Bacillus constitutive strong promoter and application thereof

Abstract

The invention discloses a bacillus constitutive strong promoter and application thereof, and relates to the technical field of biology, and the nucleotide sequence of the promoter is SEQ ID NO: 2. The mRNA transcriptional level of the reporter gene is improved; the method is used for starting a target gene to efficiently express amylase in bacillus subtilis. The method is also used for starting a target gene to efficiently express alkaline protease in bacillus licheniformis. The novel constitutive promoter with enhanced transcriptional activity is developed by taking P43 as a starting template, and under the same host and culture conditions, the absolute level of the constitutive promoter for driving expression of a reporter gene or a target product is higher than that of the current P43 promoter, so that core power is provided for performance improvement of a bacillus cell factory.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a constitutive strong promoter of Bacillus and its applications. Background Technology

[0002] Bacillus spp. ( Bacillus ), especially Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus subtilis ( Bacillus subtilis As a safe and efficient industrial microbial platform, microorganisms play a crucial role in the large-scale production of industrial enzymes, recombinant proteins, and metabolites. One of the core foundations for the successful application of this host lies in its possession of promoter elements capable of driving high-level, stable transcription of target genes.

[0003] Among numerous promoters, the P43 promoter, derived from the ribosomal protein operon of Bacillus licheniformis, has become the most representative and widely used universal promoter in this host due to its inherent constitutive strong expression characteristics. The P43 promoter can maintain high transcriptional activity throughout the cell cycle without the need for an inducer, providing a reliable basis for the expression of exogenous proteins. It is widely used in the production and research of industrial enzymes such as alkaline protease and nattokinase.

[0004] However, with the increasing demands for production efficiency and target product yield in industrial biotechnology, existing constitutive strong promoter systems, represented by P43, have gradually approached their performance limits in terms of expression intensity, becoming a key bottleneck restricting further breakthroughs in yield. Previous studies have shown that screening natural promoters based on random mutations or limited site substitutions may lead to the development of more efficient and stable strong promoters.

[0005] Therefore, this study aims to develop a novel constitutive promoter with enhanced transcriptional activity, using P43 as the starting template. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a constitutive strong promoter of Bacillus and its application, which can efficiently express proteins.

[0007] In order to achieve the objective of this invention, the following solution is proposed: A constitutive strong promoter of the genus Bacillus, named P43-SUP2, has the nucleotide sequence SEQ ID NO:2.

[0008] Firstly, the application of the constitutive strong promoter of the Bacillus genus enhances the mRNA transcription level of the reporter gene. Compared to the wild-type P43 promoter, the mRNA level of eGFP transcription driven by P43-SUP2 is increased by 3.4 times.

[0009] In a second aspect, the application of the strong constitutive promoter of Bacillus genus is used to start high-efficiency expression of amylase by a target gene in Bacillus subtilis. Compared with the wild-type P43 promoter, the expression level of amylase amyE is increased by more than 2.2 times under the premise of not affecting the normal growth of the host cell of Bacillus subtilis, and the efficiency and yield of protein production are significantly improved.

[0010] In a third aspect, the application of the strong constitutive promoter of Bacillus genus is used to start high-efficiency expression of alkaline protease by a target gene in Bacillus licheniformis. Compared with the wild-type P43 promoter, the enzyme activity of P43-SUP2 is 1.84 to 2.03 times that of the control group at each time point in the stable phase, and reaches exactly 1.99 times at the end of fermentation (72 hours).

[0011] The application has the beneficial effect that a new and transcriptionally active constitutive promoter is developed based on P43, and the absolute level (including mRNA level and protein yield) of expression of a reporter gene or a target product driven by the new constitutive promoter is higher than that of the current P43 promoter under the same host and culture conditions, which provides core power for performance improvement of Bacillus cell factories. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is a schematic diagram of the design and modification principle of the super-strong promoter P43-SUP1 in Example 1.

[0013] Figure 2 It is a comparison chart of the promoter strength based on the eGFP reporter gene in Example 2.

[0014] Figure 3 It is a DNA sequence alignment chart of the wild-type P43 promoter and the mutant promoter in Example 2.

[0015] Figure 4 It is a result chart of qRT-PCR verification of the mRNA transcription level of the reporter gene driven by P43-SUP1 in Example 2.

[0016] Figure 5 It is a process chart of the production of alpha-amylase by the Bacillus subtilis engineering strain in a 5L fermenter in Example 3; wherein (A) is an OD600 chart; and (B) is a specific enzyme activity chart.

[0017] Figure 6 It is a process chart of the production of alkaline protease by the Bacillus licheniformis engineering strain in a 5L fermenter in Example 4; wherein (A) is an OD600 chart; and (B) is a specific enzyme activity chart. DETAILED DESCRIPTION

[0018] Example 1 Construction of random mutant library based on p43 non-core sequence, as follows: First focus on the P43 promoter upstream non-core region (Upstream Non-core Region, UNR), which is located upstream of the transcription start site (TSS), contains potential transcription factor binding sites and sequences affecting DNA bending, topology, and has important regulatory effects on promoter strength, but the -35 region and -10 region core sequence directly involved in sigma factor recognition are retained in this embodiment. Figure 1 ).

[0019] According to the known P43 promoter sequence (such as SEQ ID NO: 1), the range of its core region is determined by bioinformatics analysis (from -84 to +1 site upstream of TSS) Figure 1 ). The sequence before -84 site upstream of TSS (-300 to -85) is defined as "upstream non-core region (UNR)" as the target of random mutation. A pair of specific primers is designed: Upstream primer P43-UNR-F: 5'-CTGACCGAGATTTTTTTGAGCAACTGGATCC-3' Downstream primer P43-Core-R: 5'-GGTACCGCTATCACTTTATATTTTACATAATCGCGCGCTTTTTTTCACGCCCATTTCT-3' Using the above primers, error-prone PCR was performed using the complete p43NMK plasmid as the template; the error-prone conditions were achieved by adjusting the reaction system, using low-fidelity Taq DNA polymerase (Bi Yun Tian, D0219M), MgCl2 concentration of 7 mM, MnCl2 concentration of 0.5 mM; the PCR program was set to 45 cycles to maintain high mutation density; the amplification product was purified and recovered to obtain a random mutation UNR fragment of 231 bp in length.

[0020] The linearization of p43NMK plasmid was performed using restriction endonuclease BamHI and KpnI (purchased from NEB company); according to the instructions of Gibson Assembly kit (purchased from NEB company), the linearized vector skeleton prepared in step (50 ng) was mixed with random mutation UNR fragment (molar ratio of 1:3), and assembly reaction main mix was added, and reacted at 50℃ for 60 minutes.

[0021] The assembly product was introduced into competent B. licheniformis DW2 cells by electroporation; the transformed cells were plated on LB plates containing kanamycin (20 pg / mL) and incubated at 37°C for 24 hours; 20 single colonies were randomly selected for colony PCR and sequencing verification to confirm successful recombination of the UNR region and sequence diversity; all clones were collected in LB medium containing glycerol and stored at -80°C, i.e. a P43-UNR random mutant library was successfully constructed.

[0022] Example 2 The high-expression promoter was screened using the fluorescent protein eGFP, as follows: An appropriate amount of bacterial liquid was taken from the mutant library constructed in Example 1, inoculated in a 96-deep-well plate (1 mL of TB-Kan medium per well), and incubated at 37°C with 900 rpm shaking for 24 hours; a strain carrying the original p43NMK plasmid (wild-type P43) was set as a control, and at least three replicate wells were set for each screening plate; after incubation, the OD600 (cell density) and fluorescence intensity (excitation 485 nm, emission 520 nm) of each well were determined using a multifunctional microplate reader; the specific fluorescence intensity (fluorescence intensity / OD600) of each well was calculated; after screening 6543 clones, three clones with the highest specific fluorescence intensity were screened, which were 2.89-3.42 times, 1.24-1.42 times and 1.21-1.48 times the control group, respectively. Figure 2 ).

[0023] The plasmid DNA of the three clones was extracted; the promoter region was amplified using a primer pair and subjected to Sanger sequencing; the sequencing results were compared with the wild-type P43 (SEQ ID NO: 1) to determine the specific mutations in the UNR region; Figure 3 ); thus, multiple mutants were obtained, of which the highest activity variants were named P43-SUP1, P43-SUP2 and P43-SUP3, corresponding to SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0024] qRT-PCR analysis confirmed that the mRNA level of P43-SUP1 driving eGFP transcription was increased by 3.4 times compared with wild-type P43; P43-SUP2 and P43-SUP3 had no significant difference from wild-type P43. Figure 4 ).

[0025] Example 3 The effect of P43-SUP1 in the production of B. subtilis amylase was as follows: This example aims to verify the effect of the screened promoter P43-SUP1 in the production of Bacillus subtilis industrial enzymes, taking amylase as a specific case. First, based on the Bacillus subtilis expression vector p43NMK, the primers P43-UNR-F and P43-Core-R were used to amplify P43-SUP1 and wild-type P43 as "promoter fragments". The primers amyE-F and amyE-R were used to amplify the amylase gene (amyE) from the Bacillus subtilis 168 genome α - The amylase gene (amyE) was used as the expression gene, and the primer sequences were as follows: amyE-F: 5'-GGTAAGAGAGGAATGTACGCATGTTTGCAAAACGATTCAA-3' amyE-R: 5'-ATTCTGCGAAGTGATCTTCGCCAAGCTTATTTAAATCAATGGGGAAGAGAACCGC-3' Through a Gibson Assembly reaction, linearized vector, promoter fragments (P43-SUP1 or wild-type P43), and amyE gene fragments were assembled into three fragments to obtain recombinant plasmids pHT43-SUP1-amyE and pHT43-P43-amyE (control), respectively; the correctly assembled plasmids were electroporated into Bacillus subtilis 168.

[0026] The above two engineering bacteria were inoculated into LB (containing chloramphenicol) seed culture medium, and after overnight culture, they were inoculated into starch fermentation medium at a 2% inoculation amount. The engineering bacteria were subjected to fed-batch fermentation in a 5L fermenter; the specific growth rate was controlled by glucose feeding, ammonia water was automatically fed to maintain pH 7.5, and the temperature was 37°C; samples were taken at regular intervals to monitor OD600, residual sugar, and amylase activity; the culture was incubated at 37°C, 220 rpm for 48 hours; samples were taken every 12 hours, and the supernatant was obtained by centrifugation.

[0027] The 3,5-dinitrosalicylic acid (DNS) method was used to determine the amylase activity in the fermentation supernatant; 1 μmol of reducing sugar (calculated as glucose) produced per minute of starch hydrolysis was defined as one unit of enzyme activity (U); the experiment was repeated three times.

[0028] To evaluate the effect of promoter engineering on the expression of amylase amyE in Bacillus subtilis, this study compared the cell growth and protein expression kinetics of recombinant strains driven by wild-type promoter P43 and its modified version P43-SUP1 during batch fermentation. The results are as follows Figure 5As shown, in terms of cell growth, the two groups of strains exhibited similar growth curves, indicating that promoter replacement did not significantly burden the normal proliferation of host cells. After a lag phase of 0 to 12 hours, both strains entered the logarithmic growth phase. The OD600 value of strain P43-amyE reached 97.7±4.4 at 24 hours, 185.8±1.2 at 60 hours, and then slowed down, reaching 199.9±4.0 at 84 hours. In contrast, the growth dynamics of strain P43-SUP1-amyE were highly consistent, with OD600 values ​​of 99.2±3.4, 188.9±8.3, and 204.2±4.8 at 24, 60, and 84 hours, respectively. Throughout the fermentation period, there were no statistically significant differences in biomass between the two groups of strains at each corresponding time point, confirming the compatibility of the engineered promoter with cell growth.

[0029] Regarding amylase expression, the P43-SUP1 promoter exhibited a remarkably significant and sustained enhancement effect. After 12 hours of fermentation, the enzyme activity of strain P43-SUP1-amyE reached 44365±3781 U / mL, 2.4 times that of the control group P43-amyE (18443±1961 U / mL). This expression advantage further increased over time: by the mid-fermentation stage (36 hours), the enzyme activity of the P43-SUP1 group (122119±8658 U / mL) was 2.3 times that of the control group (52975±1507 U / mL); and in the late fermentation stage (60 hours), its enzyme activity (174318±11030 U / mL) was 2.2 times that of the control group (80480±2445 U / mL). Finally, at 84 hours, the enzyme activity of strain P43-SUP1-amyE reached a peak of 211463±6752 U / mL, while that of strain P43-amyE was 96236±2534 U / mL, with the former being 2.2 times higher than the latter. The data throughout the timeline clearly demonstrate that the P43-SUP1 promoter not only significantly increased the total expression of amyE but also significantly advanced the high-level expression phase and maintained it throughout the entire fermentation cycle.

[0030] In summary, the experimental results clearly show that, compared with the wild-type P43 promoter, the engineered promoter P43-SUP1 can increase the expression level of amylase amyE by more than 2.2 times without affecting the normal growth of Bacillus subtilis host cells, thus significantly improving the efficiency and yield of protein production.

[0031] Example 4 The effects of P43-SUP1 on the production of alkaline protease from Bacillus licheniformis are as follows: This embodiment aims to verify the effectiveness of the screened promoter P43-SUP1 in Bacillus licheniformis strain 2709, using alkaline protease as a specific example. Based on the expression vector p43NMK, P43-SUP1 and wild-type P43 were amplified as "promoter fragments" using primers P43-UNR-F and P43-Core-R, respectively. The alkaline protease gene (aprE) was amplified from the Bacillus licheniformis 2709 genome by PCR using primers aprE-F and aprE-R as the expression gene. The primer sequences are as follows: aprE-F: 5'-GGTAAGAGAGGAATGTACGCATGATGAGGAAAAAGAGTTT-3' aprE-R: 5'-TTTATTCTGCGAAGTGATCTTCGCCAAGCTTATTTAAATTATTGAGCGGCAGCTTCGA-3' The linearized vector, promoter fragment (P43-SUP1 or wild-type P43), and aprE gene fragment were assembled into expression plasmids pHYS-SUP1-aprE and pHP-P43-aprE (control) using a single Gibson Assembly reaction. The plasmids were then electroporated into Bacillus licheniformis 2709 host.

[0032] The engineered bacteria were fed-batch fermented in a 5L fermenter; the growth rate was controlled by feeding glucose, and ammonia was automatically fed to maintain pH 7.5 and temperature 37℃; samples were taken at 12-hour intervals to monitor OD600, residual sugar, and protease activity; protease activity was determined by the Folin-Ciocalteu method, using casein as a substrate, at 40℃ and pH 10.5, and the amount of enzyme required to produce 1 μg of tyrosine per minute was defined as one activity unit (U).

[0033] To verify the universal enhancement effect of the engineered promoter P43-SUP1 on heterologous gene expression, this study further applied it to drive the expression of the alkaline protease gene aprE in Bacillus subtilis and compared it with the wild-type P43 promoter-driven strain in parallel fermentation.

[0034] The results are as follows Figure 6As shown, regarding cell growth, the two recombinant strains exhibited highly consistent growth kinetics throughout the fermentation cycle, indicating that promoter replacement had no significant interference with cell growth. Specifically, the OD600 of strain P43-aprE reached 60.1±3.7, 108.8±2.3, 142.1±5.6, 179.1±7.1, 183.6±5.0, and 189.6±3.0 at 12, 24, 36, 48, 60, and 72 hours, respectively. Correspondingly, the OD600 of strain P43-SUP1-aprE at the corresponding time points were 61.6±3.5, 113.6±3.1, 143.7±4.8, 180.5±4.1, 194.2±4.7, and 194.7±2.5, respectively. The two sets of data highly overlapped at all time points with no statistically significant differences, further confirming that the P43-SUP1 promoter did not introduce additional metabolic burden on the host bacteria.

[0035] Regarding protease expression, the P43-SUP1 promoter also showed a significant enhancing effect on aprE gene expression. In the early stage of fermentation (12 hours), the enzyme activity of the P43-SUP1-aprE strain reached 16181±3925 U / mL, which was 1.94 times that of the P43-aprE control strain (8358±1120 U / mL). This expression advantage persisted throughout the fermentation process: until the late logarithmic growth phase (24 hours), the enzyme activity in the P43-SUP1 group (44222±3513U / mL) was 2.03 times that of the control group (21783±1690U / mL); after entering the stationary phase, at 36, 48, 60 and 72 hours, the enzyme activities in the P43-SUP1 group were 89497±4506U / mL, 114723±11141U / mL, 139980±7541U / mL and 155325±6931U / mL, respectively, while the corresponding values ​​in the control group were 49286±4190U / mL, 61083±3943U / mL, 74189±4298U / mL and 77898±2192U / mL, respectively. The enzyme activity of the P43-SUP1 group was 1.84 to 2.03 times that of the control group at all time points during the stationary phase, and reached a precise 1.99 times at the fermentation endpoint (72 hours).

[0036] In summary, based on previous experimental results for the amyE gene, this study demonstrates that the engineered promoter P43-SUP1 significantly enhances the expression of different heterologous target genes (amyE and aprE) (1.9-2.2-fold), and this enhancement is completely independent of any negative impact on host cell growth. This confirms the application potential of P43-SUP1 as a highly efficient and universal expression element.

[0037] The above embodiments are only used to illustrate the technical concept and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the protection scope of the present invention.

Claims

1. A constitutive strong promoter of Bacillus, characterized in that, Its nucleotide sequence is SEQ ID NO:

2.

2. The application of the constitutive strong promoter of Bacillus spp. as described in claim 1, characterized in that, Increase the transcription level of reporter gene mRNA.

3. The application of the constitutive strong promoter of Bacillus spp. as described in claim 1, characterized in that, Initiate efficient expression of target gene amylase in Bacillus subtilis.

4. The application of the constitutive strong promoter of Bacillus spp. as described in claim 1, characterized in that, Initiate efficient expression of alkaline protease of the target gene in Bacillus licheniformis.

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