Double promoters for expressing sucrose phosphorylase and application of double promoters

Abstract

The invention belongs to the field of gene engineering and the technical field of molecular biology, and particularly relates to double promoters for expressing sucrose phosphorylase and application of the double promoters. The double promoters comprise any one of the following sequences: a nucleotide sequence as shown in SEQ ID NO.1 or a complementary sequence of the nucleotide sequence; 1, and a nucleotide sequence as shown in SEQ ID NO. 2 or a complementary sequence thereof. The sucrose phosphorylase is synthesized in bacillus subtilis by adopting a double-promoter expression system, the effect of the sucrose phosphorylase is obviously superior to the expression effect of a single promoter P43, the extracellular enzyme activity is improved by 403%, and efficient production is realized. Meanwhile, the DNA fragment with the promoter and RBS functions provided by the invention can realize secretory expression of protein genes under the condition of not adding an inducer, provides an effective means for secretory expression of protein by bacillus subtilis by applying the DNA fragment to expression of sucrose phosphorylase, and has a very good development prospect.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and molecular biology technology, and particularly relates to a dual promoter for expressing sucrose phosphorylase and application thereof. Background Art

[0002] Sucrose phosphorylase (LreSP-MT) is a class of 18 subfamily in the glycosyl hydrolase family GH13. Its EC number is 2.4.1.7, and it belongs to the transferase class. It has excellent substrate promiscuity and can transfer glucose groups to different acceptors using only cheap sucrose as a substrate. Glycosylation modification can improve the water solubility, stability, bioavailability and other properties of the compound, and obtain more superior performance than before. At the same time, the resulting product can be widely used in food, medicine, personal care and other fields. There are precedents in the prior art for heterologous expression of sucrose phosphorylase in genetically engineered bacteria, but the produced sucrose phosphorylase generally has the problem of poor extracellular enzyme activity, which seriously affects the industrial and efficient production of sucrose phosphorylase.

[0003] A promoter is a DNA sequence located 5' upstream of the gene coding sequence that activates RNA polymerase, enabling it to accurately bind to the template DNA and specifically initiate transcription. Transcription initiation is a critical stage in gene expression. Regulation of transcription levels is often influenced by the strength, specificity, and ability of the promoter sequence to respond to external signals. Different promoters have varying activities; some may promote efficient gene transcription, while others may inhibit it. Therefore, selecting the right promoter is crucial for efficient expression of the target protein. However, limited research has been conducted on optimizing promoters for sucrose phosphorylase production, severely limiting its efficient production. Summary of the Invention

[0004] The present invention aims to overcome the problem of poor extracellular enzymatic activity of biosynthetic sucrose phosphorylase in the prior art, and provides a dual promoter for expressing sucrose phosphorylase and its application to overcome the above-mentioned defect.

[0005] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions: In a first aspect, the present invention discloses a dual promoter for expressing sucrose phosphorylase, wherein the dual promoter comprises any one of the following sequences: (a) the nucleotide sequence shown in SEQ ID NO.1 or its complementary sequence; (b) The nucleotide sequence shown in SEQ ID NO. 2 or its complementary sequence; which is obtained by modifying the ribosome binding site of the nucleotide sequence shown in SEQ ID NO. 1.

[0006] On the one hand, the present invention significantly improves the expression level of sucrose phosphorylase by screening and optimizing the promoter for the target gene; at the same time, since the ribosome binding site (RBS) is also a key genetic element affecting gene expression, the present invention further optimizes 1 of the nucleotide sequence shown in SEQ ID NO.1, and improves the translation efficiency of mRNA at the translation initiation stage, thereby improving the expression level of sucrose phosphorylase.

[0007] In a second aspect, the present invention discloses a dual promoter for expressing sucrose phosphorylase, and its application in secretory expression of sucrose phosphorylase in Bacillus subtilis.

[0008] Bacillus subtilis is a soil-derived, Gram-positive bacterium of the genus Bacillus. Compared to Escherichia coli, it offers unique advantages as a more attractive host for exogenous gene expression. These include: the absence of endotoxin production and pathogenicity; its classification as a GRAS-safe strain; excellent secretion capacity, which greatly simplifies subsequent product isolation and purification; low nutritional requirements; a clear genetic background, facilitating genetic modification and metabolomics analysis; extensive information on its transcription and translation mechanisms, including large-scale fermentation; and a lack of significant codon usage bias. These advantages have made Bacillus subtilis an ideal host strain for the production of various industrial enzymes, such as amylases, proteases, and lipases.

[0009] In a third aspect, the present invention further discloses an expression vector carrying the above-mentioned dual promoter for expressing sucrose phosphorylase.

[0010] Furthermore, the expression vector is a plasmid, which also includes a heterologous protein nucleotide sequence operably linked to the sequence SEQ ID NO.1 or the sequence SEQ ID NO.2; the heterologous protein nucleotide sequence is located downstream of the sequence SEQ ID NO.1 or the sequence SEQ ID NO.2.

[0011] Furthermore, the heterologous protein nucleotide sequence is the nucleotide sequence of sucrose phosphorylase, and the nucleotide sequence of sucrose phosphorylase is shown in SEQ ID NO.3.

[0012] In a fourth aspect, the present invention also discloses the use of the above expression vector in the production of sucrose phosphorylase.

[0013] In a fifth aspect, the present invention further discloses a recombinant engineered cell, which is obtained by transforming or transducing a host cell with the above-mentioned expression vector.

[0014] Furthermore, the host cell is a Bacillus subtilis host cell.

[0015] Furthermore, the Bacillus subtilis host cell is Bacillus subtilis WB800.

[0016] In a sixth aspect, the present invention also discloses the use of the above-mentioned recombinant engineered cells in the production of sucrose phosphorylase.

[0017] Therefore, the present invention has the following beneficial effects: (1) The present invention uses a dual promoter expression system to synthesize sucrose phosphorylase in Bacillus subtilis, and its effect is significantly better than that of a single promoter P 43 The expression effect of the product was significant, and the extracellular enzyme activity increased by 403%, achieving efficient production. (2) The present invention further increases the extracellular enzymatic activity of sucrose phosphorylase by 14% by using the optimized RBS sequence; (3) The DNA fragment with promoter and RBS functions provided by the present invention can achieve secretory expression of protein genes without the addition of inducers. Its application in the expression of sucrose phosphorylase provides an effective means for secretory expression of proteins by Bacillus subtilis and has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 HPLC spectra of glycerol, fructose, 2-αGG, 1-αGG, glucose and sucrose standard samples.

[0019] Figure 2 Schematic diagram for comparison of extracellular enzyme activities of different single promoters.

[0020] Figure 3 The SDS-PAGE electrophoresis gel images show the expression of sucrose phosphorylase under the control of different single promoters.

[0021] Figure 4 Schematic diagram for comparison of extracellular enzyme activities of different dual promoters.

[0022] Figure 5 The SDS-PAGE electrophoresis gel images show the expression of sucrose phosphorylase under the control of different dual promoters.

[0023] Figure 6 Schematic diagram for the comparison of extracellular enzyme activities of different RBS sequences.

[0024] Figure 7 The SDS-PAGE electrophoresis gel images of sucrose phosphorylase expression with different RBS sequences.

[0025] Figure 8 Schematic diagram of the temporal changes in extracellular enzyme activity and biomass during fermentation in a 3L fermentor.

[0026] Figure 9The SDS-PAGE electrophoresis gel images of extracellular sucrose phosphorylase in the fermentation supernatant at different fermentation times.

[0027] Figure 10 For the recombinant vector pP ydjO -P yvyD -R B15 -Plasmid map of LreSP-MT. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention described below are generally only embodiments of a portion of the present invention, rather than all embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work should fall within the scope of protection of the present invention. The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents.

[0029] The culture medium involved in the following examples: LB medium: 5.0 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, deionized water, pH 7.0; TB medium: 12 g / L peptone, 24 g / L yeast powder, 5 g / L glycerol, 16 g / L K2HPO4·3H2O, 2 g / L KH2PO4, pH 7.0; Fermentation medium: peptone 16 g / L, yeast powder 20 g / L, NaCl 5 g / L, K2HPO4·3H2O 3 g / L, pH 7.0; Feed medium 1: glucose 375 g / L; Feed medium 2: yeast extract 200 g / L; like Figure 1 Shown are HPLC spectra of standard samples of glycerol, fructose, glycerol glucoside (2-αGG, 1-αGG), glucose and sucrose.

[0030] Example 1: Construction of recombinant Bacillus subtilis containing sucrose phosphorylase gene The sucrose phosphorylase lreSP-MT gene was amplified using the previously reported recombinant plasmid pET-28a-LreSPase-MT as a template (AIChE J. 2025; e18908) using primers Lre-F / Lre-R. After gel purification, the gene was stored at -20°C. The plasmid backbone was amplified using primers bone-F / R using the previously reported Escherichia coli-Bacillus subtilis shuttle vector pP43-BloSP as a template (Enzyme Microb Technol 2023, 169: 110267). After digestion with Dpn I, the gene fragment and plasmid backbone were stored at -20°C. The gene fragment and plasmid backbone were ligated using a one-step cloning kit and transformed into E. coli DH5α competent cells. The specific operation is as follows: take 10 μL of the ligation reaction product, add it to 100 μL of E. coli DH5α competent cells, flick the tube wall several times to mix, let it stand on ice for 30 minutes, heat shock at 42°C for 60 seconds, and ice bath for 2-3 minutes. Add 900 μL of sterile LB liquid without antibiotics and incubate at 37°C for 45 minutes to recover its resistance. Centrifuge at 5000 rpm for 5 minutes, concentrate the bacteria to 100 μL, and evenly spread it on an LB plate containing 100 μg / mL ampicillin. Invert the plate and culture it at 37°C overnight. Pick the clones that are positive for colony PCR, purify them by streaking and culture on a shaker, extract the plasmid, and verify by enzyme digestion and sequencing. The recombinant plasmid pP 43 -LreSP-MT, that is, the sucrose phosphorylase lreSP-MT gene is expressed by P 43 The promoter drives its transcription and expression, serving as the starting recombinant plasmid. The primer sequences in Example 1 are shown in Table 1. Table 1: Primer sequence list of Example 1

[0031] The recombinant plasmid pP was sequenced to verify the correct 43-LreSP-MT was transferred into Bacillus subtilis WB800. The specific operation was as follows: pick a fresh single colony in 50 mL LB medium, 37 ° C, 200 rpm culture overnight; transfer 6 mL seed solution to 100 mL GM medium (LB medium supplemented with 0.5 mol / L sorbitol), 37 ° C, 200 rpm culture to OD600 of 0.85-0.95; the cultured bacterial solution was ice-water bathed for 10 minutes and centrifuged at 4 ° C, 2000 g for 10 minutes to collect the bacteria, take 30 mL ice-cold ETM (0.5 mol / L sorbitol, 0.5 mol / L mannitol, 10% glycerol) to resuspend the bacteria, 4 ° C, 2000 × g centrifugation for 10 minutes, and repeat washing four times; take the bacteria after the last wash, resuspend with 1 mL of EMT, 100 μL per tube was dispensed into 1.5 mL centrifuge tubes, and stored at -80 ° C for use, which is the prepared competent state; take the competent state to melt on ice, add 10 μL vector pP 43 -LreSP-MT, ice bath for 5 minutes, transfer to a pre-cooled 2mm electroporation cuvette; 25μF, 200Ω, 2500V electric shock once, the electric shock time is about 4.5ms to 5.0ms, immediately add 1mL pre-cooled RM medium (LB containing 0.5mol / L sorbitol and 0.38mol / L mannitol); 37℃, 200prm for 3 hours, spread on a plate containing the corresponding antibiotic, culture at 37℃ overnight, pick out transformants for verification. Containing the vector pP 43 -LreSP-MT recombinant Bacillus subtilis, the engineering bacteria was named BS-P 43 -LreSP-MT.

[0032] Example 2: Screening of single promoters (1) Construction of recombinant engineered bacteria containing different single promoters The corresponding promoter was amplified using the genome of Bacillus subtilis WB800 as a template using the corresponding primer pair, and the promoter was purified by gel recovery and stored in a -20°C refrigerator. 43 -LreSP-MT was used as a template, and the plasmid backbone was obtained by amplification using primers bone-lre-F / R. The purified promoter fragment was cloned and connected with the plasmid backbone in one step, and then transformed into E. coli DH5α competent cells according to the method in Example 1. After verification by colony PCR and sequencing, recombinant vectors containing different single promoters were obtained, namely pP yvyD -LreSP-MT、pP lytR -LreSP-MT、pP nadE -LreSP-MT、pP aprE -LreSP-MT、pP spoVG -LreSP-MT、pP sigW-LreSP-MT、pP ydjO -LreSP-MT and pP yfkJ -LreSP-MT. Recombinant vectors containing different promoters were transformed into Bacillus subtilis WB800 according to the method in Example 1 to obtain recombinant Bacillus subtilis containing different recombinant vectors. The primer pairs used are shown in Table 2: Table 2: Primer sequence list of Example 2

[0033] (2) Detection of promoter expression level Single colonies of the recombinant Bacillus subtilis expressing LreSP-MT from different promoters were selected and inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin. The cultures were incubated overnight at 37°C, 200 rpm. The seed solution was transferred to 25 mL of TB medium (containing 50 μg / mL kanamycin) at a 2% (v / v) inoculum and fermented at 37°C, 200 rpm for 30 h. Protein expression levels and extracellular enzyme activity were then measured. Enzyme activity assay: The fermentation broth was centrifuged at 12000 rpm for 5 min and the supernatant was collected to obtain the crude enzyme solution. 500 μL of the supernatant was mixed evenly with 500 μL of the reaction solution (final concentration of the reaction solution was 1.5 mol / L glycerol, 1.0 mol / L sucrose, 50 mmol / L MES buffer, pH 6.5). The reaction was incubated at 50°C for 2 h and then terminated in a boiling water bath for 5 min. The reaction solution was used for HPLC analysis. Figure 2 As shown, where P yvyD The extracellular enzyme activity of the promoter was the highest, which was higher than that of the starting strain BS-P 43 -LreSP-MT (i.e. P 43 promoter) increased by 310%. HPLC detection conditions: The reaction solution was boiled in water, centrifuged at 12,000 rpm for 10 min, diluted 5-fold, filtered through a 0.22 μm filter membrane, and the filtrate was added to a liquid sample bottle; chromatographic column: YMC-Pack Polyamine II column (250 × 4.6 mm, 5 μm); column temperature: 30°C; mobile phase: acetonitrile:water = 75:25; flow rate: 1 mL / min; detector: differential detector; injection volume: 10 μL. SDS-PAGE protein electrophoresis analysis: The supernatants and cell lysates of different recombinant strains were subjected to SDS-PAGE protein electrophoresis, and 5× loading buffer was added to the samples and treated in a boiling water bath for 10 minutes. The concentration of the stacking gel was 5%, the concentration of the separating gel was 10%, and the loading volume was 7.5 μL. Figure 3 As shown, the protein gel results are consistent with the enzyme activity results, containing P yvyDThe protein expression level of the recombinant strain of the promoter was significantly higher than that of other single promoters, and a clear protein band was observed between 45-60 kDa, which was consistent with the size of sucrose phosphorylase.

[0034] Example 3: In P yvyD Construction of a dual promoter structure on a gene and its application in the expression of sucrose phosphorylase The recombinant vector pP constructed in Example 2 yvyD -LreSP-MT was used as a template, and the plasmid backbone was amplified using the primer pair bone-yvyD-F / bone-XX-R. After Dpn I digestion, the plasmid was stored in a -20°C refrigerator. The promoter fragment was amplified using the corresponding primers using the genome of Bacillus subtilis WB800 as a template, and then purified by gel recovery. The single promoter was inserted into P using a one-step cloning kit. yvyD promoter, and obtained recombinant vectors containing different dual promoter combinations, namely pP yvyD -P yvyD -LreSP-MT、pP lytR -P yvyD -LreSP-MT、pP nadE -P yvyD -LreSP-MT、pP aprE -P yvyD -LreSP-MT、pP spoVG -P yvyD -LreSP-MT、pP sigW -P yvyD -LreSP-MT、pP 43 -P yvyD -LreSP-MT、pP ydjO -P yvyD -LreSP-MT and pP yfkJ -P yvyD -LreSP-MT. According to the method of Example 1, different recombinant vectors were transformed into Bacillus subtilis WB800, and fermentation was performed to verify the protein expression level and extracellular enzymatic activity of sucrose phosphorylase. The primer sequences in Example 3 are shown in Table 3. Table 3: Example 3 primer sequence list

[0035] Comparison of extracellular enzyme activities of dual promoters: The extracellular enzyme activities of different recombinant engineered bacteria were detected according to the method in Example 2. Figure 4 As shown, the results showed that the extracellular enzyme activities of all dual promoters were higher than P 43 The enzyme activity of a single promoter, P ydjO -P yvyDThe extracellular enzyme activity of the double promoter (whose sequence is shown in SEQ ID NO.1) is higher than that of P 43 The single promoter increased by 403%. Figure 5 As shown, P ydjO -P yvyD The SDS-PAGE results of the dual promoter were consistent with the enzyme activity results, which showed that the dual promoter system could effectively increase the expression level of sucrose phosphorylase in Bacillus subtilis and achieve efficient expression of sucrose phosphorylase in Bacillus subtilis.

[0036] Example 4: RBS sequence engineering and its application in the expression of sucrose phosphorylase The dual promoter vector pP with the highest enzyme activity obtained in Example 3 was used. ydjO -P yvyD -LreSP-MT was used as a template and the corresponding primers were used to amplify the whole plasmid. B10 -R B15 ) replaces the original RBS sequence (R B0 ), and obtained 6 recombinant vectors containing different RBS sequences, namely pP ydjO -P yvyD -R B10 -LreSP-MT、pP ydjO -P yvyD -R B11 -LreSP-MT、pP ydjO -P yvyD -R B12 -LreSP-MT、pP ydjO -P yvyD -R B13 -LreSP-MT、pP ydjO -P yvyD -R B14 -LreSP-MT and pP ydjO -P yvyD -R B15 -LreSP-MT. The different recombinant vectors were transformed into Bacillus subtilis WB800 according to the method of Example 1, and the protein expression and extracellular enzymatic activity of sucrose phosphorylase were verified by fermentation. The primer sequences used are shown in Table 4: Table 4: Example 4 primer sequence list

[0037] Comparison of extracellular enzyme activities of different engineered RBS sequences: The extracellular enzyme activities of recombinant engineered bacteria containing different RBS sequences were detected according to the method in Example 2. Figure 6 As shown, the results show that R B15 The recombinant vector pPydjO -P yvyD -R B15 -LreSP-MT can further improve the extracellular enzyme activity of sucrose phosphorylase, compared with the control strain containing pP ydjO -P yvyD -R B10 -LreSP-MT recombinant engineering bacteria increased by 14%. Figure 7 As shown, R B15 The recombinant vector pP ydjO -P yvyD -R B15 -LreSP-MT protein expression level is consistent with the enzyme activity data. B15 The expression level of sucrose phosphorylase in Bacillus subtilis WB800 can be effectively increased, that is, the promoter sequence shown in SEQ ID NO.2 is obtained.

[0038] Example 5: Recombinant vector pP ydjO -P yvyD -R B15 Application of LreSP-MT in the expression of sucrose phosphorylase (1) Bacteria activation The recombinant Bacillus subtilis WB800 (pP ydjO -P yvyD -R B15 -LreSP-MT) were inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C, 200 rpm for 12 h to obtain seed solution. (2) Catalyst preparation in a 3L fermenter Inoculate 1 L of fermentation medium containing 0.05% defoamer and 50 mg / L kanamycin at a 5% volume concentration of freshly cultured seed liquid. Incubate at 36°C for 4 hours, then add feed medium 1 at a rate of 13 mL / h. After 8 hours of incubation, add feed medium 2 at a rate of 15 mL / h. Maintain the fermentation temperature at 36°C, the dissolved oxygen (DO) at greater than 30%, and the pH at 6.8 with 25% ammonia. Continue fermentation for 36 hours. Samples are taken periodically during the fermentation process and stored in a refrigerator at 4°C pending enzyme activity measurement. (3) Verification of extracellular enzyme activity and protein expression in fermentation According to the enzyme activity detection method in Example 2, the extracellular enzyme activity of samples at different time points during the fermentation process was verified. Figure 8 As shown in the results, the extracellular enzyme activity reached a maximum of 9.1U / mL at 27h of fermentation, which was nearly 10 times higher than that of the single promoter P43 shake flask culture. Figure 9As shown, SDS-PAGE results revealed a distinct protein band between 45 and 60 kDa, consistent with the size of sucrose phosphorylase, indicating normal fermentation results. Protein quantification was performed on the crude enzyme solution using a Bradford protein quantification kit, using bovine serum albumin (BSA) as the standard protein. A standard curve was constructed and samples were tested according to the instructions to calculate protein concentration. After 36 hours of fermentation, the extracellular protein concentration of sucrose phosphorylase reached 8 g / L.

[0039] Finally, it should be noted that the above experiments and embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual promoter for expressing sucrose phosphorylase, characterized in that: The dual promoter includes any of the following sequences: (a) the nucleotide sequence shown in SEQ ID NO. 1 or its complementary sequence; (b) The nucleotide sequence shown in SEQ ID NO. 2 or its complementary sequence; which is obtained by modifying the ribosome binding site of the nucleotide sequence shown in SEQ ID NO.

1.

2. Use of the dual promoter for expressing sucrose phosphorylase as claimed in claim 1 for secretory expression of sucrose phosphorylase in Bacillus subtilis.

3. An expression vector, characterized in that: It carries the dual promoter for expressing sucrose phosphorylase as claimed in claim 1.

4. An expression vector according to claim 3, characterized in that: The expression vector is a plasmid, which further comprises a heterologous protein nucleotide sequence operably linked to the sequence SEQ ID NO.1 or the sequence SEQ ID NO.2; the heterologous protein nucleotide sequence is located downstream of the sequence SEQ ID NO.1 or the sequence SEQ ID NO.

2.

5. An expression vector according to claim 4, characterized in that: The heterologous protein nucleotide sequence is the nucleotide sequence of sucrose phosphorylase, and its sequence is shown in SEQ ID NO.

3.

6. Use of the expression vector according to any one of claims 3 to 4 in producing sucrose phosphorylase.

7. A recombinant engineered cell, characterized in that: The expression vector is obtained by transforming or transducing a host cell with the expression vector according to any one of claims 3 to 5.

8. The recombinant engineered cell according to claim 7, characterized in that: The host cell is a Bacillus subtilis host cell.

9. The recombinant engineered cell according to claim 8, characterized in that: The Bacillus subtilis host cell is Bacillus subtilis WB800.

10. Use of the recombinant engineered cell according to any one of claims 7 to 9 in producing sucrose phosphorylase.

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