Promoter for improving protein expression level and application thereof

Abstract

The invention discloses a promoter for improving protein expression level and application thereof. An original promoter P43 of a cytidine deaminase gene in bacillus subtilis 168 is used as a template to construct a promoter library, GFP is used as a reporter protein, a promoter P43mut1 capable of remarkably increasing the expression of the maltogenic amylase gene is screened out through GGGGS connecting peptide and a sequence of 180bp at the 5'end of the maltogenic amylase gene before the GFP, and the nucleotide sequence of the promoter P43mut1 is as shown in SEQ ID NO: 1. Compared with an original promoter P43, the promoter P43mu1 can improve the expression level of a maltogenic amylase gene and an alpha-amylase gene in bacillus subtilis to 1.5 times, the expression of a heterologous gene in the bacillus subtilis is remarkably enhanced, and the promoter can be used as a universal promoter, has relatively strong industrial applicability, and can be widely applied to the field of bacillus subtilis. And a more efficient biocatalysis solution is provided for industries such as food, fermentation and biocatalysis.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology and relates to a promoter and an application thereof, and in particular to a promoter for improving protein expression levels in Bacillus and an application thereof. Background Art

[0002] In the fields of industrial biotechnology and synthetic biology, efficient enzyme expression is crucial for improving biocatalytic efficiency and reducing production costs. Maltogenic α-amylase (EC3.2.1.133) is an important enzyme widely used in industries such as food processing, saccharification, pharmaceuticals, and papermaking. Its main function is to catalyze the degradation of starch or related substrates to produce maltose or other oligosaccharides, thereby playing a key role in saccharification processes and food improvement. However, in actual production processes, how to improve the expression level of maltogenic amylase, especially its efficient expression in industrial microbial hosts, has always been an important topic in synthetic biology and industrial microbiology research.

[0003] Bacillus subtilis, as an important industrial microbial host, is widely used for the expression of exogenous proteins due to its efficient protein secretion ability, feasibility of genetic manipulation and strong environmental adaptability. However, the efficient expression of maltogenic amylase in Bacillus subtilis still faces many challenges, such as insufficient promoter activity, low transcription efficiency, and excessive host metabolic burden. In the gene expression regulation system, the promoter is a key element that determines the transcription efficiency, and its activity directly affects the expression level of the target gene. Therefore, screening or designing efficient promoters to enhance the transcription level of the maltogenic amylase gene in Bacillus is one of the key strategies to increase enzyme expression and optimize industrial production processes.

[0004] Currently, common promoters include strong promoters (such as T7, P43, and Pgrac) and inducible promoters (such as PxylA and Pspac), but these promoters often have problems such as limited adaptability, insufficient regulatory precision, or a heavy metabolic burden. In Bacillus subtilis, the screening and optimization of endogenous promoters and the design of artificially synthesized promoters have become important research directions for improving the expression efficiency of target proteins. Current studies have shown that promoter engineering (such as mutation screening, sequence optimization, and cis-regulatory element enhancement) can effectively increase gene transcription levels, thereby increasing the expression of exogenous proteins.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a promoter for improving protein expression levels and its application.

[0007] The present invention provides a novel promoter that can effectively enhance the transcriptional level of protease (such as maltogenic amylase) genes in Bacillus subtilis, significantly improving the expression of the enzyme. The promoter has been optimized and designed to have high transcriptional activity and can drive efficient expression of the target gene under suitable conditions. Through the application of this promoter, the industrial production level of proteases (such as maltogenic amylase) can be improved, providing more efficient biocatalytic solutions for industries such as food, fermentation, and biocatalysis.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a promoter with a nucleotide sequence as shown in any one of SEQ ID NOs: 1-4. The promoter is operably linked to a fluorescent protein gene, a maltogenic amylase gene, or an α-amylase gene to achieve efficient expression of the fluorescent protein gene, the maltogenic amylase gene, or the α-amylase gene in Bacillus subtilis, thereby improving the heterologous expression level of the fluorescent protein, the maltogenic amylase, or the α-amylase in Bacillus subtilis.

[0010] The present invention provides a promoter, wherein the promoter is selected from any one of the following (i)-(iii):

[0011] (i) a polynucleotide whose nucleotide sequence is shown in any one of SEQ ID NOs: 1-4;

[0012] (ii) a polynucleotide having a nucleotide sequence that is at least 80%, optionally at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-4, and having similar promoter activity;

[0013] (iii) a nucleotide sequence complementary to the nucleotide sequence shown in (i) or (ii).

[0014] The promoters provided by the present invention are respectively named P43mut1, P43mut2, P43mut3 and P43mut4, and their nucleotide sequences are shown in SEQ ID NOs: 1-4. P43mut1 is obtained by mutating the 109th nucleotide of the original promoter shown in SEQ ID NO: 5 from G to A, P43mut2 is obtained by mutating the 69th to 74th nucleotides of the original promoter shown in SEQ ID NO: 5 from ATGGGC to TTCCAG, P43mut3 is obtained by mutating the 69th to 74th and 104th to 109th nucleotides of the original promoter shown in SEQ ID NO: 5 from ATGGGC and ATAAG to CATCTG and TTTGAC, and P43mut4 is obtained by mutating the nucleotide sequence shown in SEQ ID NO: The original promoter shown in NO: 5 was obtained by mutating nucleotides 69-74, 81-97, and 104-109 from ATGGGC, AAAAGCGCGCGATTATG, and ATAAAG to GATCGG, TTTTTGCGTGGATATTA, and GGAAGT. The promoter of the present invention was screened for adaptability to the maltogenic amylase gene. Compared with the original, stronger promoter P43, the promoter of the present invention can enhance the transcription level of maltogenic amylase, thereby promoting the expression of amylase in Bacillus subtilis. In addition, the promoter of the present invention can significantly increase the expression level of heterologous genes (such as the α-amylase AmyS gene from Bacillus amyloliquefaciens) in Bacillus subtilis. Compared with the original promoter P43, the promoter P43mut1 of the present invention increases the expression level of maltogenic amylase or α-amylase by 1.5 times. Therefore, the promoter provided by the present invention can be used as a universal protease promoter and has strong industrial application potential.

[0015] Furthermore, the present invention provides a gene expression cassette comprising the promoter and a target gene operably linked to the promoter.

[0016] Furthermore, the present invention provides a recombinant vector comprising the promoter or the gene expression cassette.

[0017] Furthermore, the starting vector of the recombinant vector is a pBE series vector or the like.

[0018] Furthermore, the present invention provides a recombinant strain comprising the promoter, or the gene expression cassette, or the recombinant vector.

[0019] Furthermore, the host strain corresponding to the recombinant strain is selected from prokaryotic microorganisms, etc.; the prokaryotic organisms include bacteria such as Bacillus.

[0020] More specifically, the prokaryotic organism is Bacillus subtilis or Bacillus amyloliquefaciens, specifically Bacillus subtilis ATCC 6051 and its derivative strains and Bacillus amyloliquefaciens LB1ba02.

[0021] Furthermore, the present invention provides an engineered Bacillus subtilis bacterium, wherein the method for constructing the engineered Bacillus subtilis bacterium comprises the following steps:

[0022] By gene editing technology, a target gene expression cassette containing a promoter with a nucleotide sequence as shown in any one of SEQ ID NOs: 1-4 is integrated into the genome of Bacillus subtilis, and positive clones are screened.

[0023] By using gene editing technology, a gene expression cassette containing the target gene, which is the maltogenic amylase gene, is integrated into the genome of Bacillus subtilis, and positive clones are screened.

[0024] Alternatively, a plasmid containing a gene expression cassette containing the target gene, which is the maltogenic amylase gene, is transformed into Bacillus subtilis competent cells, and positive clones are screened.

[0025] Furthermore, the present invention provides the use of the promoter in promoting efficient expression of a target gene in Bacillus subtilis.

[0026] Furthermore, the gene expression cassette, or the recombinant vector, or the recombinant strain is used in preparing proteins.

[0027] Furthermore, the protein includes maltogenic amylase, α-amylase, fluorescent protein, etc.

[0028] Furthermore, the present invention provides a method for increasing protein production, comprising the step of expressing the protein using the above-mentioned gene expression cassette, recombinant vector, or recombinant strain.

[0029] The proteins include maltogenic amylase, α-amylase, fluorescent protein and the like.

[0030] The present invention has the following advantages and effects compared to the prior art:

[0031] The present invention constructs a promoter library using the original promoter P43 of the cytidine deaminase gene from Bacillus subtilis 168 as a template. Using GFP as a reporter protein, a 180-bp sequence from the 5′ end of the maltogenic amylase gene was added to the GFP via a GGGGS linker. The resulting promoter, P43mut1, was screened for significantly increased maltogenic amylase gene expression. Its nucleotide sequence is shown in SEQ ID NO: 1. Compared to the original promoter P43, the promoter P43mut1 provided by the present invention can increase the expression levels of the maltogenic amylase and α-amylase genes by 1.5 times in Bacillus subtilis, significantly enhancing the expression of heterologous genes in Bacillus subtilis. This promoter can be used as a universal promoter and has strong industrial applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The sequence of the original promoter P43 (A) and the design and construction strategy of the P43 promoter library (B).

[0033] Figure 2 This is a comparison of the fluorescence intensities of the promoter mutant prepared in Example 2 and the original promoter GFP.

[0034] Figure 3 This is a comparison of the relative activity levels of amylases in Bacillus subtilis ATCC 6051 / pBE-P43-AmyM, Bacillus subtilis ATCC6051 / pBE-P43mut1-AmyM, Bacillus subtilis ATCC6051 / pBE-P43-AmyS, and ATCC6051 / pBE-P43mut1-AmyS in Example 3. DETAILED DESCRIPTION

[0035] The present invention will be described in further detail below with reference to the examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. The operating steps or conditions not particularly noted in the following examples may be performed with reference to conventional techniques.

[0036] The term "promoter" in the present invention refers to a nucleic acid molecule that is usually located upstream of the coding sequence of the target gene, provides a recognition site for RNA polymerase, and is located upstream of the 5' direction of the mRNA transcription start site. It is a nucleic acid sequence that is not translated. After RNA polymerase binds to this nucleic acid sequence, it initiates the transcription of the target gene. In the synthesis of ribonucleic acid (RNA), the promoter can interact with the transcription factors that regulate gene transcription, control the start time and degree of gene expression (transcription), and contains a core promoter region and a regulatory region, which is like a "switch" to determine the activity of the gene and then control which protein the cell starts to produce. In the sequence of the promoter, the -35 region is the recognition site of RNA polymerase, and the -10 region is the binding site of RNA polymerase.

[0037] The promoter sequence of the present invention can be modified by conventional mutagenesis. Therefore, the promoter may include, but is not limited to, any nucleotide sequence having 70% or greater, specifically 80% or greater, more specifically 90% or greater, even more specifically 95% or greater, even more specifically 98% or greater, and most specifically 99% or greater homology to the nucleotide sequence of SEQ ID NOs: 1-4, and having similar promoter activity. Any nucleotide sequence having such homology in which a portion of the sequence is deleted, modified, substituted, or inserted should be understood to be included within the scope of the nucleic acid molecules disclosed herein, as long as the sequence has promoter activity. For example, polynucleotides having a meaningless sequence added to or at the end of the corresponding nucleotide sequence of SEQ ID NOs: 1-4, or having a portion of the sequence deleted from or at the end of the corresponding nucleotide sequence of SEQ ID NOs: 1-4, are obviously also included within the scope of the present disclosure, as long as they have the same or corresponding activity as the polynucleotide.

[0038] The promoter SEQ ID NO: 5 is referred to herein as the initial promoter or original promoter, relative to the mutant promoters SEQ ID NO: 1 to 4. For the sake of convenience, the initial promoter SEQ ID NO: 5 and its mutants such as SEQ ID NO: 1 may be collectively referred to herein as "promoters."

[0039] The term "gene expression cassette" in the present invention refers to a sequence containing a promoter, a target gene or a target gene cloning site and a terminator, in which the gene can be transcribed and translated normally. The target gene can be a protein-coding gene.

[0040] The term "vector" as used herein refers to the vehicle required to deliver a target gene into a biological cell (recipient cell) through genetic engineering. Vectors used in genetic engineering are self-replicating DNA molecules in which a segment of DNA can be removed without affecting replication. These molecules can be used to replace or insert exogenous (target) DNA, thereby introducing the target DNA into the host cell. Commonly used vectors include plasmids, bacteriophages, and viruses.

[0041] The term "derivative strain" in the present invention refers to a strain derived by any transformation, for example by one or more hybridizations and / or by mutation and / or by genetic transformation.

[0042] The gene editing technology in the present invention can adopt the Cre / loxp system, CRISPR / Cas9 system and CRISPR / Cpf1 system, etc.

[0043] The term "transformation" as used herein refers to the process of introducing a vector containing a polynucleotide encoding a target protein into a host cell, thereby enabling expression of the polynucleotide encoding the protein in the host cell. Transformation methods may include any method capable of introducing a nucleic acid into a cell, and transformation may be performed using appropriate standard techniques known in the art, depending on the host cell. Examples include, but are not limited to, electroporation, and include, but are not limited to, chemical transformation, electroporation, and other methods.

[0044] The present invention will be further described below with reference to specific embodiments.

[0045] Example 1 Preparation of promoter mutants

[0046] (1) Construction of promoter strength characterization plasmid

[0047] Using the Escherichia coli-Bacillus subtilis shuttle plasmid pBE-P43-SamyQ-AmyM as a template, primers VT-F and VT-R were designed to amplify a plasmid backbone DNA fragment, including a P43 promoter (nucleotide sequence shown in SEQ ID NO: 5), SamyQ, and the first 180 bp of the AmyM gene. The green fluorescent protein (GFP) gene sequence was synthesized by Nanjing GenScript (nucleotide sequence shown in GenBank: MN513050.1, bp 1642-2358, amino acid sequence shown in NCBI Reference Sequence L: WP_012569506.1), and GFP-F and GFP-R were designed to amplify the GFP fragment. The amplified plasmid backbone DNA fragment and GFP fragment were chemically transformed into Bacillus subtilis SCK6 competent cells, and positive transformants were selected and sequenced to obtain the pBE-P43-SamyQ-GFP recombinant plasmid. Using the plasmid pBE-P43-SamyQ-GFP as a template, primers SamyQ-F and SamyQ-R were designed to remove the signal peptide SamyQ, and finally the recombinant plasmid pBE-P43-GFP was obtained, in which GFP was used as the reporter protein, and the sequence of the 5′ end 180 bp of the AmyM gene (as shown in 1-180 bp in SEQ ID NO.6) was added in front of GFP through a GGGGS linker.

[0048] Among them, the pBE-P43-SamyQ-AmyM plasmid is disclosed in the document “Chen Y, Xin Q, Pan L, et al. Improved Recombinant Expression of Maltogenic α-Amylase AmyM in Bacillus subtilis by Optimizing Its Secretion and NADPH Production[J]. Fermentation (Basel), 2023, 9(5). DOI: 10.3390 / fermentation9050475.”

[0049] The specific amplification primers are shown in Table 1:

[0050] Table 1 Primer sequence list

[0051]

[0052] PCR amplification program: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 10 s, annealing at 64°C for 10 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0053] Reaction conditions:

[0054]

[0055] After the PCR product was purified, the amplified fragments were homologously fused using the Gib-seamless cloning (multi-fragment) kit. The ligation products were chemically transformed into Escherichia coli MachT1 competent cells, and the positive transformants were picked for sequencing and identification, and finally the promoter strength characterization vector was obtained.

[0056] (2) Construction and screening of promoter library

[0057] Using the recombinant plasmid pBE-P43-GFP prepared in step (1) as a template, primers M-p43-F-1, M-p43-R-2, M-p43-F-3 and M-p43-R-4 (Table 2) were designed to amplify the promoter fragments respectively, and PBE-P43-F and PBE-P43-R (Table 2) were used as primers to amplify the plasmid backbone. The above promoter fragments were connected to the plasmid backbone by Gib-seamless cloning (multi-fragment) cloning kit in 2 to 3 tubes, 10 μL per tube, to obtain the recombinant plasmids and transform Escherichia coli MachT1 competent cells respectively. The cells were spread on 1 piece of LB solid culture medium containing 50 μg / mL kanamycin, and the total volume of the collection was about 10 6The recombinant plasmids were extracted from the single clones of bacteria to obtain the P43 promoter library plasmids. The sequence of the original promoter P43 and the design and construction strategy of the P43 promoter library are as follows: Figure 1 shown.

[0058] Approximately 1 μg of library plasmid was transformed into Bacillus subtilis ATCC 6051 via electroporation. The cells were plated onto approximately 20 LB plates containing 25 μg / mL kanamycin, with each plate containing approximately 300-500 colonies, yielding a total of approximately 10,000 colonies. All colonies on the plates were washed with PBS buffer and subjected to flow cytometry (FACS) sorting. Colonies with enhanced fluorescence were isolated and plated onto LB plates containing 50 μg / mL kanamycin to obtain recombinant strains with enhanced fluorescence intensity.

[0059] Table 2 Primer sequence list

[0060]

[0061] Example 2 Strength Characterization of Promoter Mutation Library

[0062] The recombinant strain with enhanced fluorescence intensity obtained by screening in Example 1 and the recombinant strain constructed in Example 1 were inoculated into a 48-deep-well plate, each well containing 800 μL of TB medium containing 25 μg / mL kanamycin, and cultured at 37°C and 800 r / min for 24 h. The plates were then transferred to a new 48-deep-well plate, each well containing 900 μL of TB medium containing 25 μg / mL kanamycin. Each sample was replicated in triplicate, and the OD values were measured after culture at 37°C and 800 r / min for 24 h. 600 and fluorescence value (excitation wavelength is 490nm, emission wavelength is 530nm), and the fluorescence intensity is the measured fluorescence value / OD 600 , namely GFU / OD 600 .

[0063] See Table 3 and Figure 2 , GFU / OD 600 The promoter mutants with increased OD values were named P43mut1, P43mut2, P43mut3 and P43mut4, and their corresponding recombinant strains were named ATCC 6051 / pBE-P43mut1-GFP, ATCC 6051 / pBE-P43mut2-GFP, ATCC 6051 / pBE-P43mut3-GFP and ATCC 6051 / pBE-P43mut4-GFP, where GFU / OD 600 The promoter mutant with the highest value was P43mut1, and this recombinant strain showed at least three times the fluorescence intensity of ATCC 6051 / pBE-P43-GFP (ie, the recombinant strain with the original promoter).

[0064] Table 3 Fluorescence intensity of Bacillus subtilis

[0065]

[0066] The ATCC 6051 / pBEX-GFP strain was generated by transforming Bacillus subtilis ATCC 6051 with the recombinant vector pBEX-GFP. The recombinant vector pBEX-GFP was constructed by removing the P43 promoter from the pBE-P43-GFP recombinant vector. ATCC 6051 / pBEX-GFP served as a fluorescence control strain to eliminate possible interference from factors upstream of the promoter.

[0067] PCR amplification was performed on a single colony of ATCC 6051 / pBE-P43mut1~4-GFP using primers Test1-F and Test1-R shown in Table 2, and the amplified PCR fragment was sequenced. The results showed that the nucleotide sequences of P43mut1-4 correspond to SEQ ID NOs: 1-4, and the differences between them and the original promoter P43 are as follows: nucleotide 103 of P43mut1 (sequence shown in SEQ ID NO: 1) was obtained by mutating the G at the corresponding position in P43 (sequence shown in SEQ ID NO: 5) to A; nucleotides 69-74 of P43mut2 (sequence shown in SEQ ID NO: 2) were obtained by mutating from ATGGGC to TTCCAG; nucleotides 69-74 and 104-109 of P43mut3 (sequence shown in SEQ ID NO: 3) were obtained by mutating from ATGGGC and ATAAAG to CATCTG and TTTGAC; and nucleotides 69-74, 81-97, and 104-109 of P43mut4 (sequence shown in SEQ ID NO: 4) were obtained by mutating from ATGGGC, AAAAGCGCGCGATTATG, and ATAAAG to GATCGG, TTTTTGCGTGGATATTA, and GGAAGT.

[0068] Example 3 Evaluation of the ability of promoter mutants to express heterologous amylases

[0069] (1) Construction of recombinant strains

[0070] The maltogenic amylase AmyM gene (from Geobacillus stearothermophilus) (amino acid sequence as shown in SEQ ID NO.7, wherein the N-terminus carries 6*His; the nucleotide sequence of the gene is shown in SEQ ID NO.6, wherein the 3′ end carries a nucleotide sequence encoding 6*His) and the α-amylase AmyS gene (from Bacillus amyloliquefaciens) (amino acid sequence as shown in SEQ ID NO.9, wherein the N-terminus carries 6*His; the nucleotide sequence of the gene is shown in SEQ ID NO.8, wherein the 3′ end carries a nucleotide sequence encoding 6*His) were used as reporter proteins to construct recombinant vectors pBE-P43-AmyM, pBE-P43mut1-AmyM, pBE-P43-AmyS and pBE-P43mut1-AmyS. The plasmids were transformed into Bacillus subtilis ATCC 6051 competent cells by electroporation (2500 V, 4.5-6 ms) to construct the corresponding recombinant strains ATCC 6051 / pBE-P43-AmyM, ATCC 6051 / pBE-P43mut1-AmyM, ATCC 6051 / pBE-P43-AmyS and ATCC 6051 / pBE-P43mut1-AmyS.

[0071] Among them, the recombinant vectors pBE-P43-AmyM and pBE-P43-AmyS were constructed by replacing the "5' end 180bp of the AmyM gene + GGGGS linker + GFP gene" in the pBE-P43-GFP recombinant vector with the AmyM gene (SEQ ID NO. 6) and the AmyS gene (SEQ ID NO. 8), respectively; the recombinant vectors pBE-P43mut1-AmyM and pBE-P43mut1-AmyS were constructed by replacing the "5' end 180bp of the AmyM gene + GGGGS linker + GFP gene" in the pBE-P43mut1-GFP recombinant vector with the AmyM gene (SEQ ID NO. 6) and the AmyS gene (SEQ ID NO. 8), respectively.

[0072] (2) Fermentation of recombinant amylase expression strains and protein purification

[0073] a) inoculating the correctly identified amylase recombinant expression strain in step (1) into 10 mL of LB liquid medium (containing 50 μg / mL kanamycin) and culturing at 37° C. and 220 rpm for 8-14 h to serve as seed solution;

[0074] b) inoculating the seed solution from step a) into TB medium (containing 50 μg / mL kanamycin) at an inoculum size of 2% and a volume of 50 mL / 500 mL, and fermenting at 37° C. and 220 rpm for 24-36 hours to obtain a fermentation broth;

[0075] c) centrifuging the fermentation liquid obtained in step b) (8000 r / min, 5 min) to remove the bacterial cells, and the obtained supernatant is the crude amylase enzyme solution.

[0076] d) The crude amylase solution obtained in step c) was filtered through a 0.22 μm filter. The filtrate was purified by nickel affinity chromatography. The column was washed with 1.5 column volumes of Buffer B (500 mM NaCl, 500 mM imidazole, 20 mM Tris-HCl, pH 8.0) and rinsed with Buffer A (500 mM NaCl, Tris-HCl, pH 8.0) to a UV baseline. The supernatant was then loaded onto a His TRAP™ HP Ni-NTA prepacked column and eluted using a gradient mixture of Phase B and Phase A. The target protein was eluted at 30% Buffer B. The collected eluate was then ultrafiltered (500 mM NaCl, 20 mM Tris-HCl, 3 mM DTT (dithiothreitol), pH 8.0) to remove the imidazole and impurities, thereby obtaining the target protein.

[0077] (3) Amylase activity test

[0078] The crude amylase solution was diluted with a phosphate buffer solution having a pH of 6.0 by an appropriate multiple, and 1% soluble starch was prepared with the same buffer solution as a reaction substrate. After the substrate was prepared, it was placed in a 60°C water bath for insulation. Enzyme activity was determined using a modified DNS method: four clean 2mL centrifuge tubes were prepared, one serving as a blank control and the remaining three as experimental tubes. 200μL of the diluted crude enzyme solution was added to each of the four tubes. 800μL of 0.4M NaOH solution was added to the blank control tube. 400μL of the reaction substrate was then added to the four tubes, and the mixture was incubated at 60°C for 10 minutes. After the reaction was complete, 800μL of 0.4M NaOH solution was quickly added to the experimental tubes to terminate the reaction. 200μL of the reaction solution from each of the four tubes was transferred to a new 1.5mL centrifuge tube, and 300μL of DNS solution was added to each tube. After seven minutes of boiling water bath, the centrifuge tubes were quickly removed and cooled on ice for 15 minutes. After cooling, 200μL of the reaction solution was transferred to a 96-well microplate and the absorbance at 540nm was measured using an Infinite M200 multi-function microplate reader. Under these reaction conditions, one unit of enzyme activity was defined as the amount of enzyme required to produce 1μmol / min of maltose. Prepare maltose solutions of different concentrations and measure the corresponding OD 540value and draw the maltose standard solution curve.

[0079] The enzyme activity was calculated using the following formula:

[0080]

[0081] C: maltose concentration produced in the reaction (mg / mL);

[0082] V1: reaction system volume, mL;

[0083] D: enzyme solution dilution multiple;

[0084] 1000: convert maltose content into μg;

[0085] M: relative molecular mass of maltose;

[0086] V2: volume of enzyme solution added, mL;

[0087] T: reaction time, min.

[0088] The maltose concentration produced by each strain reaction was substituted into the above enzyme activity calculation formula, and the calculated enzyme activity was shown in Table 4; the relative enzyme activity was shown in Figure 3 .

[0089] Table 4 Amylase activity in recombinant strains of ATCC 6051

[0090]

[0091] See Table 4 and Figure 3 Amylase genes from Bacillus amyloliquefaciens and Bacillus stearothermophilus can be heterologously expressed in Bacillus subtilis. Compared with Bacillus subtilis ATCC 6051 / pBE-P43-amyM and ATCC 6051 / pBE-P43-amyS, Bacillus subtilis ATCC 6051 / pBE-P43mut1-amyM and ATCC 6051 / pBE-P43mut1-amyS showed significantly increased amylase activity, indicating that P43mut1 is a universal and potent protease promoter.

[0092] In summary, compared to the original promoter P43, the promoter mutant P43mut1 provided by the present invention can enhance the expression of heterologous amylase genes in Bacillus subtilis, achieving an enzyme activity increased by 1.5 times compared to the original promoter P43. Therefore, the promoter provided by the present invention can be used as a universal promoter and has strong industrial applicability.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A promoter, characterized in that: The nucleotide sequence of the promoter is the nucleotide sequence shown in any one of SEQ ID NOs: 1-4 or a complementary sequence thereof.

2. The promoter-related biomaterial according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the promoter according to claim 1; (b) a recombinant vector containing the promoter according to claim 1; (c) a recombinant vector containing the expression cassette described in (a); (d) a recombinant strain containing the promoter according to claim 1; (e) a recombinant strain containing the expression cassette described in (a); (f) A recombinant strain containing the recombinant vector described in (b) or (c).

3. The biomaterial according to claim 2, characterized in that: The expression cassette in (a) further contains a target gene, which is operably linked to the promoter.

4. The biomaterial according to claim 2 or 3, characterized in that: The host strain corresponding to the recombinant strains (d), (e) and (f) is selected from prokaryotic microorganisms.

5. The biomaterial according to claim 4, characterized in that: The prokaryotic microorganisms include the genus Bacillus.

6. Use of the promoter according to claim 1 or the biomaterial according to any one of claims 2 to 5 in promoting efficient expression of a target gene.

7. Use of the promoter according to claim 1 or the biological material according to any one of claims 2 to 5 in promoting high-efficiency expression of a target gene in Bacillus subtilis.

8. Use of the promoter according to claim 1 or the biomaterial according to any one of claims 2 to 5 in protein expression.

9. The use according to claim 8, characterized in that: The protein includes maltogenic amylase, α-amylase or fluorescent protein.

10. A method for increasing protein production, characterized in that: The method comprises the step of expressing the protein using the biological material according to any one of claims 2 to 5.

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