Resistance-marker-free auxotrophic bacillus subtilis as well as construction method and application thereof

Abstract

The invention provides a resistance marker-free auxotrophic bacillus subtilis and a construction method and application thereof, the newly constructed strain takes bacillus subtilis 168 as a starting strain, indole-3-glycerophosphate synthase gene trpC of the bacillus subtilis 168 is knocked out, synthesis of tryptophan in the growth and metabolism process of the strain 168 is eliminated, and the resistance marker-free auxotrophic bacillus subtilis is obtained. The shuttle vector YH46 is constructed by taking pMD-19T as a framework, so that the shuttle vector YH46 can normally grow only in the presence of exogenous tryptophan, and meanwhile, a universal auxotroph shuttle vector YH46 is constructed by taking pMD-19T as the framework, so that convenience is provided for high-efficiency expression of genes in bacillus subtilis; the tryptophan auxotrophic strain BS-TR is constructed through double exchange by using a homologous recombination principle, the construction process is simple and easy to implement, and the strain BS-TR does not contain a resistance gene selection marker, can be used as a food-grade engineering bacterium, and can be used for intracellular expression or extracellular secretion of target protein for production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a non-resistance marker-free auxotrophic Bacillus subtilis, its construction method, and its application. Background Technology

[0002] Antibody drugs are a general term for a class of drugs with an IgG structure. They have a wide range of applications and play a vital role in the clinical treatment of various diseases, including cancer and autoimmune diseases. However, the mass production of antibodies faces a series of problems that urgently need to be solved. Currently, antibody production can be carried out using different expression systems, mainly eukaryotic expression systems represented by mammalian cells, insect cells, and yeast cells, and prokaryotic expression systems represented by Escherichia coli and Bacillus subtilis. However, mammalian cells have a slow growth rate, low yield of exogenous protein expression, and high production costs. Therefore, increasing attention is being paid to yeast, E. coli, and Bacillus subtilis systems.

[0003] Yeast expression systems offer rapid growth, ease of cultivation, and modifiability, enabling high-density fermentation and secretion of exogenous proteins, thus facilitating target protein purification and reducing production costs. Bacillus subtilis is a recognized safe strain (GRAS) and is non-pathogenic and free of endotoxins and exotoxins, making it an ideal chassis cell for protein production. Bacillus subtilis expression systems offer low production and cultivation costs, ease of modification, high yields, and convenient secretory expression of target proteins, further reducing production costs, and are widely used in scientific research and industrial / agricultural production.

[0004] Antibiotic markers are widely used in basic research and applications for screening target strains during the systematic modification of yeast, Escherichia coli, and Bacillus subtilis. Antibiotics are frequently used as screening markers to obtain target strains. However, the excessive use of antibiotics is causing increasingly serious environmental damage. Simultaneously, due to antibiotic overuse, various drug-resistant bacteria have emerged in nature, which will exacerbate the harm to humans in the long run. Therefore, using functionally complementary auxotrophic markers to screen target strains has broader application prospects. Thus, establishing an antibiotic-free marker screening method for Bacillus subtilis is of great significance for both basic and applied research. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a non-resistance-marked auxotrophic Bacillus subtilis.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned non-resistance marker auxotrophic Bacillus subtilis.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned non-resistance-marked auxotrophic Bacillus subtilis.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A non-resistance-marker auxotrophic Bacillus subtilis gene, indole-3-glycerol phosphate synthase gene trpC, was knocked out in Bacillus subtilis 168 using a scarless knockout technique. The nucleotide sequence of trpC is shown in SEQ ID NO.1 of the sequence listing.

[0010] Preferably, the above-mentioned non-resistance-marker auxotrophic Bacillus subtilis further includes plasmid YH46 transformed into the strain, the nucleotide sequence of which is shown in SEQ ID NO.3 of the sequence listing.

[0011] The specific steps for constructing the above-mentioned non-resistance marker-free auxotrophic Bacillus subtilis are as follows:

[0012] (1) Using the Bacillus subtilis 168 genome as a template, the upstream and downstream fragments of the homologous arm of the indole-3-glycerol phosphate synthase gene trpC were amplified by PCR, and the upstream and downstream fragments were linked together by overlapping PCR to obtain the homologous repair arm.

[0013] (2) The homologous repair arm constructed in step (1) was linked to the pMD19-T vector by overlap PCR;

[0014] (3) Transform the vector constructed in step (2) into Bacillus subtilis 168;

[0015] (4) Transform the strain constructed in step (3) into the pWB980 vector linked with the I-SceI gene of the nuclease, express the nuclease to cut the double-stranded DNA, force the upstream and downstream fragments of trpC to undergo double exchange with trpC on the chromosome of strain 168, discard the trpC gene, and then after continuous subculture, discard the pWB980 vector linked with the I-SceI gene of the transformed strain to obtain tryptophan auxotrophic Bacillus subtilis BS-TR.

[0016] Preferably, in the above-mentioned method for constructing the non-resistance marker auxotrophic Bacillus subtilis, in step (3), the vector is based on pMD19T and contains upstream and downstream homologous arms of the indole-3-glycerol phosphate synthase gene trpC. The vector constructed in step (2) is transformed into Bacillus subtilis 168.

[0017] Preferably, in the above-mentioned method for constructing the non-resistance-marker auxotrophic Bacillus subtilis, the pWB980 vector is a vector capable of autonomous replication in Bacillus subtilis, and the nucleotide sequence of the nuclease I-SceI carried is shown in SEQ ID NO.2 of the sequence listing.

[0018] Preferably, the above-mentioned method for constructing the non-resistance marker auxotrophic Bacillus subtilis further includes step (5) transforming plasmid YH46 into the tryptophan auxotrophic Bacillus subtilis BS-TR obtained in step (4).

[0019] The above-mentioned application of non-resistance-labeled auxotrophic Bacillus subtilis in protein expression and production.

[0020] Preferably, the application of the above-mentioned non-resistance-marker auxotrophic Bacillus subtilis involves cloning the functional trpC gene into an expression vector to obtain the recombinant plasmid YH46, transforming it into non-resistance-marker auxotrophic Bacillus subtilis, inducing the expression of the target protein, and then expressing the target protein intracellularly or secreting it extracellularly.

[0021] Beneficial effects:

[0022] The aforementioned non-resistance marker-dependent auxotrophic Bacillus subtilis strain, using Bacillus subtilis 168 as the starting strain, had its indole-3-glycerol phosphate synthase gene trpC knocked out, eliminating tryptophan synthesis during the growth and metabolism of strain 168. This allowed it to grow normally only in the presence of exogenous tryptophan. Simultaneously, a universal auxotrophic shuttle vector YH46 was constructed using pMD-19T as the backbone, facilitating efficient gene expression in Bacillus subtilis. Furthermore, utilizing homologous recombination, a tryptophan auxotrophic strain BS-TR was constructed through double crossover. The construction process is simple and easy to implement. Strain BS-TR does not contain resistance gene selection markers and can be used as a food-grade engineered bacterium, capable of intracellular expression or extracellular secretion of the target protein. Attached Figure Description

[0023] Figure 1 The plasmid map for expressing plasmid YH46.

[0024] Figure 2 Spread plates of solid culture medium for Bacillus subtilis BS-TR without transformation and after transformation with plasmid YH46.

[0025] Figure 3 The image shows the sequencing results of the trpC knockout strain. The top image shows the operon models of trpD, trpC, and trpF in Bacillus subtilis; the middle image shows the tprC sequencing results of strain 168; and the bottom image shows the sequencing results of the BS-TR strain with trpC knocked out.

[0026] Figure 4SDS-PAGE electrophoresis images of fermentation by strains highly expressing cellulase and xylose isomerase. Lanes 1, 3, 5, and 6 represent pre-induction results, while lanes 2, 4, 6, and 8 represent post-induction results. Lanes 2 and 4 show cellulase gel images with and without the signal peptide, respectively, and lanes 6 and 8 show xylose isomerase gel images with and without the signal peptide, respectively. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0028] The Bacillus subtilis 168 involved in the examples was purchased from Shanghai Jiachu Bioengineering Co., Ltd. The relevant genes are listed in the sequence listing.

[0029] The culture media and transformation methods used are as follows:

[0030] Transformation medium formulation:

[0031] (1) 10×MC solution: K2HPO4 10.7g, KH2PO4 5.2g, (NH4)2SO4 20g, trisodium citrate Na3C6H5O7·2H2O 0.88g, potassium L-glutamate (C5H8KNO4·H2O) 2.2g, glucose 20g, hydrolyzed casein 1g, 1000× ferric ammonium citrate 1mL, add water to make up to 100mL, filter to sterilize, and store at -20℃.

[0032] (2) L-tryptophan solution: 10 mg / mL, filtered and sterilized, then stored away from light.

[0033] (3) 1M MgSO4 solution: 12.32g, diluted to 50mL with ultrapure water, filtered for sterilization, and stored at room temperature.

[0034] (4) 1000× Ferric ammonium citrate solution: 2.2g of ferric ammonium citrate was added to ultrapure water and brought to a final volume of 100mL. After filtration and sterilization, it was stored at 4℃ away from light.

[0035] SPIZ liquid culture medium formulation:

[0036] (1) 10×SPIZ solution: (NH4)2SO4 20g, K2HPO4 140g, KH2PO4 60g, trisodium citrate dihydrate 10g, MgSO4·7H2O 2g, add water to make up to 1L, and autoclave.

[0037] (2) 100×Metals solution: 200ml 1M MgCl2 solution, 140ml 0.5M CaCl2 solution, 5ml 1M MnCl2 solution, 10ml 10mM ZnCl2 solution, 50ml 2mg / ml vitamin B1 solution, 10ml 50mM FeCl3 solution, add water to make up to 1L, filter to sterilize, and store in the dark.

[0038] (3) 50% glucose solution: 500g D-glucose, add water to make up to 1L, filter to sterilize.

[0039] The transformation method of Bacillus subtilis is as follows:

[0040] Using an inoculation loop, pick up a Bacillus subtilis 168 bacterial suspension preserved in glycerol and streak it onto an LB agar plate. Incubate overnight at 37°C. Pick a single colony from the LB agar plate and inoculate it into 1 mL of transformation medium (100 μL 10×MC solution, 3 μL 1M MgSO4, 4 μL L-tryptophan, 893 μL ddH2O). Incubate at 37°C and 220 rpm for 4.5 h.

[0041] Take 200 μL of bacterial culture into a new test tube, add 1 μg of plasmid to competent cells, incubate at 37℃ and 220 rpm for 2 h, spread on the corresponding screening plate, and incubate overnight at 37℃.

[0042] Example 1

[0043] The specific steps for constructing the auxotrophic Bacillus subtilis strain BS-TR are as follows:

[0044] (1) Using Bacillus subtilis 168 genomic DNA as a template, the upstream fragment of the trpC gene was amplified using primers P1 and P2 as described in Table 2, and the downstream fragment of the trpC gene was amplified using primers P3 and P4. The upstream and downstream fragments of the trpC gene were linked together by overlap PCR.

[0045] (2) The chloramphenicol resistance gene (GenBank: CP140687.1) was amplified using primers P5 and P6. The fragment obtained in step (1) was linked together with the fragment in step (2) using overlap PCR, and then ligated into the pMD19-T vector (purchased from Takara) to obtain the pMD-trpC vector.

[0046] (3) Using the plasmid constructed in step (2), linearized fragments were obtained by SacI restriction enzyme digestion and transformed into Bacillus subtilis 168. The fragments were plated onto LB solid medium (5 μg / mL chloramphenicol) with the corresponding resistance, incubated overnight at 37°C, and positive clones were picked for sequencing verification.

[0047] (4) Inoculate the verified strain into LB liquid medium (5 μg / mL chloramphenicol) for culture and preserve the bacteria.

[0048] (5) The pWB980 vector (purchased from Beijing Solarbio Science & Technology Co., Ltd.) containing the I-SceI (Genebank: M12278.1) gene linking to the strain constructed in step (4) was transformed to express the endonuclease to cut the double-stranded DNA. Using the bacterial chromosome repair mechanism, the upstream and downstream fragments of trpC were forcibly double-crossed with trpC on the chromosome of strain 168, thus discarding the trpC gene and performing a traceless knockout. After continuous subculturing, the pWB980 vector linking the I-SceI gene of the transformed strain was discarded to obtain tryptophan auxotrophic Bacillus subtilis BS-TR. 10-20 single clones were picked and mixed and streaked on LB solid medium (15ug / ml kanamycin) and cultured overnight at 37°C.

[0049] (6) Select the single clones from step (5) and culture them in antibiotic-free LB solid medium and chloramphenicol-resistant LB solid medium (5 μg / mL chloramphenicol). Select antibiotic-free single clones for sequencing to verify whether the target gene has been knocked out. The sequencing results are shown in […]. Figure 3 .

[0050] (7) Inoculate the monoclonal strain with correct sequencing in step (6) into LB liquid medium without antibiotics and culture it. After dilution, spread it on LB solid medium without antibiotics and LB solid medium with kanamycin resistance (15ug / ml kanamycin) and culture it overnight. Pick the monoclonal strain without antibiotics and inoculate it into LB liquid medium. This is the strain without antibiotics BS-TR. Store it in a glycerol tube at -80℃.

[0051] The above reaction system is shown in Table 1, and the primer sequences used are shown in Table 2.

[0052] Table 1 Reaction System

[0053] reagents volume PrimeSTAR Max Premix (2×) 25μL Template DNA 1μL Upstream primer (10 μM) 1μL Downstream primer (10 μM) 1μL <![CDATA[ddH2O]]> Make up to 50 μL

[0054] The reaction procedure is as follows: pre-denaturation at 95℃ for 2 min, 95℃ for 15 s, 55℃ for 15 s, extension at 72℃ for the corresponding time, for 25 cycles, extension at 72℃ for 5 min, and holding at 4℃.

[0055] Table 2 Primer sequences

[0056]

[0057] Example 2

[0058] The specific steps for constructing the Bacillus subtilis shuttle plasmid YH46 are as follows:

[0059] (1) Using pMD19-T as a template, the Amp and ori fragments were amplified using primers P7 and P8 as described in Table 3; using pWB980 as a template, repB was amplified using primers P9 and P10 as described in Table 3; and trpC (SEQ ID NO.1) was amplified using primers P11 and P12 as described in Table 3. Using Bacillus subtilis 168 genome as a template, Pgrac was amplified using primers P13 and P14 as described in Table 3. Using Pet28a (purchased from Beijing Huayueyang Biotechnology) as a template, the lacI gene sequence was amplified using primers P15 and P16 as described in Table 3. The amplified upstream and downstream fragments were linked together by overlap PCR, transformed into E. coli, and single clones were selected for plasmid extraction. The correctly sequenced vector was named plasmid YH46. Figure 1 The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing.

[0060] (2) Using the plasmid constructed in step (1), transform the Bacillus subtilis BS-TR from Example 1. Spread it onto SPIZ basal medium ( Figure 2 ).

[0061] Table 3 Primer sequences

[0062]

[0063]

[0064] Example 3

[0065] The specific steps for constructing strains expressing cellulase and xylose isomerase are as follows:

[0066] (1) Using the genome of Bacillus subtilis as a template, the cellulase gene (uniprot: P10475) was amplified using primers P17 and P18 as described in Table 4. Its nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing, and the nucleotide sequence of the signal peptide it carries is shown in SEQ ID NO.4 of the sequence listing. Using plasmid YH46 as a template, the vector portion was amplified using primers P19 and P20. The two upstream and downstream fragments were linked together by overlap PCR and transformed into Escherichia coli DH5α. Single clones were picked, plasmids were extracted, and sequencing verification was performed.

[0067] Using the Bacillus subtilis genome as a template, the cellulase gene (without a signal peptide) was amplified using primers P17 and P21 as described in Table 4. Its nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing. Using plasmid YH46 as a template, primers P19 and P22 were used to amplify the vector portion. The two upstream and downstream fragments were linked together by overlap PCR, transformed into Escherichia coli DH5α, and single clones were selected for plasmid extraction and sequencing verification.

[0068] (2) Using the genome of Bacillus subtilis as a template, the xylose isomerase gene (uniprot: POCI80) was amplified using primers P23 and P24 as described in Table 4. Its nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing, and the nucleotide sequence of the signal peptide it carries is shown in SEQ ID NO.4 of the sequence listing. Using plasmid YH46 as a template, the vector portion was amplified using primers P19 and P20. The two upstream and downstream fragments were linked together by overlap PCR and transformed into Escherichia coli DH5α. Single clones were picked, plasmids were extracted, and sequencing verification was performed.

[0069] Using the genome of Bacillus subtilis as a template, the xylose isomerase gene (without a signal peptide) was amplified using primers P23 and P25 as described in Table 4. Its nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing. Using plasmid YH46 as a template, the vector portion was amplified using primers P19 and P22. The two upstream and downstream fragments were linked together by overlap PCR, transformed into Escherichia coli, and single clones were selected for plasmid extraction and sequencing verification.

[0070] (3) Using the plasmids constructed in steps (1) and (2), transform Bacillus subtilis BS-TR from Example 1 and plate it on SPIZ plates. Select transformants and store them in glycerol tubes at -80°C.

[0071] Table 4 Primer sequences

[0072]

[0073]

[0074] Example 4

[0075] Cellulase and xylose isomerase were produced by shake-flask fermentation of strains expressing cellulase and xylose isomerase.

[0076] The SPIZ liquid culture medium formula used is: 10ml 10×SPIZ solution, 1ml 100×Metals solution, 1ml 50% glucose solution, and brought to a final volume of 100ml.

[0077] The *Bacillus subtilis* strain constructed in Example 3 was inoculated and activated for fermentation. A portion of the bacterial culture was picked from a glycerol tube and inoculated into SPIZ medium, incubated at 37°C and 220 rpm for approximately 16 hours. Then, 2% of the inoculum was transferred to 100 ml of SPIZ liquid medium and incubated at 37°C and 220 rpm for 5 hours. IPTG was then added for induction for 3 hours, followed by protein extraction. The SDS-PAGE gel image of the protein is shown below. Figure 4 As shown. Figure 4As can be seen, the auxotrophic Bacillus subtilis strain and expression vector constructed in this invention can simultaneously express both extracellular and intracellular proteins of the target protein.

[0078] 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. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A non-resistance-marker auxotrophic Bacillus subtilis, characterized in that: The indole-3-glycerol phosphate synthase gene trpc of Bacillus subtilis 168 was knocked out using a scarless knockout technique. The nucleotide sequence of trpc is shown in SEQ ID NO.1 of the sequence listing.

2. The non-resistance-marked auxotrophic Bacillus subtilis according to claim 1, characterized in that: It also includes plasmid YH46 transformed into the strain, the nucleotide sequence of which is shown in SEQ ID NO.3 of the sequence listing.

3. The method for constructing a non-resistance-marker auxotrophic Bacillus subtilis according to claim 1, characterized in that: The specific steps are as follows: (1) Using the Bacillus subtilis 168 genome as a template, the upstream and downstream fragments of the homologous arm of the indole-3-glycerol phosphate synthase gene trpC were amplified by PCR, and the upstream and downstream fragments were linked together by overlapping PCR to obtain the homologous repair arm. (2) The homologous repair arm constructed in step (1) was linked to the pMD19-T vector by overlap PCR; (3) Transform the vector constructed in step (2) into Bacillus subtilis 168; (4) Transform the strain constructed in step (3) into the pWB980 vector linked with the I-Scel gene of the nuclease, express the nuclease to cut the double-stranded DNA, force the upstream and downstream fragments of trpC to undergo double exchange with trpc on the chromosome of strain 168, discard the trpC gene, and then after continuous subculture, discard the pWB980 vector linked with the I-Scel gene of the transformed strain to obtain tryptophan auxotrophic Bacillus subtilis BS-TR.

4. The method for constructing a non-resistance-marker auxotrophic Bacillus subtilis according to claim 3, characterized in that: In step (3), the vector is based on pMD19T and contains upstream and downstream homologous arms of the indole-3-glycerol phosphate synthase gene trpC. The vector constructed in step (2) is transformed into Bacillus subtilis 168.

5. The method for constructing a non-resistance-marker auxotrophic Bacillus subtilis according to claim 3, characterized in that: It also includes step (5) transforming plasmid YH46 into the tryptophan auxotrophic Bacillus subtilis BS-TR obtained in step (4).

6. The use of the non-resistance-labeled auxotrophic Bacillus subtilis according to claim 1 or 2 in the expression and production of proteins.

7. The application according to claim 6, characterized in that: The recombinant plasmid YH46 was obtained by cloning the functional trpC gene into an expression vector, which was then transformed into a non-resistance-marker auxotrophic Bacillus subtilis to induce the expression of the target protein, which was then expressed intracellularly or secreted extracellularly.

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