DNA molecule, promoter and use for expressing alkaline protease

Abstract

The application provides a DNA molecule, a promoter and application for expressing alkaline protease. The DNA molecule contains a promoter and a gene for expressing alkaline protease, and the sequence of the promoter comprises a nucleotide sequence shown in SEQ ID NO: 2. The application can solve the problem of low expression level of microbial alkaline protease in the prior art and is suitable for the technical field of biology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a DNA molecule, promoter, and application for expressing alkaline proteases. Background Technology

[0002] Alkaline proteases are a class of enzymes that hydrolyze peptide bonds in proteins under alkaline conditions. Their optimal pH for action is generally 9–11. The alkaline protease produced by *Bacillus licheniformis* is an endopeptide, catalyzing serine residues. Alkaline proteases are widely used in detergents, food, medical, brewing, silk, and leather industries. They are currently popular detergent additives on the market, significantly improving detergent and stain removal capabilities, especially effective against protein-based stains such as blood, sweat, milk, and oil. They constitute the largest proportion of industrial enzymes. With the rapid development of the detergent, food, and pharmaceutical industries, the demand for alkaline protease is constantly increasing. However, existing alkaline protease-producing strains suffer from low alkaline protease expression levels, insufficient to meet market demand.

[0003] Currently used alkaline proteases are mostly proteolytic enzymes derived from Bacillus licheniformis 2709, selected through mutagenesis, and produced through deep fermentation, extraction, and purification. Chinese patent application CN108570477A mentions an alkaline protease gene and a method for constructing a recombinant Bacillus subtilis strain. This patent expresses the high-activity alkaline protease gene Bmp-opt in a recombinant Bacillus subtilis strain under the regulation of different promoter signal peptides, obtaining a recombinant strain. Furthermore, Chinese patent application CN117448361A mentions a recombinant strain producing heterologous alkaline protease, containing a combined DNA fragment with two tandem promoters. The optimized dual promoters are used to regulate the expression of the exogenous gene for alkaline protease production. However, current technologies mainly focus on alkaline proteases from other sources, neglecting research on the expression of alkaline proteases from Bacillus licheniformis, and the expression level of alkaline proteases still falls short of the requirements for industrial production. Summary of the Invention

[0004] The main objective of this invention is to provide a host cell for producing alkaline protease, thereby addressing the problem of low expression levels of microbial alkaline protease in the prior art.

[0005] To achieve the above objective, according to a first aspect of the present invention, a DNA molecule for expressing an alkaline protease is provided, the DNA molecule containing a promoter and a gene for expressing the alkaline protease, the sequence of the promoter comprising the nucleotide sequence shown in SEQ ID NO: 2.

[0006] Furthermore, the aforementioned DNA molecule also contains any one or more of the following: a gene expressing a signal peptide, or a gene expressing a chaperone protein.

[0007] Furthermore, the promoter described above is located at the 5' direction of the alkaline protease gene.

[0008] To achieve the above objectives, according to a second aspect of the present invention, a promoter is provided, the promoter comprising the nucleotide sequence shown in SEQ ID NO: 2.

[0009] To achieve the above objectives, according to a third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is linked to the aforementioned DNA molecule or the aforementioned promoter.

[0010] Furthermore, the recombinant plasmids mentioned above include one or more of pHYT or PHY300PLK.

[0011] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the aforementioned DNA molecule, the aforementioned promoter, or the aforementioned recombinant plasmid.

[0012] Furthermore, the aforementioned host cells include Bacillus licheniformis or Escherichia coli.

[0013] Furthermore, the host cell contains an alkaline protease gene, and the promoter is located at the 5' position of the alkaline protease gene; the alkaline protease gene and the promoter are located in the genome of the host cell and / or on a free plasmid, and the free plasmid exists independently in the host cell.

[0014] Furthermore, the copy number of the alkaline protease gene in the aforementioned host cells is ≥2.

[0015] To achieve the above objectives, according to a fifth aspect of the present invention, a method for preparing an alkaline protease is provided, the method comprising: culturing the aforementioned host cell and collecting the alkaline protease.

[0016] Furthermore, the above-mentioned culture includes: culturing the host cells in a fermentation medium.

[0017] Furthermore, the fermentation medium mentioned above includes corn starch, soybean meal, disodium hydrogen phosphate, or sodium carbonate.

[0018] Furthermore, the above fermentation medium, in total quantity, contains 30-60 g / L of corn starch, 30-60 g / L of soybean meal, 1-3 g / L of disodium hydrogen phosphate, and 1.0-1.5 g / L of sodium carbonate; the pH of the above fermentation medium includes 7.2-7.6.

[0019] To achieve the above objectives, according to a sixth aspect of the present invention, the application of the above-described DNA molecule, the above-described promoter, the above-described recombinant plasmid, the above-described host cell, or the above-described method for preparing alkaline protease in the preparation of detergents, food, or medicine is provided.

[0020] To achieve the above objectives, according to a seventh aspect of the present invention, the application of the above-described DNA molecule, the above-described promoter, the above-described recombinant plasmid, the above-described host cell, or the above-described method for preparing alkaline protease in the detergent industry, leather industry, textile industry, food industry, feed industry, or alkaline wastewater treatment field is provided.

[0021] By applying the technical solution of the present invention, the DNA molecule used to express alkaline protease contains a promoter and a gene for expressing alkaline protease. With the help of this promoter, the gene for expressing alkaline protease can be expressed in large quantities to obtain alkaline protease, thereby efficiently preparing alkaline protease. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0023] Figure 1 A schematic diagram of the technical route for constructing a recombinant strain that highly expresses alkaline protease S8 according to Example 1 of the present invention is shown.

[0024] Figure 2 A schematic diagram of the plasmid vector structure for free expression of alkaline protease S8 according to Example 1 of the present invention is shown.

[0025] Figure 3 The diagram shows the optimization results of the plasmid vector promoter for constructing a recombinant strain that highly expresses alkaline protease S8 according to Example 2 of the present invention.

[0026] Figure 4 The diagram shows the optimization results of the plasmid vector signal peptide for constructing a recombinant strain that highly expresses alkaline protease S8 according to Example 3 of the present invention.

[0027] Figure 5 The diagram shows the optimization results of the plasmid vector chaperone protein for constructing a recombinant strain that highly expresses alkaline protease S8 according to Example 4 of the present invention. Detailed Implementation

[0028] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0029] As mentioned in the background section, existing technologies suffer from low efficiency in expressing basic proteins in microorganisms. Therefore, in this application, the inventors attempted to develop a host cell for producing alkaline protease. By optimizing aspects such as the copy number of the alkaline protease gene, signal peptide, promoter, and molecular chaperone, the target product, alkaline protease, can be efficiently prepared, making it well-suited for industrial-scale production. Thus, this application proposes a series of protection schemes.

[0030] In a first typical embodiment of this application, a DNA molecule for expressing alkaline protease is provided, the DNA molecule containing a promoter and a gene for expressing alkaline protease, the sequence of the promoter including the nucleotide sequence shown in SEQ ID NO: 2.

[0031] In this application, the inventors discovered that by analyzing and comparing the original ParprE promoter sequence SEQ ID NO:1, and obtaining the transcriptional negative regulatory factor binding site sequence based on gene sequence analysis, deleting this sequence, the modified PaprE' promoter sequence SEQ ID NO:2 was obtained, which significantly enhanced the expression level of alkaline protease in Bacillus licheniformis E417. The aforementioned DNA molecule, containing the optimized promoter sequence PaprE' and expressing alkaline protease, significantly increased the expression level of alkaline protease in host cells. In this application, by loading the optimized promoter PaprE' and expressing alkaline protease onto the DNA molecule, the transcription initiation efficiency was enhanced, achieving the technical effect of promoting efficient expression of alkaline protease. In the actual production of alkaline protease, production costs can be significantly reduced and the yield of alkaline protease increased.

[0032] SEQ ID NO: 1:

[0033] atctttcacccgtttctgtatgcgatatattgcatattttaatagatgatcgacaaggccgcaacctccttcggcaaaaaatgatctcataaaataaatgaatagtattttcataaaatgaatcagatggagcaatctcctgtcattcgcggccctcgggacctctttccctgccaggctgaagcggtcta ttcatactttcgaactgaacatttttctaaaaacagttattaataaccaaaaaattttaaattggtcctccaaaaaaataggcctaccatataattcattttttttctctataataaattaacagaataattggaatagattatattatccttctatttaaattattctgaataaagaggaggagagtgagta.

[0034] SEQ ID NO: 2:

[0035] atctttcacccgtttctgtatgcgatatattgcatattttaatagatgatcgacaaggccgcaacctccttcggcaaaaaatgatctcataaaataaatgaatagtattttcataaaatgaatcagatggagcaatctcctgtcattcgcggccctcgggacctctttccctgccagg ctgaagcggtctactaaaacagttattaataaccaaaaaattttaaattggtcctccaaaaaaaataggcctaccatataattcatttttttctataataaattaacagaataattggaatagattatattatccttctatttaaattattctgaataaagaggaggagagtgagta.

[0036] In a preferred embodiment, the alkaline protease described above includes alkaline protease S8.

[0037] In a preferred embodiment, the DNA molecule further comprises any one or more of the following: a gene expressing a signal peptide, a gene expressing a chaperone protein, or a gene expressing an alkaline protease.

[0038] In this application, the inventors discovered that when the aforementioned DNA molecule also contains, but is not limited to, genes expressing signal peptides, genes expressing chaperone proteins, or genes expressing alkaline proteases, it is possible to achieve the technical effect of improving the secretion efficiency, activity, and yield of alkaline proteases, thereby reducing the production cost of alkaline proteases and laying a good foundation for the further application of alkaline proteases in industrial production.

[0039] In a preferred embodiment, the signal peptide is SacC, YurI, YoaW, Bli0820, Bli00338 or YbdN, preferably SacC or YurI.

[0040] In this application, the inventors discovered that when the DNA molecule contains the optimized promoter PaprE', and when the DNA molecule also contains, but is not limited to, SacC, YurI, YoaW, Bli0820, Bli00338 or YbdN, preferably a gene expressing the signal peptide SacC or a gene expressing the signal peptide YurI, the activity of alkaline protease can be further improved. This indicates that by optimizing the signal peptide sequence in the DNA molecule, the technical effect of improving the expression level of alkaline protease can be achieved.

[0041] In a preferred embodiment, the chaperone protein is PrsA.

[0042] PrsA molecular chaperone proteins promote correct enzyme folding and reduce misfolding and protein aggregation during protein folding. In this application, the inventors discovered that the DNA molecule containing, but not limited to, the nucleotide sequence of the molecular chaperone PrsA shown in SEQ ID NO: 3, and with the DNA molecule containing the optimized promoter PaprE' and signal peptide gene SacC, can further enhance the expression level of alkaline protease and improve the secretion efficiency and activity stability of alkaline protease.

[0043] SEQ ID NO: 3:

[0044]

[0045] In a preferred embodiment, the sequence of the alkaline protease described above includes the amino acid sequence shown in SEQ ID NO: 4.

[0046] In this application, the inventors discovered that when the sequence of the alkaline protease includes the amino acid sequence shown in SEQ ID NO: 4 or the DNA molecule contains the alkaline protease nucleotide sequence shown in SEQ ID NO: 5, the technical effect of increasing the production of alkaline protease in host cells can be achieved by increasing the copy number of alkaline protease and by optimizing the promoter, signal peptide and molecular chaperone.

[0047] SEQ ID NO: 4:

[0048] MKKMLMLAFTFLLALTIHVGEASAMRKKSFWLGMLTALMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKA KLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAA LDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ.

[0049] SEQ ID NO: 5:

[0050]

[0051] In a preferred embodiment, the promoter in the DNA molecule is located at the 5' direction of the alkaline protease gene.

[0052] In this application, the inventors discovered that in the above-mentioned DNA molecule, the promoter is located at the 5' direction of the alkaline protease gene, and the DNA molecule consists of a promoter, a gene expressing a signal peptide, a gene expressing alkaline protease, a gene expressing the chaperone protein PrsA, and a terminator from the 5' segment to the 3' segment, which can enhance the expression level of alkaline protease in the host cell and improve the secretion efficiency and activity stability of alkaline protease.

[0053] In a second typical embodiment of this application, a promoter is provided, which includes the nucleotide sequence shown in SEQ ID NO: 2.

[0054] In this application, the inventors effectively solved the problems of low copy number and poor promoter expression efficiency of alkaline protease gene from Bacillus licheniformis by using the nucleotide sequence shown in SEQ ID NO:2 as the promoter. The original ParprE promoter sequence SEQ ID NO:1 was analyzed and compared. Based on gene sequence analysis, the binding site sequence of the transcriptional negative regulator was obtained. This sequence was deleted, resulting in the modified ParprE' promoter sequence SEQ ID NO:2, which significantly enhanced the expression level of alkaline protease in Bacillus licheniformis E417. This promoter optimization strategy not only increased the yield of alkaline protease but also reduced production costs, demonstrating the potential value of alkaline protease in industrial applications and providing strong technical support for meeting the growing demand for alkaline protease in industries such as detergents, food, and pharmaceuticals.

[0055] In a third typical embodiment of this application, a recombinant plasmid is provided, wherein the recombinant plasmid is linked to the aforementioned DNA molecule or the aforementioned promoter.

[0056] When the aforementioned plasmid is linked to the aforementioned promoter or DNA molecule, it can efficiently transcribe and translate the carried DNA molecule in the host cell, yielding a large quantity of the target product, alkaline protease. This effectively solves the problems of low yield and high production cost of alkaline protease from Bacillus licheniformis in existing technologies, providing technical support for the large-scale production and application of alkaline protease.

[0057] In a preferred embodiment, the recombinant plasmid includes one or more of pHYT or PHY300PLK.

[0058] PHY300PLK is an Escherichia coli-Bacillus shuttle expression plasmid, and its nucleotide sequence is shown in SEQ ID NO: 6. This plasmid vector is mainly used in Bacillus systems to achieve intracellular or secretory protein expression. In this application, the inventors further optimized PHY300PLK to obtain the recombinant vector pHYT shown in SEQ ID NO: 7. The pHYT vector can be used as an intermediate cloning vector for prokaryotes such as Escherichia coli due to its high copy number characteristic of the target product gene. Preferably, the recombinant vector pHYT contains the P43-SigP fragment shown in SEQ ID NO: 8. In this application, the inventors preferably selected pHYT as the expression vector for the recombinant plasmid, which can significantly improve the expression level of the alkaline protease gene in the host cell, thereby achieving the technical effect of increasing the yield of alkaline protease.

[0059] SEQ ID NO: 6:

[0060]

[0061] SEQ ID NO:7:

[0062]

[0063] SEQ ID NO:8:

[0064]

[0065] In a fourth typical embodiment of this application, a host cell is provided, wherein the host cell contains the aforementioned DNA molecule, the aforementioned promoter, or the aforementioned recombinant plasmid.

[0066] Using the aforementioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried by the recombinant plasmids can be transcribed and translated to obtain a large quantity of the target product, alkaline protease. Alkaline protease can also be obtained by using existing technologies to purify the protein from the host cells through protein disruption, crude enzyme catalysis after disruption, or other methods. The host cells used are not of plant origin.

[0067] In a preferred embodiment, the host cell includes Bacillus licheniformis or Escherichia coli.

[0068] In this application, the inventors discovered that the aforementioned host cells, including but not limited to Bacillus licheniformis or Escherichia coli, possess natural adaptability and expression systems capable of effectively carrying and expressing the target product, alkaline protease. In particular, driven by the aforementioned promoter, the expression level of alkaline protease can be significantly increased. By using the promoter PaprE' in the host cells, not only is the gene copy number of alkaline protease increased, but its expression regulation mechanism is also optimized, thereby achieving a substantial increase in alkaline protease yield. In this application, the inventors preferably selected Bacillus licheniformis E417 as the host cell, which can efficiently and stably prepare alkaline protease, suitable for large-scale fermentation preparation of highly active alkaline protease, meeting the application needs of industries such as detergents, food, and pharmaceuticals.

[0069] In a preferred embodiment, the host cell contains an alkaline protease gene, and the promoter is located at the 5' position of the alkaline protease gene; the alkaline protease gene and the promoter are located in the genome of the host cell and / or on a free plasmid, which exists independently and freely in the host cell.

[0070] In this application, the inventors discovered that when a host cell contains an alkaline protease gene, and the 5' direction of the alkaline protease gene contains the promoter described above, wherein the alkaline protease gene is located in the genome of the host cell and / or on a free plasmid, and the free plasmid exists independently in the host cell, the constructed host cell can significantly increase the yield and activity of alkaline protease, reduce the production cost of alkaline protease, and meet the market demand for high-activity alkaline protease.

[0071] In a preferred embodiment, the copy number of the alkaline protease gene in the host cell is ≥2.

[0072] In this application, the inventors discovered that by increasing the copy number of the alkaline protease gene in the host cell, wherein the copy number is ≥2 (including but not limited to copy numbers of 2, 3, 4, 5, 6, 7, 8, 9 or 10), the expression level of the target product alkaline protease can be effectively promoted, the production cost of alkaline protease can be reduced, and the market demand for alkaline protease can be met.

[0073] In a fifth typical embodiment of this application, a method for preparing an alkaline protease is provided, the method comprising: culturing the host cell described above or the host cell obtained using the construction method described above, and collecting the alkaline protease.

[0074] In this application, the inventors provide a method for preparing alkaline protease, which involves culturing the aforementioned host cells or constructing host cells using the aforementioned construction method, and then collecting the alkaline protease. Furthermore, the inventors have discovered that host cells containing, but not limited to, the optimized promoter PaprE' gene, the signal peptide SacC gene and YurI gene, the chaperone protein PrsA gene, or the alkaline protease gene can be well applied in the preparation process of alkaline protease, thus meeting market demand for alkaline protease.

[0075] In a preferred embodiment, the above-mentioned culture includes: culturing the host cells in a fermentation medium.

[0076] In a preferred embodiment, the fermentation medium includes corn starch, soybean meal, disodium hydrogen phosphate, or sodium carbonate.

[0077] In this application, the inventors discovered that when a fermentation medium containing corn starch, soybean meal, disodium hydrogen phosphate, or sodium carbonate is used, host cells can achieve highly efficient expression and secretion of alkaline protease. The nutrient components of this fermentation medium enhance the yield and activity of alkaline protease, providing a more efficient and stable alkaline protease production solution for industrial applications.

[0078] In a preferred embodiment, the fermentation medium comprises, by total amount, 30-60 g / L of corn starch, 30-60 g / L of soybean meal, 1-3 g / L of disodium hydrogen phosphate, and 1.0-1.5 g / L of sodium carbonate; and the pH of the fermentation medium is 7.2-7.6.

[0079] In this application, the inventors further discovered that the corn starch content in the fermentation medium is, for example, 30-60 g / L (including but not limited to 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, or 60 g / L), and the soybean meal content ... The concentrations of disodium hydrogen phosphate are, for example, 1-3 g / L (including but not limited to 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, or 3 g / L), and the concentrations of sodium carbonate are, for example, 1.0-1.5 g / L (including but not limited to 1.05 g / L, 1.1 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, or 60 g / L). The concentrations of the fermentation medium are 1.15 g / L, 1.2 g / L, 1.25 g / L, 1.3 g / L, 1.35 g / L, 1.4 g / L, 1.45 g / L, or 1.5 g / L. A pH of 7.2-7.6 (including but not limited to 7.2, 7.3, 7.4, 7.5, or 7.6) provides the optimal nutrient environment for host cell growth and alkaline protease synthesis. Corn starch and soybean meal are rich in various vitamins and trace elements, which are beneficial to microbial metabolism. Disodium hydrogen phosphate and sodium carbonate are essential inorganic salts for microbial growth and enzyme synthesis. They maintain the osmotic pressure and pH stability of the culture medium, promote nutrient absorption and metabolic balance in microorganisms, and thus increase the yield of alkaline protease.

[0080] In a sixth typical embodiment of this application, the application of the above-described DNA molecule, the above-described promoter, the above-described recombinant plasmid, the above-described host cell, or the above-described method for preparing alkaline protease in the preparation of detergents, food, or medicine is provided.

[0081] In this application, the inventors discovered that the alkaline protease prepared using the aforementioned DNA molecule, promoter, recombinant plasmid, host cell, or method for preparing alkaline protease exhibits good expression levels and enzyme activity, making it suitable for use in the preparation of detergents, food, or pharmaceutical products. Specifically, in the preparation of detergent products, the aforementioned alkaline protease can be used in enzyme-added laundry detergents or powders to enhance detergency, particularly for protein-based stains (including but not limited to blood or milk stains); in the preparation of food products, the aforementioned alkaline protease can be used to hydrolyze animal and plant proteins to produce yeast extract, peptones, or amino acids; in the preparation of pharmaceutical products, the aforementioned alkaline protease can be used for nucleic acid purification or anti-inflammatory and anti-swelling drugs, exhibiting high specificity and low toxicity, making it suitable for biopharmaceutical and laboratory research.

[0082] In a seventh typical embodiment of this application, the application of the above-described DNA molecule, promoter, recombinant plasmid, host cell, or method for preparing alkaline protease is provided in the fields of washing industry, leather industry, textile industry, food industry, feed industry, or alkaline wastewater treatment.

[0083] In this application, the inventors discovered that the alkaline protease prepared using the aforementioned DNA molecule, promoter, recombinant plasmid, host cell, or method for preparing alkaline protease exhibits good expression levels and enzyme activity. This alkaline protease has significant application value in the washing, leather, textile, food, feed, and alkaline wastewater treatment industries. Specifically, in the washing industry, the alkaline protease can be used in enzyme-added laundry detergents or powders to enhance detergency, particularly for protein-based stains (including but not limited to blood or milk stains). In the leather industry, the alkaline protease can be used for hair removal and softening of leather, breaking down non-collagenous proteins in the leather, compared to traditional vulcanization... In the field of animal hair removal, alkaline proteases offer advantages such as reducing pollution and improving leather quality. In the textile industry, these alkaline proteases can be used for wool anti-felting treatment, silk degumming, or removing protein impurities, improving fabric feel and luster, and enhancing fiber processing performance. In the food industry, they can be used to hydrolyze animal and plant proteins to produce products such as yeast extract, peptone, or amino acids. In the feed industry, they can be used to break down plant proteins such as soybean meal, improving the digestibility and absorption rate of protein in animals, thereby increasing feed conversion rate and reducing breeding costs. In the field of alkaline wastewater treatment, these alkaline proteases can be used to degrade organic nitrogen pollutants for wastewater treatment and bioremediation, offering advantages such as being environmentally friendly and suitable for alkaline wastewater environments.

[0084] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0085] Experimental materials and reagents:

[0086] 1. Strains and vectors:

[0087] The strains used in this application include Escherichia coli strain DH5α and Bacillus licheniformis E417 with accession number CCTCC NO: M20232199. The expression vector pHYT has the nucleotide sequence shown in SEQ ID NO: 7.

[0088] 2. Enzymes and kits:

[0089] Homologous recombinases were purchased from Nanjing Novizan Biotech Co., Ltd.; ClonExpress Ultra One Step Cloning Kit, catalog number: C115-02; specification: 50 rxns; plasmid extraction and gel purification kits were purchased from Shanghai Sangon Biotech Co., Ltd.; and restriction endonucleases were purchased from Takara Bio Inc.

[0090] 3. Culture medium:

[0091] LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0);

[0092] LB-Tc is LB medium with 15 µg / mL tetracycline added;

[0093] Fermentation medium: corn starch (30 g / L), soybean meal (30 g / L), disodium hydrogen phosphate (2 g / L), sodium carbonate (1.25 g / L), pH 7.2~7.6.

[0094] Example 1

[0095] In prokaryotic expression systems, gene copy number, promoter, signal peptide, and protein folding efficiency have the most significant impact on gene expression. Since alkaline protease S8 is secreted in *Bacillus licheniformis*, and the secretion signal peptide plays a decisive role in protein secretion, this scheme optimizes the copy number, promoter, signal peptide, and molecular chaperone of alkaline protease S8 to improve the fermentation expression level of alkaline protease S8 in *Bacillus licheniformis* E417. A simplified diagram of the technical route for constructing a recombinant strain with high alkaline protease S8 expression using *Bacillus licheniformis* as the chassis strain is shown below. Figure 1 As shown, we optimized the existing Bacillus licheniformis E417 strain expressing alkaline protease S8 by optimizing its gene copy number, promoter, signal peptide, and molecular chaperone, and constructed a recombinant strain with a high fermentation expression level of alkaline protease S8.

[0096] To increase the copy number of alkaline protease S8 in Bacillus licheniformis E417, we constructed a plasmid vector for free expression of alkaline protease S8. Using the Bacillus licheniformis E417 genome as a template, we amplified the alkaline protease S8 gene expression cassette (containing promoter, signal peptide, and terminator) by PCR. We also synthesized P43-SigP and cloned the two target fragments into pHYT using an in vitro recombination method. The specific method included: amplifying the amino acid fragment of P43-SigP shown in SEQ ID NO: 8 using primers F1 and R1 in Table 1; and simultaneously digesting the PHY300 vector with restriction endonucleases BglII and SmaI at 37 degrees Celsius. After 1 h, the vector was completely digested and the linearized vector was recovered using a product recovery kit. The two DNA fragments were assembled using an in vitro recombinant kit. The cloned product was then transformed into *E. coli* DH5α. Colony PCR and sequencing verification confirmed the formation of the plasmid vector, pHYT-P43-SigP-S8. A schematic diagram of the plasmid vector is shown below. Figure 2 As shown. pHYT-P43-SigP-S8 was transformed into Bacillus licheniformis E417 to obtain recombinant strain E417 (pHYT-P43-SigP-S8).

[0097] Table 1

[0098]

[0099] Example 2

[0100] Based on the plasmid vector for free expression of alkaline protease S8 obtained in Example 1, in order to screen promoters for efficient expression of alkaline protease S8 in Bacillus licheniformis E417, on the one hand, we replaced the P43 promoter with a dual promoter expression system, including stationary expression promoters PYkzA and PGsiB and constitutive expression promoter PserA; on the other hand, we analyzed and compared the gene sequence of the original alkaline protease S8 promoter PaprE in E417, obtained the transcription negative regulatory factor binding site sequence based on gene sequence analysis, deleted this sequence, and obtained the modified promoter sequence PaprE'.

[0101] The newly constructed recombinant vectors pHYT-PaprE-SigP-S8, pHYT-PaprE'-SigP-S8, pHYT-PYkzA-PserA-SigP-S8, and pHYT-PGsiB-PserA-SigP-S8 were transformed into Bacillus licheniformis strain E417. Verified transformants were streaked onto LB agar plates, and colonies were scraped and inoculated into fermentation medium. After 12 hours of shaking culture, the colonies were transferred to fresh fermentation medium at a ratio of 1%, and fermentation was continued with periodic sampling to determine the alkaline protease activity in the fermentation supernatant.

[0102] The results are as follows Figure 3 As shown, the experimental results indicate that the shake-flask fermentation enzyme activity of Bacillus licheniformis strain E417 (named E417-PaprE'-S8) containing the pHYT-PaprE'-SigP-S8 recombinant vector is about 50% higher than that of wild-type Bacillus licheniformis E417.

[0103] Example 3

[0104] In this invention, we further optimized the alkaline protease S8 secretion signal peptide in Bacillus licheniformis E417. Since alkaline protease S8 is secreted in Bacillus licheniformis, the signal peptide plays a crucial role. Based on the highly efficient recombinant vector for expressing alkaline protease S8 (pHYT-PaprE'-SigP-S8) obtained in Example 2, we further analyzed the effect of the alkaline protease S8 secretion signal peptide on the enzyme activity of the fermentation broth. Through a review of relevant literature (The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions.), we selected six strong signal peptides from Bacillus licheniformis, including Bli00338, YbdN, SacC, YurI, Yoaw, or Bli02820. The nucleotide sequences of these six signal peptides are shown in Table 2.

[0105] By replacing the signal peptide SigP in pHYT-PaprE'-SigP-S8 with the above signal peptides, six recombinant expression vectors were constructed: pHYT-PaprE'-Bli00338-S8, pHYT-PaprE'-YbdN-S8, pHYT-PaprE'-SacC-S8, pHYT-PaprE'-YurI-S8, pHYT-PaprE'-Yoaw-S8, or pHYT-PaprE'-Bli02820-S8.

[0106] The recombinant vectors were transformed into Bacillus licheniformis strain E417. The correct transformants were verified by streaking on LB+Tet plates, and colonies were scraped and inoculated into fermentation medium. After shaking culture for 12 h, the colonies were transferred to fresh fermentation medium at a ratio of 1%. The fermentation was carried out and samples were taken at intervals to determine the alkaline protease activity in the fermentation supernatant.

[0107] The results are as follows Figure 4 As shown, the experimental results indicate that after replacing the S8 signal peptide in strain E417 with the aforementioned six new secreted signal peptides, the shake-flask fermentation activity of alkaline protease S8 was slightly increased by SacC and YurI signal peptides. However, the fermentation activity of strains transformed with the other secreted signal peptides was reduced to some extent. Among them, the SacC signal peptide showed the greatest increase in shake-flask fermentation activity of alkaline protease S8 compared to the other signal peptides. Therefore, the recombinant expression vector (pHYT-PaprE'-SacC-S8) was selected for subsequent experiments.

[0108] Table 2

[0109]

[0110] Example 4

[0111] In this invention, we further investigated the effect of overexpression of molecular chaperones in Bacillus licheniformis E417 on the expression of alkaline protease S8. Since alkaline protease S8 is secreted in Bacillus licheniformis E417, the signal peptide is cleaved during transmembrane translocation after translation and folds into the correct protein structure with the help of molecular chaperones.

[0112] To analyze the effect of the extracellular molecular chaperone PrsA on the expression of alkaline protease S8 in Bacillus licheniformis strain E417, we cloned the complete PrsA expression cassette (including the original promoter and terminator) from Bacillus licheniformis strain E417 and cloned it into the pHYT-PaprE'-SacC-S8 recombinant plasmid in Example 3, obtaining a recombinant expression plasmid containing the PrsA expression cassette (pHYT-PaprE'-SacC-S8-PrsA). We verified that the correct recombinant vector was successfully transformed into Bacillus licheniformis strain E417, obtaining the recombinant strain. The recombinant strain was streaked on LB+Tet plates, and colonies were scraped and inoculated into fermentation medium. After shaking culture for 12 h, the culture was transferred to fresh fermentation medium at a ratio of 1%, and fermentation was continued with periodic sampling to determine the alkaline protease activity in the fermentation supernatant.

[0113] Experimental results are as follows Figure 5As shown, the experimental results indicate that the Bacillus licheniformis E417 strain containing the pHYT-PaprE-SacC-S8-PrsA recombinant plasmid exhibits approximately 10% higher shake-flask fermentation enzyme activity compared to the E417 strain containing the pHYT-PaprE'-SacC-S8 recombinant plasmid. This suggests that expression of the pHYT-PaprE'-SacC-S8-PrsA recombinant plasmid in the E4171 strain can further enhance the activity of alkaline protease S8.

[0114] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0115] This invention, through research on the copy number of alkaline protease gene from Bacillus licheniformis and the effects of signal peptides and molecular chaperones on alkaline protease expression, provides a more systematic improvement in the expression efficiency of alkaline protease in Bacillus licheniformis by increasing protein expression levels, combined with research on protein secretion, folding, and transport. By increasing the copy number of alkaline protease S8 in the genome, combined with signal peptide optimization and chaperone protein gene expression combinations, a recombinant Bacillus licheniformis alkaline protease strain was constructed and screened, with its alkaline protease expression level increased by 10-50%.

[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A host cell, characterized in that, The host cell contains DNA molecules, promoters, or recombinant plasmids; The host cell was Bacillus licheniformis E417; The promoter sequence is the nucleotide sequence shown in SEQ ID NO: 2, the DNA molecule contains the promoter and a gene expressing alkaline protease, and the recombinant plasmid is linked with the DNA molecule or the promoter.

2. The host cell according to claim 1, characterized in that, The DNA molecule also contains any one or more of the following: a gene expressing a signal peptide, or a gene expressing a chaperone protein.

3. The host cell according to claim 1, characterized in that, In the DNA molecule, the promoter is located at the 5' direction of the alkaline protease gene.

4. The host cell according to claim 1, characterized in that, The recombinant plasmid includes one or more of pHYT or PHY300PLK.

5. The host cell according to any one of claims 1 to 4, characterized in that, The host cell contains an alkaline protease gene, and the promoter of the alkaline protease gene is located at the 5' position. The alkaline protease gene and the promoter are located in the genome of the host cell and / or on a free plasmid that exists independently in the host cell.

6. The host cell according to claim 5, characterized in that, The copy number of the alkaline protease gene in the host cell is ≥2.

7. A method for enhancing the expression of alkaline protease, characterized in that, The method comprises: culturing the host cell according to any one of claims 1-4, and collecting the alkaline protease.

8. The method according to claim 7, characterized in that, The culture includes culturing the host cells in a fermentation medium.

9. The method according to claim 8, characterized in that, The fermentation medium includes corn starch, soybean meal, disodium hydrogen phosphate, or sodium carbonate.

10. The method according to claim 9, characterized in that, The fermentation medium, in total quantity, comprises 30-60 g / L corn starch, 30-60 g / L soybean meal, 1-3 g / L disodium hydrogen phosphate, and 1.0-1.5 g / L sodium carbonate; the pH of the fermentation medium is 7.2-7.

6.

11. The use of the host cell of any one of claims 1-6 or the method for increasing alkaline protease expression of any one of claims 7-10 in the preparation of detergents, food or medicine.

12. The application of the host cell of any one of claims 1-6 or the method for increasing alkaline protease expression of any one of claims 7-10 in the detergent industry, leather industry, textile industry, food industry, feed industry or alkaline wastewater treatment field.

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