Method for efficiently producing lactulose based on cellobiose epimerase and application

Abstract

The invention discloses a method for efficiently producing cellobiose epimerase through tag fusion expression and application of the cellobiose epimerase in lactulose production, and belongs to the field of biotechnology and enzyme engineering. According to the invention, the cellobiose epimerase is subjected to fusion expression on plasmid pMA5 by adopting a VNp tag, the cellobiose epimerase is produced by fermentation at a shake flask level, and the total enzyme activity of the cellobiose epimerase is improved from 16.05 U.mL <-1 > to 17.71 U.mL <-1 >. In addition, the VNp tag is subjected to truncation treatment, and the total enzyme activity of the cellobiose epimerase subjected to fusion expression is further improved to 19.31 U.mL <-1 > and 21.25 U.mL <-1 > respectively. In a 5L fermentation tank, fed-batch fermentation is adopted, and the total enzyme activity of the cellobiose epimerase reaches 38.63 U.mL <-1 >.

Description

Technical Field

[0001] The present invention relates to a method for efficiently producing lactulose based on cellobiose epimerase and its application, and belongs to the fields of biotechnology and enzyme engineering. Background Art

[0002] Bacillus subtilis is a Gram-positive rod-shaped bacterium with a clear genetic background, strong protein secretion ability, simple genetic manipulation, and a short fermentation cycle. It has been recognized as a food-grade safe strain by the U.S. Food and Drug Administration. This makes B. subtilis widely used as a model strain in the fields of food, industry, and medicine to produce recombinant proteins such as α-amylase, hyaluronic acid, riboflavin, and other compounds. Compared with commonly used industrial microorganisms such as Escherichia coli, the B. subtilis expression system has the following main advantages: (1) It does not produce toxins and heat-sensitive proteins, is not pathogenic, and is a food-grade safe strain. (2) It has a short fermentation cycle, a mature fermentation process, and is not easily infected by bacteriophages during the fermentation process. (3) It has a very strong protein secretion ability, and the product is easy to separate and purify.

[0003] Eastwood et al. accidentally discovered that recombinant expression of full-length human α-synuclein (αSyn) in E. coli leads to the extracellular release of membrane vesicles containing αSyn, which usually contain the bacterial membrane protein OmpA. After further study, they found that the 38 residues at the amino terminus of αSyn are sufficient to form OmpA-labeled extracellular membrane-bound vesicles from E. coli cells and release them into the culture medium. This αSyn-derived polypeptide was named "vesicle nucleating peptide" (VNp). Compared with existing protein secretion technologies, the technology of using "vesicle nucleating peptide" to achieve protein secretion has shown a wide range of applicability: (1) It is applicable to a variety of proteins with different molecular weight ranges. Whether it is as low as 1kDa or as high as 100kDa or more, it can be well applied. (2) This technology can effectively promote the formation of disulfide bonds between proteins and disulfide bonds within proteins. (3) It is also applicable to multi-protein complexes and can be used in conjunction with a variety of promoters, which greatly broadens its scope of application. (4) The system can also realize the export of vesicle-encapsulated proteins from E. coli. This feature provides a stable and protective environment for the separation and storage of proteins, greatly improving the stability and application potential of proteins. This simple peptide tag not only simplifies the subsequent purification process of recombinant proteins, but also enables membrane-forming vesicles to be widely used in the development of commercial products in the biotechnology and medical industries. For example, it can be used for the construction of recombinant bioreactors, the environmental dispersion of biological molecules, and as a carrier for drug delivery and vaccination. The team of Professor Jiang Min of Nanjing University of Science and Technology successfully constructed an E. coli secretion expression system by fusion expression of polyethylene terephthalate degrading enzyme Fast-PETase with VNp. Ultimately, the secretion expression level of Fast-PETase was increased to 2 g·L in a 5L bioreactor. -1 , which is currently the highest production level in the E. coli chassis. In order to further improve the expression level of recombinant enzymes (especially cellulase) in Bacillus subtilis and achieve efficient production of lactulose, this study systematically studied the efficient expression of enzymes in Bacillus subtilis using different fusion tag mutants. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method and application for efficiently producing lactulose based on cellobiose epimerase, aiming to solve the current technical problem of lack of a method for improving the expression level of recombinant enzymes (especially cellobiose epimerase) in Bacillus subtilis to achieve efficient production of lactulose.

[0005] The first technical solution provided by the present invention is a truncated VNp tag, wherein the amino acid sequence of the truncated VNp tag is shown in any one of SEQ ID NOs. 16 and 18.

[0006] SEQ ID NO.16: MDVFMKGLSKAKEGVVAAAEKTKQGV;

[0007] SEQ ID NO. 18: MDVFMKGLSKAKEGVVAAAE.

[0008] The present invention also provides a gene encoding the truncated VNp tag described in the first technical solution.

[0009] In certain embodiments, the nucleotide sequence of the gene is shown in any one of SEQ ID NO.8 and SEQ ID NO.10.

[0010] The third technical solution provided by the present invention is a recombinant Bacillus subtilis spore, wherein the recombinant Bacillus subtilis overexpresses a fusion fragment consisting of a VNp tag or a truncated VNp tag according to the first technical solution and a recombinant protein.

[0011] The invention provides a recombinant Bacillus subtilis, which is genetically modified on the basis of B. subtilis168.

[0012] In certain embodiments, plasmid pMA5 is used as an expression vector to express the fusion fragment, and the recombinant protein includes green fluorescent protein or cellobiose epimerase.

[0013] In certain embodiments, the plasmid pMA5 contains the promoter P HpaII , the promoter P HpaII The nucleotide sequence is shown as SEQ ID NO.1; it contains a VNp tag, the amino acid sequence of the VNp tag is shown as SEQ ID NO.12, and the nucleotide sequence encoding the VNp tag gene is shown as SEQ ID NO.2; it contains a truncated VNp tag, the nucleotide sequence of the truncated VNp tag is shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11.

[0014] In certain embodiments, the nucleotide sequence of the green fluorescent protein gene eGFP is shown as SEQ ID NO.3.

[0015] In certain embodiments, the nucleotide sequence of the cellobiose epimerase gene CS-CE is shown as SEQ ID NO.4.

[0016] The fourth technical solution provided by the present invention is an application method for fermenting recombinant Bacillus subtilis to prepare cellobiose epimerase, wherein the recombinant Bacillus subtilis overexpresses a fusion fragment consisting of a VNp tag or a truncated VNp tag according to the first technical solution and cellobiose epimerase.

[0017] In certain embodiments, the seed solution of the recombinant Bacillus subtilis is inoculated into TB culture medium and cultured at 37° C. and 200 rpm for 48 hours.

[0018] In certain embodiments, the inoculation is to transfer the seed solution into TB culture medium at a 1% inoculation volume.

[0019] In certain embodiments, the seed solution is obtained by culturing the recombinant Bacillus subtilis in LB medium at 37° C. and 200 rpm for 10 hours.

[0020] The fifth technical solution provided by the present invention is the use of the truncated VNp tag described in the first technical solution, or the gene described in the second technical solution, or the recombinant Bacillus subtilis described in the third technical solution, or the method described in the fourth technical solution in the production of cellobiose epimerase.

[0021] The sixth technical solution provided by the present invention is the use of the VNp tag or the truncated VNp tag described in the first technical solution in improving the expression level and extracellular secretion efficiency of recombinant Bacillus subtilis exogenous proteins.

[0022] Beneficial effects:

[0023] (1) The present invention uses Bacillus subtilis as a host and significantly improves its expression level and extracellular secretion efficiency by fusing the VNp tag with the cellobiose epimerase CS-CE.

[0024] (2) The total enzyme activity of the cellobiose epimerase of the recombinant bacterium B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE constructed in the present invention was 16.05 U·mL of the control bacterium B. subtilis BS3 amyE::QS-prkC pMA5-CS-CE. -1 Increased to 17.71 U·mL -1 The total enzyme activity of cellobiose epimerase expressed by B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE and B. subtilis BS3amyE::QS-prkC pMA5-VNp-4-CS-CE was further increased to 19.31 U·mL -1 and 21.25 U·mL-1 In a 5 L fermentor using fed-batch fermentation, the total enzyme activity of cellobiose epimerase from B. subtilis BS3amyE::QS-prkC pMA5-VNp-6-CS-CE reached 38.63 U·mL -1 The cellobiose epimerase mutants expressed by B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE and B. subtilis BS3 amyE::QS-prkC pMA5-VNp-4-CS-CE were used to catalyze 500 g·L -1 When lactose is used to produce lactulose, the lactulose yield is 405 g·L -1 and 393 g·L -1 ; The conversion rates were 81% and 78.6% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effects of VNp-tag fusion expression on eGFP expression levels. A, eGFP fluorescence intensity; B, SDS-PAGE. M, Maker (standard molecular weight protein). 1, B. subtilis 168pMA5 (control); 2, B. subtilis 168pMA5-eGFP; 3, B. subtilis 168pMA5-VNp-eGFP; 4, B. subtilis BS3 amyE::QS-prkC pMA5-VNp-eGFP. Arrows, target proteins.

[0026] Figure 2 Effects of VNp-tag fusion expression on cellobiose epimerase expression. A, Effects of VNp-tag fusion expression on cellobiose epimerase expression in B. subtilis 168; B, SDS-PAGE. M, Maker (standard molecular weight protein). 1, B. subtilis BS3 amyE::QS-prkC pMA5 (control); 2, B. subtilis BS3 amyE::QS-prkC pMA5-CS-CE; 3, B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE. Arrows, target proteins.

[0027] Figure 3Production of cellobiose epimerase from B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE in a 5-L fermentor using fed-batch fermentation. A, enzyme activity; B, SDS-PAGE of intracellular enzyme protein; C, SDS-PAGE of extracellular enzyme protein. M, Maker (marker molecular weight protein). Arrows, target proteins.

[0028] Figure 4 The effect of VNp tag truncation on eGFP expression level.

[0029] Figure 5 Effects of VNp tag truncation on cellobiose epimerase expression levels. A, Effect of VNp-6 on cellobiose epimerase expression levels; B, Effect of VNp-4 on cellobiose epimerase expression levels.

[0030] Figure 6 Cellobiose epimerase was produced by fed-batch fermentation of B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE in a 5 L fermentor. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0032] Culture medium:

[0033] LB medium: Weigh 10 g NaCl, 5 g yeast extract, and 10 g tryptone in deionized water, mix thoroughly, and dilute to 1 L. Sterilize at 121°C for 15 min. For solid medium, add 20 g agar powder.

[0034] TB medium: Weigh 24 g yeast extract, 12 g tryptone, 4 mL glycerol, 2.31 g KH2PO4, and 12.54 g K2HPO4 in deionized water, mix thoroughly, and dilute to 1 L. Sterilize at 121°C for 15 min.

[0035] B. subtilis competent cell preparation solution: Each 5 mL of BI solution consists of 500 μL 10× minimum salt solution, 50 μL 10% (v / v) yeast powder solution, 50 μL 50% (v / v) glucose solution, 25 μL 10 g·L -1Tryptophan solution, 20 μL 5% (v / v) hydrolyzed casein solution, and 4.28 mL sterile water were sterilized by high-pressure steam; each 4.5 mL of BII solution was composed of 450 μL 10× minimum salt solution, 1.8 μL 10% (v / v) yeast powder solution, 45 μL 50% (v / v) glucose solution, and 4.5 μL 10 g·L -1 Tryptophan solution, 3.6 μL of 5% (v / v) hydrolyzed casein solution, 11.25 μL of 1 M MgCl₂, 2.25 μL of 1 M CaCl₂, and 3.915 mL of sterile water were prepared by autoclaving. 10× minimum salt solution (100 mL) was prepared by autoclaving 18.34 g of K₂HPO₄·3H₂O, 6 g of KH₂PO₄, 2 g of (NH₄)₂SO₄, 1 g of sodium citrate, and 0.2 g of MgSO₄·7H₂O, with the MgSO₄·7H₂O being sterilized separately.

[0036] Preparation of E.coli competent cells and chemical transformation methods:

[0037] (1) Pipette 100 μL of bacterial suspension from a glycerol tube of E. coli JM109 stored at -80°C into 10 mL of liquid LB medium (100 mL Erlenmeyer flask) and culture for 10 h.

[0038] (2) Pipette 1 mL of bacterial solution into 100 mL of liquid LB medium (250 mL Erlenmeyer flask), shake and culture at 37°C and 200 rpm for 2 h. 600 Up to 0.6.

[0039] (3) Place on ice for 10 min and centrifuge at 4,000 × g for 5 min at 4°C.

[0040] (4) Discard the supernatant, add 30 mL of 100 mM CaCl2 to suspend the cells, incubate on ice for 30 min, and centrifuge at 4,000 × g for 5 min at 4°C.

[0041] (5) Discard the supernatant, add 3 mL of 100 mM CaCl2 + 25% glycerol to suspend the cells, and dispense into sterile EP tubes, 200 μL per tube, and store in a -80°C refrigerator.

[0042] (6) Add the enzyme-digested ligation product, the homologous ligation product, and the whole plasmid PCR digestion product to the pre-thawed competent cells and incubate on ice for 30 minutes.

[0043] (7) Heat shock at 42°C for 90 seconds and place on ice for 5 minutes.

[0044] (8) Add 800 μL of liquid LB medium and culture at 37°C, 200 rpm, and shake for 45 min.

[0045] (9) The transformation solution was centrifuged at 12,000 × g for 1 min. After discarding part of the supernatant, it was spread on a solid LB plate of the corresponding resistance and cultured at 37°C overnight.

[0046] Preparation of B. subtilis competent cells and chemical transformation methods:

[0047] (1) Thaw the B. subtilis glycerol tube stored at -80°C on ice, streak onto a solid LB plate without antibiotics, and culture at 37°C overnight.

[0048] (2) Pick a single activated colony and transfer it to 5 mL of BI medium. Culture it at 37°C and 200 rpm with shaking for 4-5 hours until the logarithmic phase.

[0049] (3) Transfer 500 μL of the above logarithmic phase bacterial solution to 4.5 mL of BII medium and culture at 37°C and 200 rpm with shaking for about 1.5 h to obtain competent cells.

[0050] (4) The competent cells were dispensed into sterile EP tubes, 500 μL per tube.

[0051] (5) Add 5-10 μL of plasmid or 2 μg of gene fragment to the competent cells and culture at 37°C and 200 rpm with shaking for 60-90 min.

[0052] (6) The transformation solution was centrifuged at 12,000 × g for 1 min. After discarding part of the supernatant, it was spread on a solid LB plate of the corresponding resistance and cultured at 37°C overnight.

[0053] eGFP fluorescence intensity measurement:

[0054] A single colony was picked from a solid LB plate and inoculated into a solution containing 25 mg·L -1 The culture was placed in 20 mL of LB medium (100 mL Erlenmeyer flask) containing kanamycin and shaken at 37°C and 200 rpm for 10 h. The culture was inoculated with 1% of the inoculum into a 25 mg·L -1 Culture the cells in 50 mL of TB medium (250 mL Erlenmeyer flask) containing kanamycin at 37°C and 200 rpm with shaking for 8 hours. Centrifuge the culture at 4°C and 12,000 × g for 10 minutes, discard the supernatant, and resuspend the cells in PBS buffer. Transfer 200 μL of the culture to a 96-well plate and measure the eGFP fluorescence intensity using a Synergy™ H4 fluorescence microplate reader. Excitation and emission wavelengths were 488 nm and 533 nm, respectively.

[0055] SDS-PAGE analysis:

[0056] Collect 1 mL of fermentation broth and centrifuge at 6,000 × g, 4°C for 10 min to collect the bacteria. Resuspend with 1 mL of 50 mM Tris-HCl buffer (pH = 7.5) and add 100 g·L -1 Lysozyme to a final concentration of 1 g·L -1 , place at room temperature for 30 minutes to break the cells. Centrifuge at 12,000×g, 4℃ for 5 minutes, and collect the supernatant as the crude enzyme solution. Take 30μL of supernatant, add 10μL 4× loading buffer (Baori Medical Biotechnology (Beijing) Co., Ltd., Beijing, China), and boil in a boiling water bath for 3 minutes. Take 5μL of sample for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. SDS-PAGE protein gel consists of 5% concentrating gel and 15% separating gel. Use Mini-PROTEAN Tetra electrophoresis instrument (Bio-Rad, USA) with the voltage set to 80V for gel electrophoresis. After completion, use 1.2g·L -1 The cells were stained with (w / v) Coomassie Brilliant Blue (R250) for 30 min, and then destained with a destaining solution (25% (v / v) ethanol and 8% (v / v) acetic acid) and observed.

[0057] Cellobiose epimerase enzyme activity assay:

[0058] Enzyme reaction system: 100 g L was prepared with 50 mM Tris-HCl buffer (pH = 7.5). -1 Lactose solution, take 900 μL 100 g L -1 Lactose solution and 100 μL of enzyme solution were mixed and reacted at 80°C for 20 minutes. The reaction was terminated by boiling for 10 minutes. After the reaction, the solution was centrifuged at 12,000 × g for 5 minutes, filtered through a 0.22 μm filter, and the lactulose content was determined by HPLC. The activity of cellobiose epimerase is defined as the amount of enzyme required to produce 1 μmol of lactulose per minute from the substrate lactose under the above conditions.

[0059] The experimental methods not specifically noted in the following examples can be completed according to conventional methods or the instructions of the manufacturers of the products used. The materials, reagents, etc. used can be obtained through commercial channels unless otherwise specified.

[0060] The primer sequences involved in the following examples are shown in Table 1.

[0061] Table 1 Primer sequences

[0062]

[0063]

[0064] Example 1: Construction of plasmid

[0065] Construction of plasmid pMA5-eGFP: Using plasmid pMA5 as a template, A5-eGFP-FW and A5-eGFP-RS were used to PCR amplify the pMA5 vector. Using the green fluorescent protein gene eGFP as a template, eGFP-A5-FW and eGFP-A5-RS were used to PCR amplify the eGFP gene fragment (shown in SEQ ID NO. 3). The PCR product was verified by agarose gel electrophoresis. Once correct, the gel was recovered and ligated using a one-step cloning kit. The successfully ligated product fragment was transformed into E. coli JM109 competent cells. The transformants were diluted and plated on LB solid medium supplemented with ampicillin and screened for positive transformants (using colony PCR). Single colonies that were correct by colony PCR were picked and inoculated into liquid LB medium supplemented with ampicillin for 8-12 hours. The recombinant plasmid was extracted using a small amount of plasmid extraction kit. The plasmid was then sequenced and analyzed. The correct plasmid was named pMA5-eGFP and the transformants were stored in a freezer at -80°C. Plasmid pMA5-VNp-eGFP was synthesized by Nanjing GenScript Biotechnology Co., Ltd., by inserting the VNp-eGFP fragment (nucleotide sequence shown in SEQ ID NO. 20) into the Nde I and BamH I sites of plasmid pMA5. Using a similar strategy as described above, the pMA5-VNp fragment was amplified by PCR using plasmid pMA5-VNp-eGFP as a template and primers A5VNP-CS-CE-FW and A5VNP-CS-CE-RS. The cellobiose epimerase gene CS-CE was amplified by PCR using primers CS-CE-A5VNP-FW and CS-CE-A5VNP-RS (shown in SEQ ID NO. 4). The recombinant plasmid pMA5-VNp-CS-CE was obtained by ligation using a cloning kit.

[0066] Example 2: Effect of VNp tag fusion expression on eGFP expression level

[0067] Construction of recombinant strain B. subtilis BS3 amyE::QS-prkC: The promoter of the gene ugtP on the genome of B. subtilis 168 was predicted using the online software Softberry. ugtP Among them, the promoter P ugtP The sequence of the -35 region is TTTAAA and the sequence of the -10 region is ATTTAAAAT. ugtP Replace the promoter P of plasmid pMA5 HpaII A similar strategy was used to construct the recombinant plasmid pMA5-P ugtP The OR2 sequence (TAACACCGTGCGTGTTG) was inserted into the promoter PugtP Downstream of the -10 region, mutant P was constructed ugtP M3 The constructed promoter mutant was used to regulate the expression of the gene eGFP, and the recombinant plasmid pMA5-P expressing green fluorescent protein eGFP was constructed. ugtP M3 -eGFP.

[0068] The constructed pMA5-P ugtP M3 -eGFP plasmid as a template, using P ugtP -L-FW、P ugtP -L-RS, lox-P ugtP -FW, lox-P ugtP -RS、ZH-P ugtP -FW、ZH-P ugtP -RS, P ugtP -R-FW、P ugtP -R-RS was used as primer to obtain the integration frame P ugtP -L-lox71-crm-lox66-P ugtP M3 -P ugtP -R, chemically transformed into B. subtilis 168 to obtain P ugtP M3 Replace P ugtP The engineered strain B. subtilis BS3.

[0069] Using the B. subtilis BS3 genome as a template, polymerase chain reaction (PCR) amplification was performed using primers amyE-L-FW and amyE-L-RS. The PCR product was verified by agarose gel electrophoresis and purified. The product was labeled amyE-L. Using the p7C6 plasmid (disclosed in the paper "Combinatorial Methylerythritol Phosphate Pathway Engineering and Process Optimization for Increased Menaquinone-7 Synthesis in Bacillus subtilis") as a template, the lox71-crm-lox66 cassette was amplified by PCR using primers lox-FW and lox-RS-1. The PCR product was verified by agarose gel electrophoresis and recovered. The product was labeled lox71-crm-lox66-1. Using lox71-crm-lox66-1 as a template and lox-FW and lox-RS-2 as primers, PCR amplified the lox71-crm-lox66 cassette. The PCR product was verified by agarose gel electrophoresis, and after verification, the PCR product was purified and labeled as lox71-crm-lox66-2. The gene-synthesized QS system gene (shown in SEQ ID NO.21) was used as a template and PCR amplified using QS-FW and QS-RS primers. The PCR product was verified by agarose gel electrophoresis, and after verification, the PCR product was purified and labeled as QS. The genome of B. subtilis BS3 was used as a template and PCR amplified using prkC-QS-FW and prkC-QS-RS primers. The PCR product was verified by agarose gel electrophoresis, and after verification, the PCR product was purified and labeled as prkC (shown in SEQ ID NO.22). Using the genome of B. subtilis BS3 as a template, PCR amplification was performed using primers amyE-R-FW and amyE-R-RS. The PCR product was verified by agarose gel electrophoresis and purified after verification. The product was labeled amyE-R. Fusion PCR amplification was performed using the previously constructed fragment amyE-L, lox71-crm-lox66-2, QS, and R6 promoter sequences (shown in SEQ ID NO. 23), prkC, and amyE-R as templates, using primers amyE-L-FW and amyE-R-RS. The PCR product was verified by agarose gel electrophoresis and recovered after verification. The product was labeled amyE-L-lox71-crm-lox66-QS-prkC-amyE-R cassette.

[0070] The integration cassette was chemically transformed into B. subtilis BS3 competent cells and plated on solid LB medium (containing 5 μg / mL chloramphenicol). Positive transformants were screened by colony PCR, and the pDG148-Cre plasmid was then transformed into positive clones and plated on solid LB medium (containing 50 μg / mL kanamycin). Several transformants were selected and cultured in 20 mL of liquid LB medium containing 50 μg / mL kanamycin at 37°C with 0.2 mM IPTG for 12 hours to eliminate the resistance gene crm. These were transferred to 1 mL of antibiotic-free liquid LB medium and shaken at 50°C for 12 hours to eliminate the plasmid pDG148-Cre, which was then plated on solid LB medium without antibiotics. After individual colonies grew, they were simultaneously transferred to solid LB medium without resistance, solid LB medium containing 5 μg / mL chloramphenicol, and solid LB medium containing 50 μg / mL kanamycin. The strains that grew only on the solid LB medium without resistance were selected, indicating that both the resistance gene and the pDG148-Cre plasmid had been eliminated. Colony PCR confirmed that a positive clone, B. subtilis BS3 amyE::QS-prkC, was obtained, with amyE as the integration site (shown in SEQ ID NO. 24).

[0071] The recombinant plasmids pMA5-eGFP and pMA5-VNp-eGFP in Example 1 were chemically transformed into B. subtilis 168, and the recombinant plasmid pMA5-VNp-eGFP was chemically transformed into the competent cells of the recombinant strain B. subtilis BS3 amyE::QS-prkC. The cells were coated with 25 mg·L -1 The kanamycin-resistant LB plate was cultured at 37°C for 12 hours. The single colonies on the plate were verified by colony PCR, and the correct single colonies were selected and plated with 25 mg·L -1 The cells were cultured in 20 mL of liquid LB medium containing kanamycin resistance at 37°C for 8 h, and the glycerol tubes were stored in a -80°C ultra-low temperature freezer. Recombinant strains B. subtilis168pMA5-eGFP, B. subtilis 168pMA5-VNp-eGFP, and B. subtilis BS3 amyE::QS-prkC pMA5-VNp-eGFP were obtained.

[0072] The constructed recombinant bacteria were cultured in TB medium at 37°C and 200 rpm, and the expression level of the fluorescent protein eGFP was detected.

[0073] like Figure 1As shown, at 48 h, the unit cell fluorescence intensity of B. subtilis 168pMA5-VNp-eGFP was 1.62×10 4 au increased to 4.65×10 5 The results showed that the fusion expression of VNp tag significantly enhanced the expression level of eGFP in B. subtilis 168. The fluorescence intensity per unit cell of the constructed recombinant strain B. subtilis BS3amyE::QS-prkC pMA5-VNp-eGFP was further increased to 5.17×10 5 au, which was significantly higher than that of B. subtilis 168pMA5-VNp-eGFP, further verifying that the quorum sensing system regulatory gene prkC expression system has a promoting effect on the increase of recombinase expression level.

[0074] Example 3: Effect of VNp tag fusion expression on recombinase expression level

[0075] To further verify the effect of tag VNp fusion expression on the enzyme production level of B. subtilis, cellobiose epimerase was used as a model enzyme protein for heterologous expression. The specific steps were as follows:

[0076] The recombinant plasmid pMA5-CS-CE (the construction method of the recombinant plasmid is disclosed in patent CN118421546A) and the plasmid pMA5-VNp-CS-CE in Example 1 were chemically transformed into the competent cells of the strain B. subtilis BS3 amyE::QS-prkC and coated with 25 mg·L -1 The kanamycin-resistant LB plate was cultured at 37°C for 12 hours. The single colonies on the plate were verified by colony PCR, and the correct single colonies were selected and plated with 25 mg·L -1 The cells were cultured in 20 mL of liquid LB medium containing kanamycin resistance at 37°C for 8 h, and the glycerol tubes were preserved and stored in a -80°C ultra-low temperature freezer. Recombinant strains B. subtilis BS3 amyE::QS-prkC pMA5-CS-CE and B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE were obtained.

[0077] like Figure 2 As shown, the total enzyme activity of cellobiose epimerase of B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE was 16.05 U·mL -1Increased to 17.71 U·mL -1 The results showed that the fusion expression of the tag VNp could significantly enhance the expression level of cellobiose epimerase in B. subtilis.

[0078] Example 4: Production of Cellobiose Epimerase by Fed-Batch Fermentation in a 5 L Fermentor

[0079] The strain B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE constructed in Example 3 was subjected to fed-batch fermentation in a 5 L fermentor to produce cellobiose epimerase. The specific steps were as follows:

[0080] The strain was cultured in the presence of 25 mg·L -1 Streak on LB solid medium containing kanamycin and culture at 37℃ for 10h. Pick a single colony and inoculate it into 25mg·L -1 In 50mL of liquid LB culture medium containing kanamycin, shake and culture at 37°C and 200rpm for 12h to obtain seed liquid. The seed liquid was inoculated into a 5L fermenter at a rate of 2.5%. The initial culture medium of the fermenter was TB culture medium with a liquid volume of 1.95L, a culture temperature of 37°C, a ventilation volume of 1.5vvm, and an initial stirring speed of 400rpm. After the dissolved oxygen rebounded, the stirring was set to be linked to the dissolved oxygen, the stirring speed was 300-800rpm, the dissolved oxygen range was 40%-50%, and the pH was adjusted to 7.0 with 20% phosphoric acid and ammonia water, and 5g·L -1 ·h -1 (500g·L -1 Glucose was added to the fermenter at a constant flow rate at the feed rate of glucose mother liquor.

[0081] like Figure 3 As shown in Figure 2, at 22 h, the total enzyme activity of cellobiose epimerase of the engineered strain B. subtilis BS3 amyE::QS-prkC pMA5-VNp-CS-CE reached a maximum of 32.20 U·mL -1 Compared with shake flask fermentation, the total enzyme activity of cellobiose epimerase increased by 81.82%. At 24h, the OD 600 It reached its maximum at 51.30.

[0082] Example 5: Effect of VNp tag truncation on eGFP expression levels

[0083] The VNp tag of the plasmid pMA5-VNp-eGFP constructed in Example 1 was truncated by 3, 6, 9, 12, 15, 18, and 21 amino acids (the sequences of VNp1 to 7 are shown in Table 2). Plasmids pMA5-VNp-1-eGFP, pMA5-VNp-2-eGFP, pMA5-VNp-3-eGFP, pMA5-VNp-4-eGFP, pMA5-VNp-5-eGFP, pMA5-VNp-6-eGFP, and pMA5-VNp-7-eGFP were synthesized by Nanjing GenScript Co., Ltd. and chemically transformed into competent cells of the recombinant strain B. subtilis BS3 amyE::QS-prkC and plated with 25 mg L -1 The kanamycin-resistant LB plate was cultured at 37°C for 12 hours. The single colonies on the plate were verified by colony PCR, and the correct single colonies were selected and plated with 25 mg·L -1 The cells were cultured in 20 mL of liquid LB medium containing kanamycin resistance at 37 °C for 8 h, and the glycerol tubes were preserved and stored in a -80 °C ultra-low temperature freezer. The recombinant bacteria B.subtilis BS3amyE::QS-prkC pMA5-VNp-1-eGFP, B.subtilis BS3amyE::QS-prkC pMA5-VNp-2-eGFP, B.subtilis BS3 amyE::QS-prkC pMA5-VNp-3-eGFP, and B.subtilis BS3 were obtained. amyE::QS-prkCpMA5-VNp-4-eGFP, B.subtilis BS3 amyE::QS-prkC pMA5-VNp-5-eGFP, B.subtilis BS3amyE::QS-prkC pMA5-VNp-6-eGFP, B.subtilis BS3amyE::QS-prkC pMA5-VNp-7-eGFP.

[0084] Table 2 VNp tag and truncated tag sequences

[0085]

[0086]

[0087] like Figure 4 As shown, the fluorescence intensity per unit cell of the recombinant strain B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-eGFP was significantly higher than that of the control strain B. subtilis BS3 amyE::QS-prkC pMA5-VNp-eGFP (5.17×10 5au increased to 6.06×10 5 At the same time, the unit cell fluorescence intensity of the recombinant strain B. subtilis BS3amyE::QS-prkC pMA5-VNp-4-eGFP increased from 5.17×10 5 au increased to 5.83×10 5 The results showed that truncation of the VNp tag by 18 or 12 amino acids (amino acid sequences are shown as 16 and 18, respectively) significantly enhanced the expression level of eGFP in B. subtilis BS3amyE::QS-prkC.

[0088] Example 6: Effect of VNp tag truncation on recombinase expression levels

[0089] The plasmid pMA5-VNp-6-eGFP and the cellobiose epimerase gene CS-CE constructed in Example 5 were double-digested using restriction endonucleases BamH I and Sal I, and the digested products were verified by agarose gel electrophoresis. After verification, the gel was recovered. The gene CS-CE was ligated to the pMA5 vector using Solution I ligase. The successfully ligated product fragments were transformed into E. coli JM109 competent cells, and the transformants were diluted and coated on solid LB medium (containing 100 mg·L -1 Ampicillin) and screen the positive transformants. Pick the correct single colony from the colony PCR and inoculate it in a medium containing 100 mg·L -1 The cells were cultured in 20 mL of liquid LB medium (100 mL Erlenmeyer flask) containing ampicillin for 8-12 h, and the recombinant plasmid was extracted and digested with restriction enzymes BamH I and Sal I for verification. The plasmid was then sequenced and analyzed. The correct plasmid was named pMA5-VNp-6-CS-CE and stored in a -80°C refrigerator. The transformants were chemically transformed into competent cells of the recombinant strain B. subtilis BS3amyE::QS-prkC and plated with 25 mg·L -1 The kanamycin-resistant LB plate was cultured at 37°C for 12 hours. The single colonies on the plate were verified by colony PCR, and the correct single colonies were selected and plated with 25 mg·L -1Culture the culture in 20 mL of kanamycin-resistant LB medium at 37°C for 8 h. Store the glycerol tube in a -80°C freezer. This yields the recombinant B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE. Similarly, construct the recombinant B. subtilis BS3 amyE::QS-prkC pMA5-VNp-4-CS-CE.

[0090] like Figure 5 As shown, the total enzyme activity of cellobiose epimerase of B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE was 17.71 U·mL -1 Increased to 21.25 U·mL -1 At the same time, the total enzyme activity of cellobiose epimerase of B. subtilis BS3 amyE::QS-prkC pMA5-VNp-4-CS-CE was 17.71 U·mL -1 Increased to 19.31 U·mL -1 The results showed that truncation of the tag VNp by 18 or 14 amino acids could significantly enhance the expression level of cellobiose epimerase in B. subtilis.

[0091] Example 7: Production of Cellobiose Epimerase by Fed-Batch Fermentation in a 5 L Fermentor

[0092] The strain B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE constructed in Example 6 was subjected to fed-batch fermentation in a 5 L fermentor to produce cellobiose epimerase, specifically by the following steps:

[0093] The strain was cultured in the presence of 25 mg·L -1 Streak on LB solid medium containing kanamycin and culture at 37℃ for 10h. Pick a single colony and inoculate it into LB medium containing 25mg·L -1In 50mL of liquid LB culture medium containing kanamycin, shake and culture at 37°C and 200rpm for 12h to obtain seed liquid. The seed liquid was inoculated into a 5L fermenter at a rate of 2.5%. The initial culture medium of the fermenter was TB culture medium with a liquid volume of 1.95L, a culture temperature of 37°C, a ventilation volume of 1.5vvm, and an initial stirring speed of 400rpm. After the dissolved oxygen rebounded, the stirring was set to be linked to the dissolved oxygen, the stirring speed was 300-800rpm, the dissolved oxygen range was 40%-50%, and the pH was adjusted to 7.0 with 20% phosphoric acid and ammonia water, and 5g·L -1 ·h -1 (500g·L -1 Glucose was added to the fermenter at a constant flow rate at the feed rate of glucose mother liquor.

[0094] like Figure 6 As shown in Figure 2, at 24 h, the total enzyme activity of cellobiose epimerase of the engineered strain B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE reached a maximum of 38.63 U·mL -1 Compared with shake flask fermentation, the total enzyme activity of cellobiose epimerase increased by 81.79%. At 24h, the OD 600 It reached its maximum at 53.30.

[0095] Example 8: Cellobiose epimerase catalyzes the production of lactulose from lactose

[0096] The cellobiose epimerases produced by recombinant bacteria B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE and B. subtilis BS3 amyE::QS-prkC pMA5-VNp-4-CS-CE were respectively catalyzed by 500 g·L -1 Lactose produces lactulose. The reaction system is 5 mL and the enzyme amount is 5 U·mL -1 , substrate concentration 500 g·L -1 The reaction temperature was 80°C, pH 7.5, and catalysis for 20 h, and the lactulose conversion rate was determined. The results showed that the lactulose yield of B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE expressing cellobiose epimerase was 405 g·L -1 The lactulose conversion rate was 81%. Meanwhile, the lactulose yield of B. subtilis BS3 amyE::QS-prkC pMA5-VNp-6-CS-CE expressing cellobiose epimerase was 393 g·L -1, the lactulose conversion rate was 78.6%.

[0097] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A truncated VNp tag, characterized in that The amino acid sequence of the truncated VNp tag is shown in any one of SEQ ID NOs. 16 and 18.

2. A gene encoding the truncated VNp tag according to claim 1.

3. A recombinant subtilis spore, characterized in that: The recombinant Bacillus subtilis overexpresses a fusion fragment consisting of a VNp tag or the truncated VNp tag according to claim 1 and a recombinant protein, and the amino acid sequence of the VNp tag is shown in SEQ ID NO.

12.

4. The recombinant subtilis spore according to claim 3, characterized in that Plasmid pMA5 is used as an expression vector to express the fusion fragment, and the recombinant protein includes green fluorescent protein or cellobiose epimerase.

5. The recombinant Bacillus subtilis spore according to claim 4, characterized in that The plasmid pMA5 contains the promoter P HpaII , the promoter P HpaII The nucleotide sequence is shown in SEQ ID NO.

1.

6. The recombinant subtilis spore according to claim 4, characterized in that The nucleotide sequence of the green fluorescent protein gene eGFP is shown in SEQ ID NO.3; the nucleotide sequence of the cellobiose epimerase gene CS-CE is shown in SEQ ID NO.

4.

7. A method for preparing cellobiose epimerase, characterized in that: The method comprises fermenting and preparing cellobiose epimerase using recombinant Bacillus subtilis; the recombinant Bacillus subtilis overexpresses a fusion fragment consisting of a VNp tag or the truncated VNp tag according to claim 1 and cellobiose epimerase.

8. The method according to claim 7, characterized in that The recombinant Bacillus subtilis was cultured in TB medium at 35-37° C. for at least 48 hours.

9. Use of the truncated VNp tag according to claim 1, or the gene according to claim 2, or the recombinant Bacillus subtilis according to any one of claims 3 to 6, or the method according to claim 7 or 8 in the production of cellobiose epimerase.

10. Use of a VNp tag or the truncated VNp tag according to claim 1 in improving the expression level and extracellular secretion efficiency of exogenous proteins in recombinant Bacillus subtilis.

Images