A bacillus subtilis strong promoter and application thereof in preparation of allulose

Abstract

This invention discloses a strong promoter for Bacillus subtilis and its application in the preparation of allulose, belonging to the fields of genetic engineering and enzyme engineering. The strong promoter is obtained by replacing one or more sequences in the -10, -16, and -35 regions of the wild-type endogenous promoter of Bacillus subtilis with the conserved sequences TATAAT, ATG, and TTGACA, or by further truncating the 5' end of the substituted promoter. The resulting strong promoter mutant P... VGt1 The transcriptional activity of the promoter was significantly enhanced, and the intracellular enzyme activity reached 23.6 U / mg after driving DAE gene expression, which is 35% higher than that of the original promoter, and the equilibrium conversion rate reached 32.75%. The promoter modification method provided by this invention is simple to operate and has wide applicability. It can significantly enhance the expression level of exogenous proteins in Bacillus subtilis and has important application value in the field of rare sugar biosynthesis.

Description

(I) Technical Field

[0001] This invention relates to a strong promoter of Bacillus subtilis and its application in the preparation of allulose. (II) Background Technology

[0002] Bacillus subtilis is a Gram-positive bacterium widely used in the industrial production of recombinant proteins. Due to its outstanding characteristics such as non-toxicity, high secretion rate, ease of genetic manipulation, and high cell density, it has been used as a standard host strain. Although many heterologous proteins have been successfully expressed in Bacillus subtilis and theoretically offer significant advantages, applying Bacillus subtilis to produce proteins in practical environments faces several challenges, such as typically low plasmid copy numbers and low protein expression levels, making promoter modification crucial.

[0003] D-Allulose is a rare sugar found in very little nature. It is characterized by low calories and a variety of biological activities. Due to its advantages in regulating blood sugar, protecting oral health, and weight management, it is widely used in the food and medical fields, demonstrating enormous market potential.

[0004] Traditional promoters are modified in strength through random mutagenesis, but this method is cumbersome, labor-intensive, and unsuitable for high-throughput screening. Furthermore, the modification methods are difficult to apply to other promoters, making it challenging to find promoters with further functional enhancements. In recent years, rational design has become a trend, which involves enhancing promoter strength by replacing key conserved sequences to make them closer to "consensus sequences." Meanwhile, excessively long promoter sequences may contain repressive elements and hinder the modular assembly of multi-gene pathways. Therefore, truncating promoters to obtain minimal core promoters while enhancing strength has significant application value.

[0005] Currently, natural endogenous promoters have drawbacks such as relatively low activity and poor stability. Based on this background, a simple and feasible method for modifying Bacillus subtilis promoters is needed. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a strong promoter for Bacillus subtilis and its application in the preparation of allulose. The promoter is modified by substituting a base in a certain region or truncating the core region of the endogenous promoter of Bacillus subtilis. The modification method is simple, can be applied to different types of promoters, and can obtain a large number of promoter mutants in a short time. This results in a superior promoter mutant that enhances the expression of exogenous proteins, meets different application needs, and has played a positive role in actual protein production.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a strong promoter for Bacillus subtilis, wherein the strong promoter for Bacillus subtilis is obtained by replacing one or more sequences in regions -10, -16, and -35 of the wild-type endogenous promoter derived from Bacillus subtilis with conserved sequences, or by truncating the replaced promoter; wherein region -10 (referring to the nucleic acid sequence approximately 10 bp upstream of the transcription start site) is replaced with TATAAT, region -16 (referring to the nucleic acid sequence approximately 16 bp upstream of the transcription start site) is replaced with ATG, and region -35 (referring to the nucleic acid sequence approximately 35 bp upstream of the transcription start site) is replaced with TTGACA.

[0009] Furthermore, the truncation is performed using BDGP software (Berkeley Drosophila Genome Project) to predict the promoter, where promoter fragments with a score of 0.8-1 are used as the core region, and the last 10 bp of each core region fragment is used as the transcription start site (TSS). All core region fragments and TSSs are retained, and the promoter is truncated.

[0010] Furthermore, the wild-type promoters include constitutive promoters (also known as non-specific promoters, which are promoters that can continuously express the target protein without the addition of any inducer) or self-inducible promoters (a type of promoter whose gene transcription activity is regulated by specific physical or chemical signals), and further include P43, P... aprE P amyE P gsiB P hag P lytR , P sigx 、 P spoVG P y1bp P yvyD P abrB P asd P ffh P vals P HpaⅡ The nucleotide sequences are shown in SEQ ID NO.1-14 and SEQ ID NO.49, respectively, or nucleotide sequences that have more than 95% homology with them.

[0011] Furthermore, the preferred nucleotide sequence of the Bacillus subtilis strong promoter is shown in one of SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.36, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, or SEQ ID NO.48.

[0012] Secondly, the present invention provides an expression vector containing the strong promoter of Bacillus subtilis. The expression vector is suitable for Bacillus subtilis expression, preferably based on the pMA5 plasmid.

[0013] Thirdly, the present invention provides a recombinant bacterium that expresses the D-allulose 3-epimerase (DAE) gene using the strong promoter of the aforementioned Bacillus subtilis. The host of the recombinant bacterium includes, but is not limited to, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus fragilis, Bacillus claurifolius, Bacillus alkalophilus, or Bacillus thuringiensis. Preferably, the host is Bacillus subtilis. WB800 .

[0014] Furthermore, the recombinant bacteria are obtained by inserting the strong promoter into the pMA5 plasmid carrying the DAE gene and then transforming it into the host bacteria.

[0015] Fourthly, this invention provides an application of the recombinant bacteria constructed using the aforementioned strong promoter in the preparation of D-allulose. The method of application involves using the wet bacterial cells obtained from the fermentation of the recombinant bacteria as a catalyst, D-fructose as a substrate, and Co... 2+ Using ions as auxiliaries and PB buffer (0.05 mol / L, pH 6.5) as the reaction medium, the reaction system was constructed and reacted at 1000 rpm and 70 °C to obtain a reaction solution containing D-allulose.

[0016] Furthermore, the substrate concentration in the reaction system is 100 g / L, the wet bacterial cell concentration is 100 g / L, and Co... 2+ The concentration of added ions was 0.1 mol / L.

[0017] Furthermore, the catalyst is prepared by streaking the recombinant bacteria onto LB solid medium, activating it at 37°C, transferring it to LB liquid medium, culturing it in test tubes at 37°C for 13-14 hours, inoculating it into LB liquid medium at a volume concentration of 1-20%, fermenting it at 28-40°C and 100-250 rpm (preferably 37°C and 200 rpm), and collecting the wet cells.

[0018] The LB liquid medium consists of 10 g / L peptone, 5 g / L yeast extract and 10 g / L NaCl, with water as the solvent; the LB solid medium is the LB liquid medium with an additional 10 g / L agar.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] This invention, based on sequence analysis of wild-type promoters derived from Bacillus subtilis, establishes a method for promoter modification. This method involves targeted substitution or truncation of base classes in the wild-type promoter, improving σ factor recognition efficiency and expression stability, thereby enhancing the expression intensity of exogenous genes using Bacillus subtilis promoters. The method described in this invention for promoter optimization is simple to design and operate, easily yielding promoters with enhanced expression intensity, and achieving increased expression intensity of exogenous genes using both inducible and constitutive promoters. The modified promoter P... VGt1 It exhibits the best performance at an optimal temperature of 70℃, an optimal pH of 7.0, and with Co as the optimal metal ion. 2+ D-allulose was prepared using a whole-cell method with 100 g / L D-fructose as a substrate. The reaction reached equilibrium within 1.5 h, with a conversion rate of 32.75% and enzyme activity increased to 23.6 U / mg, a 10.4-fold increase. Applying this promoter to the expression of proteins such as DAE enzymes effectively increased their expression levels, demonstrating its practical value. This invention also provides promoter variants generated by this method, which can be applied to protein production research. (iv) Description of the attached drawings

[0021] Figure 1 A bar chart showing the fluorescence intensity values ​​of recombinant expression strains with wild-type promoters.

[0022] Figure 2 Schematic diagram of wild-type promoter core region replacement.

[0023] Figure 3 For wild-type promoters (P spoVG P abrB P vals and P asd Fluorescence intensity bar graph of mutants after modification of the -35, -16, and -10 regions in the image.

[0024] Figure 4 P spoVG SDS-PAGE analysis before and after modification; lane 1 is P spoVG Lane 2 is P VG35 Lane 3 is P VG16 Lane 4 is P VG10 Lane 5 is P VG3510 Lane 6 is P VG3516 Lane 7 is P VG351610 Lane 8 is P VG1610 .

[0025] Figure 5 P abrB SDS-PAGE analysis before and after modification; lane 1 is P abrB Lane 2 is P rB35 Lane 3 is PrB16 Lane 4 is P rB10 Lane 5 is P rB3516 Lane 6 is P rB351610 Lane 7 is P rB3510 Lane 8 is P rB1610 .

[0026] Figure 6 P valS SDS-PAGE analysis before and after modification; lane 1 is P valS Lane 2 is P lS35 Lane 3 is P lS16 Lane 4 is P lS10 Lane 5 is P lS3510 Lane 6 is P ls3516 Lane 7 is P lS351610 Lane 8 is P lS1610 .

[0027] Figure 7 P asd SDS-PAGE analysis before and after modification; lane 1 is P asd Lane 2 is P sd35 Lane 3 is P sd16 Lane 4 is P sd10 Lane 5 is P sd3510 Lane 6 is P sd3516 Lane 7 is P sd351610 Lane 8 is P sd1610 .

[0028] Figure 8 Schematic diagram of promoter truncation (using P) sd3510 (For example).

[0029] Figure 9 A bar graph showing the fluorescence intensity of the mutant promoter with its core region truncated and the wild-type promoter.

[0030] Figure 10 SDS-PAGE analysis of the promoter mutant after truncating the core region; lane 1 is P VG1 Lane 2 is P VG2 Lane 3 is P ls1 Lane 4 is P ls2 Lane 5 is P rB3510 Lane 6 is P sd3516 .

[0031] Figure 11 , Bar graphs of enzyme activity in DAE-driven expression before and after promoter modification.

[0032] Figure 12A bar chart showing the relative expression levels of DAE mRNA driven by the modified promoter.

[0033] Figure 13 The conversion rate of DAE enzyme expression driven by the modified promoter mutant. (V) Detailed Implementation Methods

[0034] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0035] As used herein, a “promoter” refers to a nucleic acid sequence that is typically located upstream (5' end) of the coding sequence of a target gene and guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter provides a recognition site for RNA polymerase and other factors necessary for proper transcription initiation. In this invention, “promoter” includes promoters of defined length identified in commercially available systems of Bacillus subtilis, as well as endogenous or exogenous promoters commonly used in Bacillus subtilis research, and also includes unidentified DNA sequences within 1000 bp upstream of any gene in Bacillus subtilis or other species that may be promoters.

[0036] As used in this article, “modification” or “modification” refers to the replacement of all or part of a base class in a “promoter” with another base class.

[0037] As used herein, "target gene" refers to a gene that can be transcribed / expressed under the guidance of the promoter of this invention.

[0038] Suitable target genes in this invention are diverse, including but not limited to: structural genes, genes encoding proteins with specific functions, enzymes, and reporter genes (such as green fluorescent protein). It is known in the art that yeast cells can be used for large-scale transcription / expression of target genes, and such genes suitable for transcription / expression by Bacillus subtilis can be used as the "target genes" described in this invention.

[0039] As used herein, a "strong promoter" refers to a promoter that significantly enhances the intensity of target gene transcription / expression compared to a wild-type promoter, for example, by more than 5%, preferably more than 10%, even better by more than 15%, such as more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, and so on. The intensity of target gene transcription / expression can be determined by detecting the amount of target gene transcription / expression, a technique well-known to those skilled in the art. For example, SDS-PAGE can detect protein expression levels.

[0040] As used herein, the terms "high-intensity promoter" or "enhanced promoter" are relative. In some embodiments, a high-intensity or enhanced promoter is one that significantly increases the strength of the transcription of the target gene or the expression of the target protein compared to a wild-type promoter.

[0041] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0042] As used in this article, “driving the transcription / expression of the target gene”, “initiating the transcription / expression of the target gene”, and “guiding the transcription / expression of the target gene” can be used interchangeably.

[0043] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0044] As used herein, the promoter variants formed by the modification methods described above, as known in the art, can alter the promoter sequence through the substitution, deletion, or insertion of a few bases or base sequences, but in some cases, substantially change the function and strength of the promoter. This invention relates to homologous sequences of the aforementioned promoter variants, preferably with homology of 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 97% or more; more preferably, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. These homologous sequences can have functions similar to the aforementioned promoter sequences.

[0045] The pMA5 plasmid carrying the GFP gene in this invention is derived from... Aequorea victoria The green fluorescent protein (GFP) gene (NCBI accession number YDB82640) was inserted between the BamHI and MluI restriction sites of the original pMA5 plasmid. The pMA5 plasmid carrying the DAE gene was derived from... Christensenellaceae bacterium The D-allulose 3-epimerase DAE gene (NCBI accession number No. MBQ9941839.1) was inserted into the pMA5 plasmid between the BamHI and MluI restriction sites.

[0046] Example 1: Amplification and screening of wild-type promoters derived from Bacillus subtilis

[0047] 1. Amplification of wild-type promoters derived from Bacillus subtilis

[0048] In this embodiment, the wild-type promoters selected and modified are constitutive promoters or self-inducible promoters derived from Bacillus subtilis that do not require the addition of an inducer: P43, P aprE P amyE P gsiB P hag P lytR P sigx P spoVG P y1bp P yvyD P abrB P asd P ffh P vals P HpaⅡ The above wild-type promoter sequence was obtained from Bacillus subtilis by PCR. WB800 The genome was amplified, and the primers are shown in Table 3. The base sequences are shown in SEQ ID NO.1-SEQ ID NO.14 and SEQ ID NO.49.

[0049] Table 1. PCR amplification system

[0050]

[0051] Table 2. PCR amplification program:

[0052]

[0053] Table 3. Primers for wild-type promoter amplification

[0054]

[0055] 2. Construction and screening of engineered bacteria with wild-type promoter-driven GFP gene

[0056] (1) After recovering the promoter fragment from step 1, insert it into the plasmid pMA5 carrying the GFP gene, between the BamHI and MluI restriction sites, to obtain recombinant plasmids mediated by different promoters.

[0057] (2) Preparation and transformation of chemically competent cells: After preparing competent cells using the modified Spizizen salt induction method, recombinant plasmids mediated by different promoters were transformed into Bacillus subtilis. WB800 The recombinant expression strain was obtained from competent cells. The specific steps are as follows: 1) Frozen and stored at -80℃. B. subtilis WB800Streaking of the strain's glycerol tubes onto LB non-resistant plates. Then, incubate in an inverted 37°C incubator for 10-12 h. 2) Pick a single colony from the incubated LB plate and inoculate it into 5 mL of GM I medium, incubating at 37°C and 220 r / min for 12-14 h. GM I medium: 1 mL 10x Spizizen salts, 0.1 mL, 10% yeast extract, 0.25 mL 20% glucose, 0.2 mL 1% hydrolyzed casein, 0.2 mL 0.25% required amino acids, and add sterile distilled water to a total volume of 10 mL. 10x Spizizen salts stock solution: 15% K2HPO4·3H2O, 6% KH2PO4, 2% (NH4)2SO4, 0.2% MgSO4, 1% sodium citrate, dissolved in distilled water, 6.6 x 10 4 Autoclave at room temperature and store at room temperature. 3) Use a pipette to take 250 μL of bacterial culture and inoculate it into a new GMⅠ medium. Incubate at 37℃ and 220 r / min for 4-5 h until OD500 reaches zero. 600 ≈0.8-1.0 (late logarithmic growth phase). 4) Transfer 2 mL of the cultured bacterial solution to 20 mL of GM II medium and incubate at 37℃ and 220 r / min for 1-2 h. GM II medium: 1 mL 10x Spizizen salts, 0.05 mL 10% yeast extract, 0.25 mL 20% glucose, 0.04 mL 1% hydrolyzed casein, 0.2 mL 0.25% required amino acids, 0.05 mL 0.1 mol / L CaCl2, 1 mL 25 mmol / L MgCl2, and add sterile distilled water to a total volume of 10 mL. 5) Pre-cool the cultured bacterial solution on ice for 10 min, transfer it to a pre-cooled 50 mL centrifuge tube, centrifuge at 4000 rpm and 4℃ for 7 min, remove the supernatant and collect the bacterial precipitate. 6) Thoroughly resuspend the bacterial pellet in 2 mL of GMⅡ medium and add 1 mL of the original medium supernatant. Dispense the suspension evenly into four pre-cooled 1.5 mL centrifuge tubes (750 μL per tube). 6) Slowly inject the plasmid from step (1) into competent cells, mix gently, incubate at 37°C for 30 min, then transfer to a shaker at 37°C and 220 r / min for 1.5 h. Centrifuge at 4000 r / min for 5 min to concentrate the bacterial culture. Spread the culture onto Kan-resistant LB solid medium and incubate at 37°C for 16-20 h.

[0058] (3) The recombinant expression strain was streaked onto LB solid medium and activated at 37°C for 12 h. It was then transferred to LB liquid medium and incubated at 37°C for 13-14 h. The bacterial culture was then inoculated into LB liquid medium at a volume concentration of 1% and incubated at 37°C and 200 rpm for 45 h. Cell fluorescence intensity (reflecting GFP expression level) was detected using a multi-functional microplate reader. The results are shown in [Figure number missing]. Figure 1 (P) y1bp P yvyD P43, P sigx P ffh Exhibiting negligible fluorescence, wild-type promoter P capable of expressing the GFP gene was screened. aprE 、 P amyE P gsiB P hag P lytR P spoVG P abrB P asd P vals P HpaⅡ The nucleotide sequences are shown in SEQ ID NO.2-SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.11-12, SEQ ID NO.14, and SEQ ID NO.49, respectively. Four of these are wild-type promoters P that can efficiently express the GFP gene. spoVG P abrB P asd P vals .

[0059] (4) Regarding P gsiB P HpaⅡ P lytR P aprE P hag P spoVG P amyE P43, P abrB P asd P vals During the culture process of the recombinant expression strains in step (3) above, samples were taken every 5 hours to detect the cell fluorescence intensity of the selected strong wild-type promoter recombinant expression strains using a multifunctional microplate reader, and the OD was detected using a UV spectrophotometer. 600 Growth curves were plotted, and the results showed that P spoVG It exhibited the strongest transcriptional activity, with a fluorescence intensity of 2.00 × 10⁻⁶. 6 au; P abrB The second highest was 6.61 × 10⁻⁶. 5 au, approximately P spoVG 33.0%. P valS and Pasd It exhibited moderate to high fluorescence intensity, at 4.60 × 10⁻⁶. 5 au and 4.11×10 5 au, approximately 23.0% and 20.5% of the maximum value. P lytR It exhibits moderate activity, with a fluorescence intensity of 3.00 × 10⁻⁶. 5 au, approximately P spoVG 15.0%. P HpaII P gsiB and P amyE The activity of P was further reduced, while P aprE and P hag It exhibits negligible fluorescence, indicating that its transcriptional activity is extremely low or completely silent.

[0060] Combination Figure 1 The results show that P spoVG P abrB P vals and P asd These are the preferred candidate promoters for efficient heterologous protein expression. Overall, all four promoters support biomass accumulation during the logarithmic growth phase and maintain or reach peak fluorescence expression during the stationary phase. Among them, P… spoVG Its transcriptional strength and persistence are significantly superior to the other three promoters, making it the preferred candidate promoter for subsequent heterologous protein expression.

[0061] Example 2: Construction and screening of promoter mutants

[0062] Reference Figure 3 The four wild-type promoters P selected in Example 1 were used. spoVG (abbreviated P) VG ), P abrB (abbreviated P) rB ), P asd (abbreviated P) sd ), P vals (abbreviated P) ls Targeted substitutions were performed on the bases in the -35, -16, and -10 regions of the sequence, and overlapping PCR primers were designed (Table 4) to sequentially replace the bases with "TTGACA", "ATG", and "TATAAT". The primer design principle was to introduce the target base change at the mutation site and retain 15-20 bp homologous arms on both sides.

[0063] Using the recombinant plasmids of the four wild-type promoters constructed in Example 1 as templates, PCR amplification of promoter mutants was performed using the primers in Table 4. These mutants were then inserted upstream of the reporter gene GFP carried by the pMA5 plasmid containing the GFP gene to construct single-point mutants (such as P...). VG10 (representing P) spoVGThe bases in the -10 region are replaced with TATAAT), P rB35 (representing P) abrB (e.g., base substitution in the -35 region to TTGACA), and two-region mutants (e.g., P) sd3510 (representing P) asd The bases in the -35 and -10 regions are replaced with TTGACA and TATAAT, respectively, and P rB3516 (representing P) abrB (base substitutions in the -35 and -16 regions to TTGACA and ATG, etc.), three-region mutants (P sd351610 (representing P) asd (The bases in the -35, -16, and -10 regions were replaced with TTGACA, ATG, and TATAAT, respectively). Recombinant expression strains were constructed using the method described in Example 1, and fermentation and fluorescence intensity were detected. The relative fluorescence intensity of each mutant was calculated with the pre-mutation fluorescence intensity as 100%. The results are shown in […]. Figure 3 .

[0064] SDS-PAGE analysis: The bacterial suspensions of the recombinant expression strains constructed from the above promoter mutants were resuspended in PB buffer (0.05 mol / L, pH 6.5) to a concentration of 35 g / L. 10 μL of 4×LDS protein loading buffer and 30 μL of bacterial suspension were mixed, boiled for 10 min, and then subjected to SDS-PAGE analysis. The results are shown in the figure. Figures 4-7 As shown, the results indicate that there is a distinct band in the 25-35 kDa range, which corresponds to the size of the target protein, and the thickness of the protein band is consistent with the enzyme activity.

[0065] Screening yielded the superior promoter mutant P VG10 (nucleotide sequence as shown in SEQ ID NO.29), P rB3516 (nucleotide sequence as shown in SEQ ID NO.18), P sd10 (nucleotide sequence as shown in SEQ ID NO.22), P sd3510 (nucleotide sequence as shown in SEQ ID NO.26), P sd3516 (nucleotide sequence as shown in SEQ ID NO.27), P ls10 (The nucleotide sequence is shown in SEQ ID NO.36).

[0066] Table 4. Promoter mutation primers (numbers 35, 10, and 16 represent single, double, or triple region mutations, respectively).

[0067]

[0068]

[0069] Example 3: Truncated mutations in dominant promoter mutants

[0070] 1. BDGP software prediction and core area analysis

[0071] The dominant promoter mutants (P) screened in Example 2 were analyzed using the Neural Network Promoter Prediction online tool in BDGP software. rB3516 P ls10 P sd3510 P VG10 Each sequence was scanned and analyzed. All fragments with prediction scores between 0.8 and 1.0 were collected and defined as core candidate regions for that promoter variant. The downstream 10 bp position of each core candidate region fragment was recorded and labeled as the predicted transcription start site (TSS). (The last sentence appears to be incomplete and possibly refers to a different sequence.) sd3510 For example, see Figure 8 As shown, the core candidate region and the corresponding transcription start site are retained, and the truncated mutant is denoted as P. sdt .

[0072] 2. Construction of promoter truncated variants

[0073] Starting from the 5' end of the core fragment in step 1, primers were designed with an endpoint approximately 20-50 bp downstream of the predicted transcription start site (ensuring the complete 5'UTR is included) (Table 5) for PCR amplification to obtain the truncated mutant promoter. sd3510 The shortened version is denoted as P. sdt P rB3516 The shortened version is denoted as P. rBt P lS10 The shortened version is denoted as P. lSt1 and P lSt2 P VG10 The shortened version is denoted as P. VGt1 Sum of P VGt2 .

[0074] The truncated promoter fragment was cloned into plasmid pMA5 carrying the GFP gene. A recombinant expression strain was constructed using the method described in Example 1, and fermentation culture was performed. Fluorescence intensity was detected, with the wild-type strain as a control. Results are shown in [Figure 1]. Figure 9 .

[0075] Using the method in Example 2, the SDS-PAGE analysis diagram of the truncated mutant is shown below. Figure 10 As shown, the results indicate that there is a distinct band in the 25-35 kDa range, which corresponds to the size of the target protein, and the thickness of the protein band is consistent with the enzyme activity.

[0076] Figure 9 This indicates that, compared to the wild-type promoter, P spoVG After replacement and truncation, PVGt1 The fluorescence intensity ranged from 1.8 × 10⁻⁶. 6 au increased to 2.28×10 6 au, and P VGt2 It is comparable to its original level. P valS The effects of replacement and truncation are most significant: P lSt2 The fluorescence intensity ranged from 4.3 × 10⁻⁶. 5 au surged to 1.86×10 6 au, P lSt1 It also increased by about 60%. asd and P abrB The activity increased by approximately 240% and 100% after replacement and truncation, respectively, indicating that truncation has a universal enhancing effect on promoters with different genetic backgrounds. Figure 9 The dominant mutant P was obtained through screening. VGt1 (nucleotide sequence as shown in SEQ ID NO.45), P rBt (nucleotide sequence as shown in SEQ ID NO.43), P sdt (nucleotide sequence as shown in SEQ ID NO.44), P lst2 (The nucleotide sequence is shown in SEQ ID NO.48).

[0077] Table 5. Promoter mutation primers

[0078]

[0079] Example 4: Enzyme activity detection of dominant promoter mutants

[0080] 1. Enzyme activity detection

[0081] wild-type promoter P spoVG P abrB P asd P vals and the superior promoter mutant P screened in Example 3 lst2 P VGt1 P sdt P rBt A recombinant vector was constructed by inserting the original promoter site of the pMA5 plasmid carrying the DAE gene, and then transformed into Bacillus subtilis using the method described in Example 1. WB800 The recombinant expression strain was obtained from competent cells, cultured using the method in Example 1, and the bacterial solution was collected by centrifugation at 8000 rpm for 10 min.

[0082] The final concentration composition of the 1 mL enzyme activity assay system is: D-fructose 100 g / L, Co 2+A 1 mL reaction system was prepared using 0.1 mol / L ions, 100 g / L wet bacterial cells, and 0.05 mol / L PB buffer (pH 6.5). The reaction was carried out at 70°C for 30 min at 1000 rpm, cooled on ice, boiled for 10 min, and centrifuged at 12000 rpm for 1 min. The supernatant was diluted 25-fold with ddH₂O, filtered through a 0.22 μm membrane, and the D-allulose content of the filtrate was determined by HPLC to calculate enzyme activity. Results are shown below. Figure 11 The results showed that P VGt1 It exhibited optimal driving capacity, with enzyme activity reaching 23.6 U / mg, compared to wild-type P. spoVG The difference was approximately 35%, consistent with the fluorescence intensity screening results. This confirms that the truncated mutant exhibits a stable transcriptional enhancement effect in both reporter gene and actual enzyme gene expression. rBt and P abrB Secondly, the enzyme activities were 18.05 U / mg and 15.93 U / mg, respectively, with the latter showing a slight improvement compared to the wild-type promoter. lSt2 and P lSt2 The enzyme activities were 10.6 U / mg and 8.8 U / mg, respectively. Although P lSt2 It exhibited extremely high transcriptional activity in fluorescent selection, but its efficiency in driving DAE enzyme expression was significantly reduced. Notably, P... valS No enzyme activity was detected, indicating that although this promoter can drive GFP expression, it cannot effectively transcribe or translate the DAE gene. This may be related to the rare codon at the 5' end of the DAE coding sequence or mRNA stability. VGt1 The optimal promoter for DAE enzyme expression was determined to be promoter P. VGt1 The DAE gene was expressed at a high level in Bacillus subtilis WB800 cells under mediated reaction. The optimal temperature for whole-cell recombinant DAE reaction was 70℃, the optimal pH was 7.0, and the optimal metal ion was Co. 2+ D-allulose was prepared using a whole-cell method with 100 g / L D-fructose as a substrate. The reaction reached equilibrium within 1.5 h, with a conversion rate of 32.75% and an enzyme activity of 23.6 U / mg.

[0083] Enzyme activity (U) is defined as the amount of enzyme required to generate one micromole of D-allulose per minute.

[0084] HPLC detection conditions: Agilent 1260 HPLC system, Agilent autosampler, Sugar-Park column, Agilent differential detector, mobile phase: calcium disodium ethylenediaminetetraacetate, column temperature: 80℃, flow rate:

[0085] 0.5 mL / min, injection volume 10 μL.

[0086] 2. Conversion rate

[0087] Promoter mutant P VGt1 A recombinant vector was constructed by inserting the original promoter site of the pMA5 plasmid carrying the DAE gene, and then transformed into Bacillus subtilis using the method described in Example 1. WB800 The recombinant expression strain was obtained from competent cells, cultured using the method in Example 1, and the bacterial solution was collected by centrifugation at 8000 rpm for 10 min.

[0088] The final concentration composition of a 1 mL reaction system is: D-fructose 100 g / L, Co 2+ A 1 mL reaction system was prepared using 0.1 mol / L ions, 100 g / L wet bacterial cells, and 0.05 mol / L PB buffer (pH 6.5). The reaction was carried out at 1000 rpm and 70 °C for 80 min. Samples were taken every 2 min, and the D-allulose content was determined using the method in step 1. The D-fructose conversion rate was calculated. The results are shown in [Figure 1]. Figure 12 It can be seen that it basically reaches equilibrium in 1.5 hours, and the final equilibrium conversion rate is 32.75%.

[0089] Example 5: Detection of relative fluorescence intensity of mRNA

[0090] wild-type promoter (P) spoVG P abrB P asd P vals collectively referred to as P Wt ) and its mutant P rBt 、 P VGt1 、 P lst2 、 P sdt Recombinant expression strains constructed by inserting the pMA5 plasmid carrying the GFP gene were used to extract RNA using an RNA extraction kit. RNA purity was assessed using a Nanodrop 2000c micro spectrophotometer (Thermofisher Scientific), and RNA with an A260 / 280 ratio of 1.8–2.2 was selected for further analysis. RNA was reverse transcribed into cDNA using HiScript® Ⅱ Q RT SuperMix (Vazyme), and quantification was performed using the primers listed in Table 6 using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The reaction was performed in a qTOWER3G IVD real-time PCR instrument (Analytik jena), with rpoB as an internal control gene. The target gene was relatively quantified using the ΔΔCt method, and the results are shown in the table below. Figure 13This indicates that, compared with the wild-type promoter, the relative mRNA expression levels of each truncated mutant were significantly increased: P vgt1 It increased by about ten times, while P sdt P lSt2 and P rbt It increased two to three times. Furthermore, fluorescence intensity was highly positively correlated with changes in mRNA levels.

[0091] Table 6. Primers (CB-F / R are used to replace the GFP gene in the pMA5 plasmid carrying the GFP gene with the DAE gene)

[0092]

Claims

1. A strong promoter for Bacillus subtilis, characterized in that, The Bacillus subtilis strong promoter is obtained by replacing one or more sequences in regions -10, -16, and -35 of the wild-type endogenous promoter derived from Bacillus subtilis with conserved sequences, or by truncating the replaced promoter; the -10 region is replaced with TATAAT, the -16 region with ATG, and the -35 region with TTGACA.

2. The Bacillus subtilis strong promoter as described in claim 1, characterized in that, The truncation is performed using BDGP software to predict the promoter, where promoter fragments with a fraction of 0.8-1 are used as the core region, and the last 10 bp of each core region fragment is used as the transcription start site. All core region fragments and transcription start sites are retained, and the promoter is truncated.

3. The Bacillus subtilis strong promoter as described in claim 1, characterized in that, The wild-type promoter includes P amyE P gsiB P lytR P spoVG P abrB P asd P vals P HpaⅡ .

4. The Bacillus subtilis strong promoter as described in claim 1, characterized in that, The nucleotide sequence of the Bacillus subtilis strong promoter is shown in one of SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.36, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, or SEQ ID NO.

48.

5. An expression vector containing the Bacillus subtilis strong promoter as described in claim 1.

6. A recombinant bacterium expressing the D-allulose 3-epimerase gene using the Bacillus subtilis strong promoter of claim 1.

7. The application of a recombinant bacterium constructed using the strong promoter of claim 1 in the preparation of D-allulose.

8. The application as described in claim 7, characterized in that, The application method is as follows: using the wet bacterial cells obtained by fermentation of the recombinant bacteria as a catalyst, D-fructose as a substrate, and Co... 2+ Using ions as an auxiliary agent, a reaction system was constructed using 0.05 mol / L, pH 6.5 PB buffer solution as the reaction medium. The reaction was carried out at 1000 rpm and 70 °C to obtain a reaction solution containing D-allulose.

9. The application as described in claim 8, characterized in that, The reaction system contained 100 g / L of substrate and 100 g / L of wet bacterial cells. Co 2+ The concentration of added ions was 0.1 mol / L.

10. The application as described in claim 8, characterized in that, The catalyst is prepared by streaking the recombinant bacteria onto LB solid medium, activating it at 37°C, transferring it to LB liquid medium, culturing it in test tubes at 37°C for 13-14 hours, inoculating it into LB liquid medium at a volume concentration of 1-20%, fermenting it at 28-40°C and 100-250 rpm, and collecting the wet cells.

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