A method for improving the heterologous expression level of D-psicose 3-epimerase

Abstract

The present invention discloses a method for improving the heterologous expression level of D-psicose 3-epimerase, which belongs to the field of bioengineering technology. amyE Catabolite responsive element (CRE) region of the promoter is used to improve the expression level of foreign proteins in Bacillus subtilis, and its application in heterologous expression of D-psicose 3-epimerase (DPEase). amyE Molecular modification of the CRE region of B. subtilis can alleviate the carbon catabolite repression (CCR) effect of B. subtilis, improve carbon source utilization and the expression level of the target protein, and the DPEase expression level is 97.9% to 349.5% of the unmodified group.

Description

Technical Field

[0001] The invention relates to a method for improving the heterologous expression level of D-psicose 3-epimerase, and belongs to the technical field of bioengineering. Background Art

[0002] In recent years, the incidence of chronic diseases such as obesity, diabetes, hyperlipidemia, and hypertension has skyrocketed due to excessive consumption of high-sugar, high-fat foods. D-allulose is a new functional rare sugar, designated GRAS (Grade As Safe for All) by the U.S. Food and Drug Administration (FDA). D-allulose has 70% of the sweetness of sucrose, but only 10% of its calories. It is also difficult to digest and absorb, offering great potential for applications in food, nutrition, and health.

[0003] D-psicose is extremely rare in nature. Currently, its industrial production primarily relies on D-psicose 3-epimerase (DPEase), which reversibly epimerizes the hydroxyl group at the C3 position of D-fructose. DPEases have been discovered from various sources, including Agrobacterium tumefaciens, Clostridium cellulolyticum, Desmospora sp., and Ruminococcus sp.

[0004] B. subtilis is an aerobic, spore-forming, endotoxin-free Gram-positive bacterium. It was the first Bacillus species to be used as a host for genetic engineering. Designated GRAS by the FDA, B. subtilis offers advantages such as simple and rapid cultivation, a well-established fermentation infrastructure, and production technology, making it an ideal expression host for industrial enzymes. However, during fermentation, recombinant bacteria are affected by carbon catabolite repression (CCR), preventing them from efficiently utilizing carbon sources. In B. subtilis, carbon catabolite control is achieved through the global regulatory protein carbon catabolite protein A (CcpA). CcpA binds to DNA as a formyl-phosphorylated complex of histidine-containing proteins. Cis-acting elements of genes are located in promoter regions or within the open reading frames of regulated genes and operons, and are termed catabolite responsive elements (CREs). In 1990, Weickert et al., based on genetic analysis, deduced a 14-bp consensus sequence for amyO with a certain degree of symmetry: T G W A ANC * G NTNW C A (the most important bases are underlined, N is any base, W represents adenine or thymine, and the asterisk represents the axis of symmetry) (Proc Natl Acad Sci. 1990, 87(16): 6238-42). In 1997, Jeong-Ho Kim et al. found that CcpA protects a 26 bp region centered on the axis of symmetry of the amyO consensus sequence and has a higher affinity for guanine than for deoxynucleotides near the axis of symmetry. Therefore, the guanine deoxynucleotides at -2 and +5 in the coding strand and +4 and +10 in the template strand, as well as their symmetrical positioning in the DNA sequence, are crucial for the binding of CcpA to amyO (Nucleic Acids Research, 1997, 25(17): 3490-3496).

[0005] Through molecular modification of the CRE region, the CCR effect is alleviated to improve the carbon source utilization of B. subtilis and increase the expression level of the exogenous protein DPEase, which is of great significance to the D-psicose enzymatic preparation industry. Summary of the Invention

[0006] The present invention aims to provide a method for heterologously expressing DPEase in recombinant strain B.subtilis by modifying the promoter P. amyE A scheme to improve carbon source utilization and DPE expression by targeting the CRE region.

[0007] The present invention provides a promoter, wherein the promoter is a nucleotide sequence as shown in SEQ ID NO. amyE The promoter is the parent, amyE Single mutations were performed at the -2, +3, +4, +5, +6, or +10 position of the CRE region of the promoter.

[0008] In one embodiment, after a single mutation at the +6 position of the CRE region, a base deletion is introduced at the -3, -6 or -9 position.

[0009] In one embodiment, the CRE region refers to the amyO-like nucleotide region TGTAAGCGTTAACA having symmetry.

[0010] In one embodiment, the -2 position of the CRE region is mutated from G to C, designated G2C.

[0011] In one embodiment, the -2 position of the CRE region is mutated from G to A, designated G2A.

[0012] In one embodiment, the -2 position of the CRE region is mutated from G to T, designated G2T.

[0013] In one embodiment, the +3 position of the CRE region is mutated from G to C, designated G3C.

[0014] In one embodiment, the +3 position of the CRE region is mutated from G to T, designated G3T.

[0015] In one embodiment, the +3 position of the CRE region is mutated from G to A, designated G3A.

[0016] In one embodiment, the +4 position of the CRE region is mutated from C to G, designated C4G.

[0017] In one embodiment, the +4 position of the CRE region is mutated from C to A, designated C4A.

[0018] In one embodiment, the +4 position of the CRE region is mutated from C to T, designated C4T.

[0019] In one embodiment, the +5 position of the CRE region is mutated from G to C, designated G5C.

[0020] In one embodiment, the +5 position of the CRE region is mutated from G to A, designated G5A.

[0021] In one embodiment, the +5 position of the CRE region is mutated from G to T, designated G5T.

[0022] In one embodiment, the +6 position of the CRE region is mutated from T to A, designated T6A.

[0023] In one embodiment, the +6 position of the CRE region is mutated from T to G, designated T6G.

[0024] In one embodiment, the +6 position of the CRE region is mutated from T to C, designated T6C.

[0025] In one embodiment, the +10 position of the CRE region is mutated from C to G, designated C10G.

[0026] In one embodiment, the +10 position of the CRE region is mutated from C to T, designated C10T.

[0027] In one embodiment, the T at the +6 position of the CRE region is mutated to an A, and a base deletion is introduced at the -3 position, designated as T6AΔ3.

[0028] In one embodiment, the T at the +6 position of the CRE region is mutated to an A, and a base deletion is introduced at the -6 position, designated as T6AΔ6.

[0029] In one embodiment, the T at the +6 position of the CRE region is mutated to an A, and a base deletion is introduced at the -9 position, designated as T6AΔ9.

[0030] The present invention also provides an expression vector carrying the above-mentioned promoter.

[0031] In one embodiment, the expression vector further comprises a target gene; the target gene is downstream of the promoter.

[0032] In one embodiment, the recombinant plasmid uses a Bacillus subtilis expression vector as a backbone, including but not limited to pHT01, or pHT304, or pHY300PLK, or pMA09, or pDG1663 plasmids.

[0033] In one embodiment, the target gene is the gene dpe encoding D-psicose 3-epimerase.

[0034] In one embodiment, the nucleotide sequence of the gene dpe is shown as SEQ ID NO.2.

[0035] Microbial cells carrying the above-mentioned recombinant plasmid.

[0036] The present invention provides a method for increasing the expression level of a target gene.

[0037] In one embodiment, the method is to use the above-mentioned promoter to induce the expression of the target gene.

[0038] The present invention also provides a method for preparing DPEase, which comprises inoculating the above-mentioned microbial cells into a culture medium, heating at 35-38°C, 180-220 r·min -1 After 1.5-2.5h of incubation, transfer to 31-34℃, 180-220r·min -1 Fermentation 45 to 50 hours.

[0039] The present invention provides the use of DPEase prepared by the above method in the preparation of D-psicose.

[0040] The present invention provides use of the above-mentioned expression vector and microbial cell in expressing DPEase and preparing D-psicose.

[0041] Beneficial effects:

[0042] P amyEThe relatively conservative -2, +3, +4, +5, +6, and +10 sites in the CRE region were mutated ( Figure 1 ), as well as introducing a point mutation at the +6 position (T to A) and base deletions at the -3, -6, and -9 positions. A ΔCre mutant with a CRE region deleted was also constructed as a control. Under the same fermentation time, the ΔCre mutant with a direct deletion of the CRE region showed little growth. With the exception of C10A, the promoter concentrations and enzyme activities of the other mutants were higher than those of the original strain. Most of the designed CRE region mutants have a positive effect on alleviating the CCR effect, and the present invention has great potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Is the promoter P amyE CRE area. DETAILED DESCRIPTION

[0044] 1. LB medium (g / L): yeast powder 5.0%, tryptone 10.0%, NaCl 10.0%.

[0045] 2. TB culture medium (g·L -1 ): Yeast powder 24.0, glycerol 5.0, tryptone 12.0, K2HPO4·3H2O, KH2PO4 2.31.

[0046] Example 1: Construction of CRE mutants

[0047] (1) Construction of recombinant plasmid pHY300PLK-P amyE -dpe

[0048] Synthesized P amyE promoter (SEQ ID NO.1) and a gene fragment of the D-psicose 3-epimerase gene dpe (SEQ ID NO.2), wherein P amyE The promoter is located upstream of the gene dpe and regulates the expression of the gene dpe. The gene fragment is inserted into the shuttle vector pHY300PLK and expressed in Bacillus subtilis to obtain the recombinant plasmid pHY300PLK-P amyE -dpe.

[0049] (2) Construction of mutant plasmid

[0050] Design site-directed mutagenesis primers to carry P amyE Recombinant plasmid pHY300PLK-P containing the promoter and the gene encoding D-psicose 3-epimerase dpe amyE -dpe was used as a template to introduce mutations at different sites in the CRE region.

[0051] Taking mutant G2C as an example, the construction method is as follows:

[0052] 1) Single-Primer PCR: PCR amplification was performed using either the upstream or downstream primer of G2C. The reaction system consisted of 12.5 μL 2× Super Pfx MasterMix, 25 ng template, and 0.5 μL G2C-F / 0.5 μL G2C-R, made up to 25 μL with water. The reaction procedure was as follows: initial denaturation at 98°C for 3 min 30 s, followed by three cycles of (98°C for 30 s; 55°C for 30 s; 72°C for 1 min), with an incubation period of 4°C. Two PCR products were obtained.

[0053] 2) After the reaction, the two PCR products obtained in step 1) were mixed and pre-denatured at 98°C for 3 min 30 s; then 15 cycles were performed (98°C, 30 s; 55°C, 30 s; 72°C, 1 min / kb); annealed at 72°C for 5 min, and then incubated at 4°C to obtain the amplified product.

[0054] 3) Take 7.5 μL of the amplified product obtained in step 2), add 1.5 μL of Dpn I and 1 μL of CutSmart, mix well, and incubate in a 37°C water bath for 9 h. The template in the digestion system is used for subsequent transformation. The transformation product is transformed into Escherichia coli JM109 competent cells and spread onto LB solid medium (containing 30 μg mL -1 Ampicillin) and cultured overnight at 37°C. Positive clones were selected, plasmids were extracted, and sequencing was performed for verification.

[0055] 4) The plasmid sequenced correctly in step 3) was electroporated into a B. subtilis competent cell for expression to obtain a recombinant Bacillus subtilis containing the mutant G2C.

[0056] Similarly, mutants ΔCre, G2C, G2A, G2T, G3C, G3T, G3A, C4G, C4T, C4A, G5C, G5A, G5T, T6A, T6G, T6C, C10G, C10A, C10T, T6AΔ3, T6AΔ6, and T6AΔ9 were constructed.

[0057] Table 1 Primer sequences

[0058]

[0059]

[0060] Example 2: Shake flask fermentation of recombinant Bacillus subtilis

[0061] 1) Shake flask fermentation: A single colony of the recombinant Bacillus subtilis prepared in step 1 or a glycerol tube was inoculated into 10 mL of LB (containing 30 μg mL -1 Tetracycline) at 37°C, 200 r·min -1 The obtained seed solution was inoculated into TB (containing 30 μg·mL -1 Tetracycline) medium, 37°C, 200 r·min -1 After culturing for 2 h, transfer to 33 °C and 200 r·min -1 Ferment for 48 hours.

[0062] 2) Preparation of crude enzyme solution: After the shake flask fermentation is completed, centrifuge at 8000g for 15 minutes at 4°C, discard the supernatant, and collect the cells. 2+ ) buffer. High-pressure homogenization at 1000 bar at 4°C disrupts the bacterial cell wall. Repeat this process three times. Centrifuge the resulting suspension at 8000 g for 20 minutes at 4°C. The supernatant is the crude enzyme solution.

[0063] Example 3: Mutant enzyme activity and equilibrium conversion rate of catalytic D-fructose

[0064] Bacterial concentration determination: dilute the bacterial solution with deionized water, mix well and evenly sample, place the sample in a 1cm glass cuvette, read the absorbance at 600nm wavelength, and ensure that the absorbance is within the effective range of 0.2-0.8. 600 ) = absorbance (0.2-0.8) × dilution factor.

[0065] Enzyme activity assay: At 60°C, pH 7.5, 800 μL of 100 g L -1 D-fructose was used as the substrate. 200 μL of the crude enzyme solution obtained in Example 2, diluted with buffer, was added. After mixing evenly, the reaction was allowed to proceed for 10 minutes. The reaction was terminated by boiling in a water bath for 10 minutes. The sample was centrifuged at 12,000 g for 5 minutes, the supernatant was removed, and the supernatant was diluted with deionized water to an appropriate dilution. The supernatant was then filtered through a 0.22 μm filter to remove impurities. The D-psicose and D-fructose contents were determined by high-performance liquid chromatography (HPLC).

[0066] Equilibrium conversion of D-fructose: At 60°C and pH 7.5, 8 mL of 100 g·L -1D-fructose was used as the substrate, and 2 mL of the crude enzyme solution obtained in Example 2, diluted with buffer, was added. After mixing evenly, the reaction was allowed to proceed for 4 hours, and the reaction was terminated by boiling in a water bath for 10 minutes. The sample was centrifuged at 12,000 g for 5 minutes, and the supernatant was removed. The supernatant was diluted with deionized water to an appropriate dilution and then filtered through a 0.22 μm filter to remove impurities. The D-psicose and D-fructose contents and equilibrium conversion rate were determined by high-performance liquid chromatography (HPLC).

[0067] Buffer: 20 mM HEPES, pH 7.5, containing 0.1 mM Co 2+ .

[0068] HPLC chromatographic conditions were as follows: Agilent 1200 HPLC chromatograph, ShodexTM Asahipak NH2P-504E column, column temperature set at 35°C, Agilent differential detector, Agilent autosampler, mobile phase 75% acetonitrile (organic membrane filtration followed by ultrasonic treatment for 5 min), mobile phase flow rate 0.8 mL·min -1 The enzyme activity of DPE and the equilibrium conversion rate of D-fructose were calculated based on the absorption peak area of ​​D-psicose and the peak area of ​​the standard.

[0069] Table 2 The bacterial mass and CcDPE enzyme activity of the recombinant strain after 48 h of fermentation in shake flask

[0070]

[0071]

[0072] 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. SEQUENCE LISTING <110> Jiangnan University <120> A method for improving the heterologous expression level of D-psicose 3-epimerase <130> BAA210753A <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 280 <212> DNA <213> Artificial sequence <400> 1 cttacagaag agcggtaaaa gaagaaataa aaaagaaatc atcttttttg tttggaaagc 60 gagggaagcg ttcacagttt cgggcagctt tttttatagg aacattgatt tgtattcact 120 ctgccaagtt gttttgatag agtgattgtg ataattttaa atgtaagcgt taacaaaatt 180 ctccagtctt cacatcggtt tgaaaggagg aagcggaaga atgaagtaag agggattttt 240 gactccgaag taagtcttca aaaaatcaaa taaggagtgt 280 <210> 2 <211> 879 <212> DNA <213> Artificial Sequence <400> 2 atgaagcacg gcatctacta cgcctactgg gagcaggagt gggaggccga ctacaagtac 60 tacatcgaga aggtggccaa gctgggcttc gacatcctgg agatcgccgc cagccccctg 120 cccttctaca gcgacatcca gatcaacgag ctgaaggcct gcgcccacgg caacggcatc 180 accctgaccg tgggccacgg ccccagcgcc gagcagaacc tgagcagccc cgaccccgac 240 ​​​​gacaagaagg gcgactggga gaggagcgtg gagagcgtga gggaggtggc caaggtggcc 420 gaggcctgcg gcgtggactt ctgcctggag gtgctgaaca ggttcgagaa ctacctgatc 480 aacaccgccc aggaggggcgt ggacttcgtg aagcaggtgg accacaacaa cgtgaaggtg 540 atgctggaca ccttccacat gaacatcgag gaggacagca tcggcggcgc catcaggacc gccggcagct acctgggcca cctgcacacc ggcgagtgca acaggaaggt gcccggcagg 660 ggcaggatcc cctgggtgga gatcggcgag gccctggccg acatcggcta caacggcagc 720 gtggtgatgg agcccttcgt gaggatgggc ggcaccgtgg gcagcaacat caaggtgtgg 780 agggacatca gcaacggcgc cgacgagaag atgctggaca gggaggccca ggccgccctg gacttcagca ggtacgtgct ggagtgccac aagcacagc

Claims

1. A promoter, characterized in that The nucleotide sequence is shown in SEQ ID NO. amyE The promoter is the parent, amyE Single mutation is performed at the -2, +3, +4, +5 or +6 position of the CRE region of the promoter; the nucleotide sequence of the CRE region is TGTAAGCGTTAACA.

2. The promoter according to claim 1, characterized in that After a single mutation at the +6 site of the CRE region, base deletions were introduced at the -3, -6, or -9 sites.

3. An expression vector, characterized in that The expression vector carries the promoter according to claim 1 or 2.

4. The expression vector according to claim 3, characterized in that The expression vector further comprises a target gene; the target gene is located downstream of the promoter.

5. The expression vector according to claim 4, characterized in that The expression vector uses a Bacillus subtilis expression vector as a backbone, including but not limited to pHT01, pHT304, pHY300PLK, pMA09, or pDG1663 plasmids.

6. The expression vector according to claim 4, characterized in that The target gene is the gene dpe encoding D-psicose 3-epimerase.

7. A microbial cell carrying the expression vector according to any one of claims 3 to 6, wherein the microbial cell is Bacillus subtilis.

8. A method for increasing the expression level of the D-psicose 3-epimerase gene dpe in Bacillus subtilis, characterized in that: The method is to use the promoter described in claim 1 or 2 to induce the expression of the target gene dpe.

9. A method for preparing D-psicose 3-epimerase, characterized in that: The microbial cells according to claim 7 are inoculated into the culture medium and heated at 35-38°C and 180-220 r·min. -1 After 1.5-2.5h of incubation, transfer to 31-34℃, 180-220r·min -1 Fermentation 45 to 50 hours.

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