A bacillus subtilis strain for efficiently expressing cutinase and application thereof

Abstract

The application discloses a high-efficiency expression bacillus subtilis strain of cutinase and application thereof, and particularly, based on bacillus subtilis WS9CΔH, P csn As a promoter, sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA) or sp(csn) is used as a signal peptide to obtain a bacillus subtilis genetically engineered bacterium by overexpressing cutinase BhrPETase. The application uses a csn promoter gene on a bacillus subtilis genome and a signal peptide gene such as ydjM to design a new expression element, and through 60 h of flask fermentation, the whole-cell enzyme activity of the fermentation liquor of cutinase BhrPETase reaches 42.9 U·mL ‑1 , the extracellular enzyme activity reaches 40.3 U·mL ‑1 , and high-efficiency expression of cutinase BhrPETase is realized.

Description

Technical Field

[0001] This invention relates to a Bacillus subtilis strain that efficiently expresses keratinase and its application, particularly a Bacillus subtilis chassis strain constructed by optimizing the promoter and signal peptide of keratinase and its application, belonging to the fields of genetic engineering, enzyme engineering and food engineering technology. Background Technology

[0002] Cutinase is a carboxylic acid hydrolase (EC 3.1.1) belonging to the serine hydrolase class and the carboxylic acid hydrolase subfamily (Kawai F, Kawabata T, Oda M. Current state and perspectives related to the polyethylene terephthalate hydrolases available for biorecycling [J]. AcsSustainable Chemistry & Engineering, 2020, 8(24): 8894-8908.). This type of enzyme has a wide range of hydrolytic abilities and can act on the cuticle of plant leaves and fruits, as well as various esters and polyester compounds such as triglycerides and polyethylene terephthalate (PET).

[0003] Structurally, keratinase exhibits typical α / β hydrolase folding characteristics: its core consists of a β-sheet composed of five β chains, which alternate with five α helices to form a compact three-dimensional conformation. Its active site is composed of the classic catalytic triplet—Ser120, His188, and Asp175. Unlike conventional lipases, the serine catalytic site of keratinase is not deeply embedded in a hydrophobic pocket inside the molecule, but is exposed on the protein surface, allowing direct contact with the solvent. Therefore, keratinase does not possess the "interfacial activation" property (Martinez C, Degeus P, Lauwereys M, et al. Fusarium-solanicutinase is a lipolytic enzyme with a catalytic serine accessible to solvent[J]. Nature, 1992, 356(6370): 615-618).

[0004] In 2005, Müller et al. first reported a highly efficient hydrolase from the thermophilic actinomycete Thermobifida fusca, which can depolymerize aromatic polyester PET at a significant rate, exhibiting degradation performance superior to that of traditional lipases (Müller RJ, Schrader H, Profe J, et al. Enzymatic degradation of poly(ethyleneterephthalate): Rapid hydrolyse using a hydrolase from T-fusca [J]. Macromolecular Rapid Communications, 2005, 26(17): 1400-1405.). Subsequently, in 2008, Chen Sheng et al. first identified the coding gene for Tf Cutinase and revealed that its optimal reaction temperature is approximately 60°C, and it possesses good thermal stability. This marked the formal beginning of research on bacterial cutinases and laid the foundation for subsequent functional exploration and application development (Chen S, Tong X, Woodard RW, et al. Identification and characterization of bacterial cutinase [J]. Journal of Biological Chemistry, 2008, 283(38): 25854-25862). Since then, various bacteria have been found to possess cutinase activity, including *Streptomyces scabies*, *Pseudomonas putida*, and *Thermobifida fusca*.

[0005] It is worth noting that BhrPETase, a keratinase derived from the thermophilic strain bacterium HR29, has become one of the current research hotspots due to its excellent heat resistance and high efficiency in degrading PET under high temperature conditions (CuiY, Chen Y, Sun J, et al. Computational redesign of a hydrolase for nearlycomplete PET depolymerization at industrially relevant high-solids loading[J]. Nature communications, 2024, 15(1): 1417; Xingxiang X, Kefeng N, Helong H, et al. Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalatehydrolase from bacterium HR29 [J]. Enzyme and Microbial Technology, 2021,143: 109715).

[0006] In terms of expression system selection, *Bacillus subtilis* has become an ideal host for protein secretion expression due to its clear genetic background, well-defined metabolic pathways, non-pathogenicity, lack of significant codon bias, short fermentation cycle, and ease of achieving high-density cell culture. Furthermore, this strain possesses a strong extracellular protein secretion capacity, capable of directly releasing target proteins into the culture medium, making it widely used in the production of industrial enzymes. However, the efficient heterologous protein expression system of *Bacillus subtilis* still has certain limitations, currently only supporting the large-scale preparation of some industrial enzymes. Therefore, achieving efficient expression of keratinase in this system is of significant practical importance for promoting its industrial application.

[0007] Efficient expression of exogenous proteins, especially efficient extracellular secretion, in Bacillus subtilis is a key challenge in the field of industrial enzyme production. Optimizing promoters (which regulate transcription) and signal peptides (which guide protein transmembrane transport) through rational design or high-throughput screening are two core and effective strategies for improving the expression level of target enzymes.

[0008] The strength of a promoter directly determines the mRNA abundance of a target gene. Optimization strategies include using strong constitutive / inducible promoters, constructing tandem (dual) promoters, and rationally modifying the core sequence of the promoter. Many studies have significantly increased expression by replacing or combining strong promoters. For example, one systematic study constructed a promoter containing the inducible promoter P. aprE and P acoA The toolbox for improving secretory protein overproduction in Bacillus subtilis was compared and found to yield higher protein production under certain conditions (Kriger A, Welsch N, Durwald A, et al. A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis [J]. Applied Microbiology and Biotechnology, 2022, 106:5137-5151). Furthermore, site-directed mutations or replacement with consensus sequences in conserved regions such as -35 and -10 of the promoter significantly enhanced transcription efficiency. A classic study on the P... ylb The promoter was engineered, and by optimizing its -35, -10 regions and upstream sequence, the resulting engineered promoter NBP3510 had a transcriptional activity 340 times stronger than the wild type, resulting in a 26-fold and 195-fold increase in the expression levels of reporter proteins β-galactosidase and sfGFP, respectively (Zhou C, Ye B, Cheng S, et al. Promoterengineering enables overproduction of foreign proteins from a single copyexpression cassette in Bacillus subtilis [J]. Microbial Cell Factories, 2019,18:111).

[0009] The compatibility between the signal peptide and the target protein is crucial to secretion efficiency. The optimal signal peptide varies depending on the target protein and usually requires screening through a library. Constructing a signal peptide library for high-throughput screening is the most common strategy. For example, in expressing thermostable α-cyclodextrin glucosidase (α-CGTase), researchers screened the optimal signal peptide citH from a library of 173 signal peptides derived from Bacillus subtilis and further saturated its key amino acids, ultimately increasing the extracellular enzyme activity by 47.9% compared to the original signal peptide. Similarly, in expressing thermostable β-glucosidase (PfuBGL), CitHSP, screened from 173 signal peptides, increased enzyme secretion by 16-fold (Khadye V S., Sawant S Shaikh K, Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization [J]. Protein Expression and Purifcation, 2021,182:105843).

[0010] The applicant previously obtained BhrPETase, a keratinase that efficiently degrades PET. Subsequently, using molecular biology techniques and homologous recombination, the BhrPETase gene was constructed into the pHY300PLK vector, and its heterologous recombination expression in Bacillus subtilis was achieved. However, since the expression level of BhrPETase is still low, this invention aims to screen and optimize its expression elements to achieve efficient expression of BhrPETase in the Bacillus subtilis system. Summary of the Invention

[0011] Purpose of the invention: This invention addresses the problem of low expression levels of keratinase in Bacillus subtilis in existing technologies by providing a Bacillus subtilis strain that expresses keratinase efficiently and its applications.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: One of the technical solutions provided by this invention is a genetically engineered strain of Bacillus subtilis, wherein the engineered strain is based on Bacillus subtilis WS9CΔH, with P csn The signal peptides sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), or sp(csn) are obtained by overexpressing the keratinase BhrPETase.

[0013] Furthermore, the amino acid sequence of the keratinase BhrPETase is shown in SEQ ID No. 1.

[0014] Furthermore, the nucleotide sequence of the keratinase BhrPETase is shown in SEQ ID No. 2.

[0015] Furthermore, the nucleotide sequence of the csn promoter gene is shown in SEQ ID No. 3.

[0016] Furthermore, the nucleotide sequences of the sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), or sp(csn) signal peptide genes are shown in SEQ ID No.4-SEQ ID No.12.

[0017] Furthermore, the present invention replaces the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), or sp(csn) signal peptide gene.

[0018] Furthermore, the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain is replaced with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), or sp(TasA).

[0019] Furthermore, the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain is replaced with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), or sp(YwtF).

[0020] Furthermore, the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain is replaced with sp(ydjM), sp(amyE), or sp(PenP).

[0021] Furthermore, the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain is replaced with sp(ydjM).

[0022] Furthermore, the methods for overexpressing the keratinase BhrPETase using expression elements include, but are not limited to, methods such as constructing recombinant plasmids.

[0023] The second technical solution provided by this invention is the application of the above-mentioned Bacillus subtilis genetically engineered bacteria, especially its application in expressing the keratinase BhrPETase.

[0024] The beneficial effects of this invention are as follows: (1) This invention utilizes the csn promoter gene and signal peptide genes such as ydjM on the Bacillus subtilis genome to design novel expression elements, thereby obtaining a Bacillus subtilis expression system that can efficiently express the keratinase BhrPETase, laying the foundation for efficient protein expression and promoting the efficient expression and industrial production of the keratinase BhrPETase.

[0025] (2) This invention provides a Bacillus subtilis chassis strain capable of high production of BhrPETase. When carrying a BhrPETase expression cassette, after 60 h of shake-flask fermentation, the whole-cell BhrPETase activity in the fermentation broth reaches as high as 42.9 U·mL. -1 The extracellular enzyme activity reached 40.3 U·mL -1 . Attached Figure Description

[0026] Figure 1 Overexpression vector pHY300PLK(E)-P csn -SP ydjM - The construction process of BhrPETase.

[0027] Figure 2 Whole-cell enzyme activity of recombinant strains expressing the keratinase BhrPETase with different single promoters.

[0028] Figure 3 Whole-cell and extracellular enzyme activities of recombinant strains expressing BhrPETase with different signal peptides.

[0029] Figure 4 Electrophoresis images of proteins from recombinant strains (M: protein molecular weight standard; from left to right: fermentation supernatant, intracellular supernatant, and intracellular precipitate of WS9CΔH-P1SP1, WS9CΔH-P1SP2, and WS9CΔH-P1SP7). Detailed Implementation

[0030] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0031] According to a specific embodiment of the present invention, the construction method includes: in the starting strain Bacillus subtilis WS9CΔH, using promoter P... csn The signal peptide sp(aprE) was used to express the keratinase BhrPETase, and the WS9CΔH-P1 strain was constructed.

[0032] Furthermore, the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain was replaced with the sp(ydjM) signal peptide gene to obtain the recombinant strain WS9CΔH-P1SP1.

[0033] This invention provides a genetically engineered bacterium that expresses keratinase, obtained by expressing keratinase BhrPETase using the above-mentioned expression element in the above-mentioned Bacillus subtilis genetically engineered bacterium. The amino acid sequence of the keratinase BhrPETase is shown in SEQ ID No. 2, and the nucleotide sequence of the gene encoding the keratinase BhrPETase is shown in SEQ ID No. 1.

[0034] According to a preferred embodiment of the present invention, the promoter or signal peptide gene sequence can be cloned using conventional methods in the art. For example, an overexpression vector can be constructed by one-step cloning and chemically transformed into Bacillus subtilis WS9CΔH to achieve overexpression of the target gene. The overexpression vector includes, but is not limited to, the pHY300PLK(E) plasmid.

[0035] According to the present invention, the overexpression of keratinase BhrPETase can be carried out by conventional means in the art, for example, by chemically transferring an expression vector containing an expression cassette encoding the keratinase BhrPETase gene into the basal bacteria using conventional means in the art, wherein the expression vector includes, but is not limited to, the pHY300PLK(E) plasmid.

[0036] According to a more preferred embodiment of the present invention, the construction method includes the following steps: (1) Materials and reagents p-Nitrophenol butyrate (pNPB) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; antibiotics and lysozyme were purchased from Shanghai Sangon Biotech Co., Ltd.; agarose was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.; 2×Phanta Max Master Mix, 2×Phanta Flash Master Mix (Dye Plus), and One-Step PAGE GelFast Preparation Kit (12%) were purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid miniprep kit (DP103) and ordinary agarose gel DNA recovery kit (DP209) were purchased from Beijing Tiangen Biotech Co., Ltd.; corn steep liquor was purchased from Hubei Angel Yeast Co., Ltd.; soybean peptone was purchased from Shanghai Yuantai Biotechnology Co., Ltd.; other common reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Information on the strains and plasmids involved in this invention and its embodiments is shown in Table 1. The strain B. subtilis WS9C was first disclosed in the article: Luo H, Zhu X, Wu J, et al. Identification and optimization of genes potentially related to protein expression for enhancing α-amylase production in Bacillus subtilis [J]. Systems Microbiology and Biomanufacturing, 2024, 4:1121-1129.

[0037] Table 1. Strains and plasmids Information on the strains and plasmids involved in this invention and its embodiments is shown in Table 2.

[0038] Table 2 Primer Sequences (2) Plasmid extraction Take 5 mL of overnight cultured bacterial culture into a centrifuge tube, centrifuge at 12000 rpm for 1 min, remove the supernatant, and extract plasmids according to the instructions provided with the plasmid mini-prep kit.

[0039] (3) Amplification and ligation of target gene and vector fragment Plasmids were constructed using a one-step cloning method, which utilizes the principle of homologous recombination and is characterized by its simplicity, speed, and efficiency. The PCR amplification system is shown in Table 3.

[0040] Table 3 PCR amplification system The PCR reaction conditions are shown in Table 4.

[0041] Table 4 PCR reaction conditions *: The denaturation, annealing, and extension processes are repeated 25-35 times. Primers were designed according to the ClonExpress II One-Step Cloning method. After gel recovery of the PCR products, the target gene fragment and vector backbone were obtained and ligated according to the system in Table 2-7. The one-step cloning enzyme ligation system is shown in Table 5.

[0042] Table 5. Enzyme ligation system for one-step cloning Note: The specific amounts of DNA fragments and vector fragments added to the system need to be calculated based on the base counts of both. Refer to ClonExpress for details. ® The calculations were performed according to the instructions for the IIOne Step Cloning Kit, and the connection reaction conditions were determined to be 37°C for a precise reaction time of 30 min. (4) Agarose gel electrophoresis and gel recovery a) Preparation of nucleic acid gel. Weigh 1 g of agarose and add it to 100 mL of 1×TAE electrophoresis buffer (1% agarose gel solution), shake well, and heat in a microwave oven to dissolve. Once the agarose gel solution has cooled to 50-60℃, add 10 μL of GoldView gel. TM Nucleic acid dye (1 μL·10 mL) -1 Gently swirl and shake to mix well, pour into the glue-making tank, and remove the comb after it solidifies.

[0043] b) Electrophoresis. Place the prepared nucleic acid gel into the electrophoresis tank, and add 1×TAE electrophoresis buffer until the buffer just covers the gel surface. Mix the DNA sample and 10×Loading Buffer, add them to the sample wells, and simultaneously add an appropriate amount of DNA Marker to the left or right side. Perform electrophoresis at 80 V for 20-30 min, and record the results using a GelDoc-it gel imaging system.

[0044] c) Gel recovery. After electrophoresis, cut the correctly sized bands from the gel imaging system and weigh them. Then, strictly follow the instructions of the agarose gel DNA recovery kit to recover the PCR products.

[0045] (5) Colony PCR PCR was performed using transformant colonies as templates to verify whether the target gene was successfully ligated into the vector.

[0046] The PCR amplification system is the same as in Table 3. The PCR reaction conditions are the same as in Table 4.

[0047] (6) Preparation and transformation of B. subtilis WS9C and B. subtilis WS9CΔH competent cells a) Thaw the B. subtilis WS9C or B. subtilis WS9CΔH bacterial suspension stored at -80℃ in an ice box, then streak the bacterial suspension onto an LB antibiotic-free plate and incubate upside down at 37℃ for 8-10 h. b) Pick a single colony and inoculate it into 10 mL of LB medium, and incubate at 37 ℃ for about 8 h; c) Take 500 μL of bacterial culture and 250 μL of 400 g·L⁻¹ solution. -1 The xylose solution was transferred to 50 mL of LB medium and cultured for 4 h to induce the conk gene (Rahmer R, Heravi KM, Altenbuchner J. Construction of a super-competent Bacillus subtilis 168 using the pmt / a-comks inducible cassette [J]. Frontiers in Microbiology, 2015, 6: 1431-1442.). d) After the culture is prepared, place it on an ice bath for 20 min. Take 170 μL of the culture and mix it with 30 μL of 70% glycerol, then aliquot the mixture into 1 mL centrifuge tubes and store at -80℃.

[0048] e) Inject 10 μL of plasmid (about 1 μg) into competent cells (placed on ice), mix gently, and let stand in an ice bath for 20-30 min; f) Place the ice-baked bacterial mixture in a 37°C water bath for 20-30 min, then incubate at 200 rpm in a 37°C shaker for 2 h. g) Spread the bacterial culture onto a plate with plasmid-corresponding resistance and incubate upside down at 37°C for 10-12 h.

[0049] (7) Preparation of B. subtilis WS9CΔH strain a) Inject the constructed plasmid pHY300PLK(E)-PE194-N20-HrcA-cas9 into B. subtilisWS9C competent cells (placed on ice), mix gently, and incubate on ice for 20-30 min; b) Place the ice-baked bacterial mixture in a 37°C water bath for 20-30 min, then incubate at 200 rpm in a 37°C shaker for 2 h; spread the bacterial mixture onto a plate containing tetracycline (25 μg / mL) and incubate upside down at 37°C for 10-12 h.

[0050] c) Pick a single colony from the plate and perform colony PCR using primers hrcA-up-F and hrcA-down-R.

[0051] d) Verify the obtained PCR product by agarose gel electrophoresis.

[0052] e) Pick positive colonies with the target band at 2000bp and streak them onto LB plates without resistance. Incubate at 51℃ with the plates upside down for 8 h.

[0053] f) Randomly select single colonies and transfer them to antibiotic-free LB plates and plates containing tetracycline (25 μg / mL), respectively, and incubate them upside down at 37°C for 10-12 h.

[0054] g) Select single colonies that grew on antibiotic-free LB plates and did not grow on plates containing tetracycline (25 μg / mL) as B. subtilis WS9CΔH strain.

[0055] (8) Shake flask fermentation culture Seed culture: Take the glycerol tube containing the strain from the -80℃ freezer, add the bacterial culture to 10 mL LB liquid medium at an inoculation rate of 2‰ (v / v), and incubate at 37 ℃ and 200 rpm for 12 h.

[0056] Shake flask fermentation: Add seed culture to 50 mL TB liquid medium at an inoculum of 5% (v / v) and incubate at 37 ℃ and 200 rpm for 60 h.

[0057] (9) Determination of bacterial cell concentration Dilute the fermented culture medium with deionized water and pour it into a 1 cm thick colorless transparent cuvette. Adjust the wavelength of the UV spectrophotometer to 600 nm and measure the absorbance of the solution, i.e., OD. 600 Note that the OD value should fall between 0.2 and 0.8 (the linear range is more accurate).

[0058] (10) SDS-polyacrylamide gel electrophoresis a) Preparation of protein gel Prepare the SDS-PAGE gel according to the instructions of the kit.

[0059] b) Sample pretreatment. Mix 5 μL of fermentation supernatant with 20 μL of 5× Loading Buffer and heat at 95℃ for 10–15 min.

[0060] c) Sample loading and electrophoresis. Place the solidified gel plate into the electrophoresis tank, adding electrophoresis buffer to both the inner and outer tanks (the liquid level in the inner tank should be higher than the gel). Then, use a pipette to add the sample and protein marker to the wells, avoiding air bubbles. Initially, use a constant voltage of 80 V for electrophoresis. Once the bromophenol blue leaves the stacking gel and enters the separating gel, switch to a constant voltage of 120 V. After approximately 1-1.5 hours, the bromophenol blue will have moved to the bottom of the gel, indicating the end of electrophoresis. Finally, use a multi-functional gel imaging system to take photos and record the results.

[0061] (11) Assay of keratinase esterase activity Esterase activity assay: First, accurately weigh 0.0139 g of p-nitrophenol butyrate (pNPB) and use 100 mmol·L⁻¹ esterase activity assay. -1 Dilute the pH 8.0 phosphate buffer to 1 L to prepare a 100 μmol·L⁻¹ solution. -1 The p-nitrophenol stock solution. Then, 1.5 mL of 100 mmol·L⁻¹... -1 Potassium phosphate buffer (preheated to 60°C for 10 min) was added to a 0.5 cm glass cuvette, and the sample was zeroed at 405 nm. 1.44 mL of the same buffer was accurately pipetted, followed by 30 μL of the diluted sample and 30 μL of 50 mmol·L⁻¹ potassium phosphate buffer. -1The pNPB solution was rapidly mixed until homogeneous. The mixture was immediately placed in a spectrophotometer, and the reaction kinetics were monitored at 405 nm for 1 minute, with absorbance values ​​recorded every 5 seconds. Esterase activity was defined as the amount of enzyme required to catalyze the hydrolysis of pNPB to produce 1 μmol of p-nitrophenol per minute at 60°C; this is one unit of enzyme activity (U).

[0062] Example 1: Preparation of the starting strain Bacillus subtilis WS9CΔH First, since hrcA is a chaperone protein inhibitor, its knockout is beneficial to the expression and folding of the keratinase BhrPETase. In this example, the starting strain was Bacillus subtilis WS9CΔH, and the preparation method is as follows: Bacillus subtilis WS9C was obtained from our laboratory. The hrcA-up fragment was obtained using conventional PCR with primers hrcA-up-F and hrcA-up-R; the hrcA-down fragment was obtained using primers hrcA-down-F and hrcA-down-R; the Cas9 fragment was obtained using primers Cas9-F and Cas9-R; the pHY ligation fragment 1 was obtained using primers pHY-cas9-F and pHY-PE194-R; the replicon PE194 was obtained using primers PE194-F and PE194-R; the N20 sequence of hrcA was obtained using primers N20-F and N20-R; and the pHY ligation fragment 2 was obtained using primers pHY-F and pHY-R. The obtained fragments were ligated using a one-step cloning enzyme method to obtain the target plasmid pHY300PLK(E)-PE194-N20-HrcA-cas9. The constructed plasmid pHY300PLK(E)-PE194-N20-HrcA-cas9 was transformed into WS9C competent cells, and a positive hrcA knockout strain was obtained by colony PCR. The knockout strain was streaked and cultured at high temperature to eliminate the plasmid pHY300PLK(E)-PE194-N20-HrcA-cas9, thus obtaining the target strain Bacillus subtilis WS9CΔH.

[0063] Example 2: To investigate the effects of different promoters on the activity of the keratinase BhrPETase expressed by the strain, this example uses the starting strain B. subtilis WS9CΔH and different promoters to perform BhrPETase expression. csn P amyQ P sunA P nprE P spovG P HpaII P amyE P gapAP gsiB P dhaS P srf P GroESL The signal peptide sp(aprE) was used to express the keratinase BhrPETase, and the following strains of *B. truncatum* were constructed: WS9CΔH-P1, WS9CΔH-P2, WS9CΔH-P3, WS9CΔH-P4, WS9CΔH-P5, WS9CΔH-P6, WS9CΔH-P7, WS9CΔH-P8, WS9CΔH-P9, WS9CΔH-P10, WS9CΔH-P11, or WS9CΔH-P12. The aim was to investigate the effect of different signal peptides on the activity of the keratinase BhrPETase expressed by these strains. The specific methods are as follows: In this embodiment of the invention, a genetically engineered Bacillus subtilis strain was prepared. This engineered strain specifically targets the P gene on the Bacillus subtilis WS9CΔH genome. csn P amyQ P sunA P nprE P spovG P HpaII P amyE P gapA P gsiB P dhaS P srf and P GroESL The promoter gene was cloned to obtain the P. csn P amyQ P sunA P nprE P spovG P HpaII P amyE P gapA P gsiB P dhaS P srf and P GroESL The nucleotide sequences of the promoter genes are shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, respectively.

[0064] The vector fragment was obtained by conventional PCR using primers pHY-RBS-F and pHY-RBS-R. The target sequence of the obtained promoter was then ligated to the obtained vector fragment using a one-step cloning enzymatic method to obtain the target plasmid. This plasmid was then transformed into WS9CΔH competent cells to obtain recombinant strains WS9CΔH-P1, WS9CΔH-P2, WS9CΔH-P3, WS9CΔH-P4, WS9CΔH-P5, WS9CΔH-P6, WS9CΔH-P7, WS9CΔH-P8, WS9CΔH-P9, WS9CΔH-P10, WS9CΔH-P11, or WS9CΔH-P12.

[0065] The obtained recombinant strains WS9CΔH-P1, WS9CΔH-P2, WS9CΔH-P3, WS9CΔH-P4, WS9CΔH-P5, WS9CΔH-P6, WS9CΔH-P7, WS9CΔH-P8, WS9CΔH-P9, WS9CΔH-P10, WS9CΔH-P11, or WS9CΔH-P12 were cultured in shake flasks at 37°C, and the activity of keratinase BhrPETase was measured after 60 h of fermentation.

[0066] Figure 2 The whole-cell enzyme activities of recombinant strains expressing the keratinase BhrPETase were shown. The results indicated that, at 37°C, using aprE as the signal peptide, the whole-cell enzyme activities of the twelve recombinant strains were 18.6 U·mL⁻¹. -1 11.3 U·mL -1 11.2 U·mL -1 10.8 U·mL -1 10.6 U·mL -1 10.2 U·mL -1 9.5 U·mL -1 8.3 U·mL -1 7.6 U·mL -1 7.5 U·mL -1 5.3 U·mL -1 and 3.8 U·mL -1 The results showed that intracellular enzyme activity was relatively low (all below 2.3 U·mL). -1 The measured extracellular enzyme activity was almost equal to the whole-cell enzyme activity, so the extracellular enzyme activity was not measured again in subsequent experiments in this embodiment. The results show that the whole-cell enzyme activity of strain WS9CΔH-P1 was significantly higher than that of other groups, indicating that when aprE is used as the signal peptide, selecting P... csn The promoter is pHY300PLK(E)-P, i.e., the plasmid is pHY300PLK(E)-P. csn-sp(aprE)- BhrPETase can effectively enhance the activity of the keratinase BhrPETase.

[0067] Example 3: P obtained in Example 2 csn Based on the optimal promoter, and using strains with the PamyQ promoter as a control group, this example further modifies sp(aprE) from the WS9CΔH-P1 strain. The signal peptide genes were replaced with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), sp(csn), sp(Epr), and sp(Yncm) signal peptide genes, resulting in recombinant strains WS9CΔH-P1SP1, WS9CΔH-P1SP2, WS9CΔH-P1SP3, WS9CΔH-P1SP4, WS9CΔH-P1SP5, WS9CΔH-P1SP6, WS9CΔH-P1SP7, WS9CΔH-P1SP8, WS9CΔH-P1SP9, WS9CΔH-P1SP10, and WS9CΔH-P1SP11, respectively. The aim was to investigate the effect of different signal peptides on the activity of the keratinase BhrPETase expressed in these strains. The specific methods are as follows: The genetically engineered bacteria expressing keratinase in this embodiment of the invention were prepared by amplifying the sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), sp(csn), sp(Epr), sp(Yncm), and sp(aprE) signal peptide genes on the Bacillus subtilis WS9CΔH genome using a signal peptide library kit. The nucleotide sequences of the sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), sp(csn), sp(Epr), sp(Yncm), and sp(aprE) signal peptide genes are shown in SEQ ID No. 4, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, and SEQ ID No. 10, respectively. No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25 and SEQ ID No. 26 are shown.

[0068] The vector fragment was obtained by conventional PCR using primers pHY-SP-F and pHY-SP-R. The target sequence of the obtained promoter was then ligated to the obtained vector fragment using a one-step cloning enzymatic method to obtain the target plasmid, which was then transformed into WS9CΔH competent cells to obtain recombinant strains WS9CΔH-P1SP1, WS9CΔH-P1SP2, WS9CΔH-P1SP3, WS9CΔH-P1SP4, WS9CΔH-P1SP5, WS9CΔH-P1SP6, WS9CΔH-P1SP7, WS9CΔH-P1SP8, WS9CΔH-P1SP9, WS9CΔH-P1SP10, and WS9CΔH-P1SP11.

[0069] The obtained recombinant strains WS9CΔH-P1SP1, WS9CΔH-P1SP2, WS9CΔH-P1SP3, WS9CΔH-P1SP4, WS9CΔH-P1SP5, WS9CΔH-P1SP6, WS9CΔH-P1SP7, WS9CΔH-P1SP8, WS9CΔH-P1SP9, WS9CΔH-P1SP10 and WS9CΔH-P1SP11 were cultured in shake flasks at 37℃, and the activity of keratinase BhrPETase was measured after 60 h of fermentation.

[0070] Figure 3 The whole-cell and extracellular enzyme activities of the recombinant strain expressing the keratinase BhrPETase are shown. The results indicate that at 37°C, with P csn Using this promoter, the whole-cell enzyme activities of the eleven recombinant strains were 44.8 U·mL⁻¹. -1 43.9 U·mL -1 39.3 U·mL -1 33.9 U·mL -1 45.1 U·mL -1 30.3 U·mL -1 34.6 U·mL -1 24.2 U·mL -1 34.1 U·mL -1 53.6 U·mL -1 and 6.8 U·mL -1 It can be seen that, in the recombinant strains obtained after replacing the signal peptide sp(aprE), only the enzyme activities of the sp(Epr) and sp(Yncm) signal peptide gene groups were reduced. The whole-cell enzyme activities of other recombinant strains were significantly increased. Among them, the strain with the highest extracellular enzyme activity was WS9CΔH-P1SP1, reaching 40.3 U·mL. -1 The rest are WS9CΔH-P1SP2 (32.6 U·mL) in descending order. -1), WS9CΔH-P1SP3 (30.1 U·mL -1 ), WS9CΔH-P1SP4 (27.8 U·mL -1 ), WS9CΔH-P1SP5 (20.2 U·mL -1 ), WS9CΔH-P1SP6 (18.5 U·mL -1 ), WS9CΔH-P1SP7 (24.8 U·mL -1 ), WS9CΔH-P1SP8 (21.5 U·mL -1 ), WS9CΔH-P1SP9 (16.1 U·mL -1 Therefore, with P csn When the promoter is used, the optimal signal peptide genes are sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), and sp(csn).

[0071] in conclusion: In summary, this invention, through optimizing the expression element of the keratinase BhrPETase in Bacillus subtilis, obtained the optimal recombinant strain WS9CΔH-P1SP1, whose promoter is P. csn The signal peptide is SP(ydjM), and its whole-cell enzyme activity reaches 44.8 U·mL. -1 The extracellular enzyme activity reached 40.3 U·mL -1 The next two are: The recombinant strain WS9CΔH-P1SP2 has a promoter of P. csn The signal peptide is sp(amyE), and its whole-cell enzyme activity reaches 35.8 U·mL. -1 The extracellular enzyme activity reached 32.6 U·mL -1 .

[0072] The recombinant strain WS9CΔH-P1SP3 has a promoter of P. csn The signal peptide is sp(PenP), and its whole-cell enzyme activity reaches 41.4 U·mL. -1 The extracellular enzyme activity reached 30.1 U·mL -1 .

[0073] The recombinant strain WS9CΔH-P1SP4 has a promoter of P. csn The signal peptide is sp(AbnA), and its whole-cell enzyme activity reaches 33.9 U·mL. -1 The extracellular enzyme activity reached 27.8 U·mL -1 .

[0074] The recombinant strain WS9CΔH-P1SP5 has a promoter of P. csn The signal peptide is sp(YwtF), and its whole-cell enzyme activity reaches 33.6 U·mL. -1 The extracellular enzyme activity reached 20.2 U·mL -1 .

[0075] The recombinant strain WS9CΔH-P1SP6 has a P promoter. csn The signal peptide is sp(YhcR), and its whole-cell enzyme activity reaches 24.1 U·mL. -1 The extracellular enzyme activity reached 18.5 U·mL -1 .

[0076] The recombinant strain WS9CΔH-P1SP7 has a promoter of P. csn The signal peptide is sp(TasA), and its whole-cell enzyme activity reaches 45.8 U·mL. -1 The extracellular enzyme activity reached 24.8 U·mL -1 .

[0077] The recombinant strain WS9CΔH-P1SP8 has a promoter of P. csn The signal peptide is sp(YqgA), and its whole-cell enzyme activity reaches 30.7 U·mL. -1 The extracellular enzyme activity reached 21.5 U·mL -1 .

[0078] The recombinant strain WS9CΔH-P1SP9 has a promoter of P. csn The signal peptide is sp(csn), and its whole-cell enzyme activity reaches 33.9 U·mL. -1 The extracellular enzyme activity reached 16.1 U·mL -1 .

[0079] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A genetically engineered Bacillus subtilis strain that efficiently expresses keratinase, characterized in that, The genetically engineered bacteria are derived from Bacillus subtilis WS9CΔH, by replacing the sp(aprE) signal peptide gene in the WS9CΔH-P1 strain with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), or sp(csn) signal peptide genes to obtain the chassis bacteria, using P csn The promoter is used to express keratinase in the chassis bacteria.

2. The genetically engineered bacterium according to claim 1, characterized in that, The keratinase is BhrPETase, and its amino acid sequence is shown in SEQ ID No.

1.

3. The genetically engineered bacterium according to claim 1, characterized in that, The promoter P csn The nucleotide sequence is shown in SEQ ID No.

3.

4. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequences of the sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), sp(TasA), sp(YqgA), or sp(csn) signal peptide genes are shown in SEQ ID No.4-SEQ ID No.12, respectively.

5. The genetically engineered bacterium according to claim 1, characterized in that, The sp(aprE) signal peptide gene in the WS9CΔH-P1 strain is replaced with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), sp(YwtF), sp(YhcR), or sp(TasA).

6. The genetically engineered bacterium according to claim 5, characterized in that, The sp(aprE) signal peptide gene in the WS9CΔH-P1 strain was replaced with sp(ydjM), sp(amyE), sp(PenP), sp(AbnA), or sp(YwtF).

7. The genetically engineered bacterium according to claim 6, characterized in that, The sp(aprE) signal peptide gene in the WS9CΔH-P1 strain was replaced with sp(ydjM), sp(amyE), or sp(PenP).

8. The genetically engineered bacteria according to claim 7, characterized in that, The sp(aprE) signal peptide gene in the WS9CΔH-P1 strain was replaced with sp(ydjM).

9. The application of the genetically engineered bacteria expressing keratinase according to any one of claims 1-8, characterized in that, It is used in the production of keratinase.

10. The application according to claim 9, characterized in that, The keratinase includes BhrPETase.

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