chromatogram obtained by analyzing the experimental data using SECIX OS software is shown in the following figure. Figure 9 As shown in the figure, the peak elution time of L-Met standard is about 1.1 min according to the secondary scanning results. After qualitative and quantitative analysis of L-Met standard at 0, 0.25 ppm, 0.5 ppm, 0.75 ppm and 1 ppm, the regression equation Y = 2858*X - 53.76 and R2 = 0.9975 were obtained.

[0162] The specific parameters of HPLC-MS are shown in Tables 17 and 18.

[0163] Table 17 HPLC-MS Instrument Detection Parameters

[0164] High performance liquid chromatography-mass spectrometry parameter chromatographic column F5 mobile phase 0.1% formic acid + acetonitrile Flow rate 0.2 mL / min Sample loading amount 5μL ion source ESI Ion source temperature 500℃ Ionization Positive ion mode Ion spray voltage (ISVF) 5500V Positive ion mode declustering voltage (DP) 70V Collision energy (CE) 35V Collision energy variation range (CES) 15V Electron release delay (IRD) 67ms Electron beam width (IRW) 25ms SCIEX triple quadrupole mass spectrometer parameter Gas1 atomized gas Gas2 Auxiliary heating gas Atomizer pressure (CUR) 35psi Desolvation gas N2 Capillary voltage 3800V Scan type TOF MS Cycle number 538 Primary mass spectrometry scanning ion range 100-300m / z Ion scanning range 50-250m / z

[0165] Table 18 Elution Procedure

[0166] power 200W Ultrasonic working time 3-5s Ultrasonic gap time 3-5s Alarm temperature 27℃ Total duration 15min

[0167] The sample preparation process includes: lysozyme digestion, ultrasonic disruption, and protease hydrolysis (using trypsin combined with streptase E for sample digestion) of the fermentation broth. The specific steps are as follows: (1) Lysozyme lysis: Take 4 mL of bacterial solution at 12 h of induction and centrifuge at 4000 rpm for 12 min. Discard the supernatant and resuspend the precipitate with 2 mL of 25 mM Tris-HCl. Mix well by pipetting and repeat 3 times. Add 5 mg of lysozyme powder to each tube and lyse at 37℃ and 90 rpm for 2 h; (2) Ultrasonic disruption: Centrifuge at 5000 rpm for 12 min and resuspend 3 mL of 25 mM Tris-HCl 3 times; Sonicate the bacterial solution in ice water. See Table 19 for specific parameters; (3) Protease hydrolysis: After disruption, take 1 mL of the disruption solution and centrifuge at 12500 rpm for 2 min. Take 1 mL of supernatant and resuspend the bacterial precipitate 3 times with 1 mL of 25 mM Tris-HCl. The supernatant of the lysate and the bacterial precipitate were simultaneously divided into two groups for treatment: one group was treated with 5 mg trypsin and 5 mg streptoprotein E, and the other group was treated with 5 mg trypsin. The mixture was digested overnight at 37°C and 70 rpm to hydrolyze the proteins into amino acids. After labeling each tube, the mixture was centrifuged at 12500 rpm for 3 min, and 100 μL of the supernatant was transferred to a new EP tube. 900 μL of ultrapure water was added. The mixture was then filtered through a 0.22 μm filter membrane into a sample vial and analyzed by HPLC-MS.

[0168] Table 19 Ultrasonic Disruption Parameters for Bacillus subtilis

[0169] power 200W Ultrasonic working time 3-5s Ultrasonic gap time 3-5s Alarm temperature 27℃ Total duration 15min

[0170] Qualitative and quantitative analysis of each group of samples was performed using HPLC-MS in positive ion mode, and experimental data were analyzed using SECIXOS software. Specifically, SECIX software was used to predict the C5H group under positive ion mode ionization of formic acid. 11 The mass-to-nucleus ratio of NO2S is 150.0583, which corresponds to the molecular weight of the compound detected in the fermentation broth sample.

[0171] The results are as follows Figure 10 As shown, the Met content of the wild-type Bacillus subtilis (WB) fermentation broth samples (LB, 1% MCC, 1% MCC + 0.1 mMMEt) were 21.55 mg / L, 23.39 mg / L, and 26.45 mg / L, respectively, while the Met content of the recombinant Bacillus subtilis (RB) fermentation broth samples (LB, 1% MCC, 1% MCC + 0.1 mMMEt) were 29.23 mg / L, 43.06 mg / L, and 51.36 mg / L, respectively. The Met content of the recombinant strain under the two induction methods was increased by 84.1% and 94.2% compared with that of the wild-type strain, respectively.

[0172] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A recombinant expression vector, characterized in that, The recombinant expression vector comprises an upstream homologous arm sequence for knocking out an endo-1,4-beta-D-glucanase gene coding region, a downstream homologous arm sequence for knocking out the endo-1,4-beta-D-glucanase gene coding region, and a gene sequence encoding a high-sulfur zein; wherein the upstream homologous arm sequence for knocking out the endo-1,4-beta-D-glucanase gene coding region, the gene sequence encoding the high-sulfur zein, and the downstream homologous arm sequence for knocking out the endo-1,4-beta-D-glucanase gene coding region are connected in series; wherein the gene sequence encoding the high-sulfur zein is shown in SEQ ID NO:

3. The upstream homologous arm sequence for knocking out the endo-1,4-beta-D-glucanase gene coding region is shown in SEQ ID NO: 1; and / or the downstream homologous arm sequence for knocking out the endo-1,4-beta-D-glucanase gene coding region is shown in SEQ ID NO: 2; the recombinant expression vector uses the integrative plasmid PHY300PLK for homologous recombination.

2. Biomaterial, characterized in that, The recombinant expression vector of claim 1, the biological material comprises a gene expression kit or an engineered cell.

3. A recombinant Bacillus subtilis, characterized in that, The endo-1,4-beta-D-glucanase gene in Bacillus subtilis is knocked out, and then the recombinant expression vector of claim 1 is introduced into Bacillus subtilis to obtain a recombinant Bacillus subtilis.

4. The recombinant B. subtilis of claim 3, wherein, The preservation number of Bacillus subtilis is ATCC 23857.

5. A fermentation broth or culture broth, characterized in that, The recombinant expression vector of claim 1, the biological material comprises a gene expression kit or an engineered cell. Fermented or cultured by the recombinant Bacillus subtilis of claim 3 or 4.

6. A method of preparing high-sulfur zein, characterized by, Fermented or cultured by the recombinant Bacillus subtilis of claim 3 or 4. The high-sulfur zein is obtained by culturing the recombinant Bacillus subtilis of claim 3 or 4 using a culture medium, and the culture medium comprises cellulose.

7. The production method according to claim 6, wherein The preparation method comprises one or more of the following conditions: (a) the cellulose comprises one or more combinations of sodium carboxymethyl cellulose, microcrystalline cellulose, or hay powder; (b) the mass fraction of the cellulose is 1%; (c) the culture time is 12 h.

8. The recombinant expression vector of claim 1, the biological material of claim 2, or the recombinant Bacillus subtilis of claim 3 or 4, the fermentation broth or culture broth of claim 5, for use in increasing the content of methionine in animal feed.

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