Chassis bacterium capable of efficiently expressing heterologous protease and application of chassis bacterium

Abstract

The invention belongs to the field of gene engineering and enzyme engineering, and relates to breeding of industrial microorganisms, in particular to a chassis strain for producing protease as well as a construction method and application of the chassis strain. According to the invention, point mutation is carried out on a coding gene rsbX of deoxyribonucleic acid regulatory protein on a bacillus amyloliquefaciens genome, so that a chassis strain capable of realizing efficient heterologous expression of protease (such as aminopeptidase and alkaline protease) is obtained, and the capability of knowing the production of heterologous protein of bacillus amyloliquefaciens is improved; meanwhile, high-efficiency extracellular expression of aminopeptidase and alkaline protease in bacillus amyloliquefaciens is realized, and the method has important significance on industrial production of the bacillus amyloliquefaciens.

Description

Technical Field: The present invention belongs to the fields of genetic engineering and enzyme engineering, and relates to the breeding of industrial microorganisms, particularly to a chassis strain for producing protease, its construction method and application. Background Art: The σB factor (Sigma B) in Bacillus Bacillus is an alternative σ factor that is mainly responsible for regulating the stress response of cells under environmental stress. The σB factor binds to RNA polymerase to initiate the transcription of stress-related genes, helping the cells to cope with adverse conditions such as heat shock, oxidative stress, and osmotic changes. Its activity is regulated by proteins such as the anti-σ factor RsbW and the anti-σ factor RsbV. The σB factor plays an important role in enhancing the environmental adaptability of cells, spore formation, and the virulence regulation of certain pathogenic bacteria, and is a key factor in studying the stress mechanism of bacteria.

[0003] rsbX The gene encodes a sigmaB regulatory factor (one of the seven Rsb proteins), which is co-transcribed with sigmaB and negatively regulates the stress-dependent activation pathway. RsbX is essential for sigmaB-mediated stress adaptation. It can not only turn off the stress response after the stress of the strain, but also inhibit the activity of sigmaB under non-stress conditions, reducing unnecessary gene expression, saving the metabolic flux and energy in the bacterial cell. Strains lacking RsbX show impaired motility and biofilm formation, and also have increased survival rate at low pH values.

[0004] Alkaline protease ( Alkaline protease ) hydrolyzes peptide bonds, amide bonds, and ester bonds in the range of slightly alkaline pH values, and has the functions of transesterification and transpeptidation. Alkaline protease has a wide range of uses in industries such as food, washing, and leather making. Since microbial proteases are all extracellular enzymes, compared with animal and plant-derived proteases, they have the advantages of relatively simple downstream technology processing, low price, wide source, easy cultivation of bacteria, high yield, etc. They have all the characteristics of animal and plant proteases, and have stronger hydrolysis ability and alkali resistance compared with neutral proteases, have relatively high heat resistance and certain esterase activity, and are easy to realize industrial production.

[0005] Aminopeptidase ( Aminopeptidase ) as an exopeptidase can specifically recognize the amino acid residues at the N-terminus of proteins and polypeptide chains and hydrolyze them one by one. It can cooperate with alkaline protease, carboxypeptidase, etc. to effectively reduce the bitterness in the protein hydrolysate and prepare a variety of bioactive peptides, which has broad application prospects in industries such as food and medicine. However, the current yield of aminopeptidase is relatively low. Therefore, improving the expression level of aminopeptidase is of great value for improving the application effect of aminopeptidase and broadening its application fields.

[0006] Keratinase ( keratinase) is a protease that can specifically degrade keratin. Keratinase can be produced by a variety of microorganisms, can specifically degrade keratin, and has broad application prospects in industries such as feed, leather, medicine, food, and environmental governance. However, the current yield of keratinase is relatively low. Therefore, improving its expression level is of great value for further enhancing its application effect and expanding its application fields.

[0007] As a type of aerobic, spore-forming Gram-positive bacterium, Bacillus amyloliquefaciens has the characteristics of a clear genetic background, fast growth rate, strong adaptability, non-pathogenicity, strong ability to secrete proteins, and relatively easy transfer of recombinant DNA. Moreover, it has a good fermentation basis and production technology, and has been recognized as a safe production strain by the US Food and Drug Administration (FDA), and can be used as an important heterologous protein production host.

[0008] During the production of heterologous proteins, maintaining the activity of the production strain is the basis for ensuring high yields of heterologous proteins. However, during the fermentation process, due to factors such as nutrient deficiency, growth environment stress, accumulation of secondary metabolites and product enzymes, and limitations of natural cell growth, the stress response of the strain is activated, leading to cell death. Bacterial death affects the production of heterologous proteins and greatly limits the ability to continuously express foreign proteins. Summary of the Invention: In view of the above problems, the purpose of the present invention is to provide a Bacillus amyloliquefaciens chassis strain that is beneficial to the heterologous production of protease by modifying the genetic engineering host.

[0010] To achieve the above purpose, the present invention adopts the following technical solutions: One of the technical solutions provided by the present invention is a deoxyribonucleic acid regulatory protein RsbX mutant, which is obtained by introducing a T165I mutation on the basis of the wild-type RsbX shown in SEQ ID NO.1; Furthermore, the amino acid sequence of the RsbX mutant is as shown in SEQ ID NO.3; Even further, the coding gene of the RsbX mutant rsbX * , and its nucleotide sequence is as shown in SEQ ID NO.4.

[0011] Another technical solution provided by the present invention is the application of the RsbX mutant described in Technical Solution 1, especially its application in the highly efficient expression of heterologous protease.

[0012] A further technical solution provided by the present invention is a Bacillus amyloliquefaciens genetic engineering chassis bacterium, in which the deoxyribonucleic acid regulatory protein RsbX expressed by the chassis bacterium has a T165I mutation; Furthermore, the deoxyribonucleic acid regulatory protein coding gene on the chassis bacterium genome is as shown in SEQ ID NO. 4; Furthermore, the chassis bacterium does not express the following genes on the Bacillus amyloliquefaciens host genome: six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr , extracellular polysaccharide gene cluster eps , polyglutamic acid gene cluster pgs , and sacB genes.

[0013] The fourth technical solution provided by the present invention is the application of the chassis bacterium described in the third technical solution in the expression of heterologous proteins; Furthermore, the heterologous proteins include, but are not limited to: alkaline protease, aminopeptidase, keratinase, etc.

[0014] The fifth technical solution provided by the present invention is a genetically engineered bacterium for producing alkaline protease, which is obtained by heterologous overexpression of alkaline protease on the basis of the genetically engineered chassis bacterium described in the third technical solution; Furthermore, the coding gene of the alkaline protease is as shown in SEQ ID NO. 5.

[0015] The sixth technical solution provided by the present invention is a genetically engineered bacterium for producing aminopeptidase, which is obtained by heterologous overexpression of aminopeptidase on the basis of the genetically engineered chassis bacterium described in the third technical solution; Furthermore, the coding gene of the aminopeptidase is as shown in SEQ ID NO. 6.

[0016] The seventh technical solution provided by the present invention is a genetically engineered bacterium for producing keratinase, which is obtained by heterologous overexpression of keratinase on the basis of the genetically engineered chassis bacterium described in the third technical solution; Furthermore, the coding gene of the aminopeptidase is as shown in SEQ ID NO. 7.

[0017] The eighth technical solution provided by the present invention is the application of the genetically engineered bacterium described in the fifth to seventh technical solutions above, especially the application in alkaline protease, aminopeptidase, and keratinase respectively.

[0018] In the eighth aspect, the present invention provides a method for producing alkaline protease, aminopeptidase, and keratinase, the method comprising: culturing the genetically engineered bacterium described in the fifth to seventh technical solutions in a medium to produce protease; and collecting the protease from the genetically engineered bacterium and / or the medium. Wherein, the protease is selected from alkaline protease, aminopeptidase, and keratinase.

[0019] The beneficial effects of the present invention are as follows: In previous studies, the growth vitality of Bacillus amyloliquefaciens obtained (see Patent CN116218750A) decreased in the late stage of fermentation, the number of viable bacteria decreased, and the ability to produce proteins weakened. Therefore, it is necessary to transform the strain to improve the ability to produce heterologous proteins. In the present invention, by performing point mutations on the coding gene of the deoxyribonucleic acid regulatory protein on the genome of Bacillus amyloliquefaciens rsbX a chassis strain capable of highly heterologously expressing proteases (such as aminopeptidase and alkaline protease) was obtained, which improved the ability of Bacillus amyloliquefaciens to produce heterologous proteins. At the same time, the high-efficiency expression of aminopeptidase and alkaline protease outside the cells of Bacillus amyloliquefaciens was achieved, which is of great significance for its industrial production.

[0020] The present invention provides a Bacillus amyloliquefaciens chassis strain capable of highly producing aminopeptidase, alkaline protease, and keratinase. When carrying the expression cassettes of aminopeptidase, alkaline protease, and keratinase, flask fermentation was carried out separately. The activity of recombinant alkaline protease in the fermentation supernatant was 109% of the expression activity of the control strain, the activity of recombinant aminopeptidase was 138% of the expression activity of the control strain, and the activity of recombinant keratinase was 122% of the expression activity of the control strain. Description of the drawings: Figure 1 For the construction process of the temperature-sensitive point mutation plasmid pWH-T2- rsbX * in Example 1.

[0022] Figure 2 For the single-exchange verification of gene point mutation in Example 1; wherein, M: maker; 1: Δ acfi - rsbX * .

[0023] Figure 3 For the rsbX gene point mutation verification in Example 1; wherein, M: maker; 1: Δ acfi - rsbX * .

[0024] Figure 4 For the determination of the growth conditions of the genetically engineered bacteria Δ acfi - rsbX * and Δ acfi in Example 1. Detailed implementation manners: The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all well-known methods to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples without departing from the essence and scope of the present invention also fall within the protection scope of the present invention.

[0026] The present invention provides a deoxyribonucleic acid regulatory protein RsbX mutant, which is obtained by introducing a T165I mutation on the basis of the wild-type RsbX shown in SEQ ID NO.1; the amino acid sequence of the RsbX mutant is as shown in SEQ ID NO.3; the coding gene of the RsbX mutant rsbX * , and the nucleotide sequence is as shown in SEQ ID NO.4.

[0027] Among them, the wild-type RsbX is from Bacillus amyloliquefaciens, and the amino acid sequence is as follows: MIQVEENEHVQTLVYQLNKEGKSICGDSFFIKANEEELVCAVADGLGSGSLANESSSAIKDIVKTYADEDVESIIERCNQAMRNKRGATASILKFNFTKRELTYCSIGNVRFFLHSPSGEVFHPLPISGYLSGKPQKYKTYTSSYEKGATFIIYTDGLEVPHIRTCLKQGHSIEVISKSLHPYTTSRRDDLTYILGQLLS*.

[0028] Among them, the RsbX mutant (T165I), the amino acid sequence is as follows: MIQVEENEHVQTLVYQLNKEGKSICGDSFFIKANEEELVCAVADGLGSGSLANESSSAIKDIVKTYADEDVESIIERCNQAMRNKRGATASILKFNFTKRELTYCSIGNVRFFLHSPSGEVFHPLPISGYLSGKPQKYKTYTSSYEKGATFIIYTDGLEVPHIRICLKQGHSIEVISKSLHPYTTSRRDDLTYILGQLLS*.

[0029] The present invention provides a Bacillus amyloliquefaciens genetic engineering chassis bacterium carrying the encoding gene of the above-mentioned deoxyribonucleic acid regulatory protein RsbX mutant. According to the present invention, the above gene can be carried by conventional means in the art. For example, the gene can be point-mutated by conventional means in the art.

[0030] According to a preferred embodiment of the present invention, a point mutation is introduced into the deoxyribonucleic acid regulatory protein encoding gene of the Bacillus amyloliquefaciens. rsbX The point mutation can be carried out by conventional means in the art. For example, by means of homologous recombination, a point mutation vector is constructed and electrotransformed into Bacillus amyloliquefaciens, and the above gene is point-mutated from the genome through single and double exchanges. Preferably, the point mutation vector is the pWH-T2 plasmid containing the Kana resistance gene.

[0031] According to a preferred embodiment of the present invention, the starting strain of the genetically engineered bacterium is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) on the basis of CGMCC No. 11218 without expressing the following genes on the genome: six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr , extracellular polysaccharide gene cluster eps , polyglutamic acid gene cluster pgs , and sac gene B (the construction and acquisition information of this strain has been disclosed in patent application CN116218750A).

[0032] According to the present invention, the above-mentioned Bacillus amyloliquefaciens genetic engineering bacterium can be used as a chassis strain for heterologous protein production.

[0033] The present invention provides a genetically engineered bacterium for producing aminopeptidase, which is based on the above-mentioned Bacillus amyloliquefaciens genetic engineering bacterium chassis and heterologously overexpresses the aminopeptidase gene ywaD (GenBank: QJR48409.1).

[0034] The present invention provides a genetically engineered bacterium for producing alkaline protease, which is based on the above-mentioned Bacillus amyloliquefaciens genetic engineering bacterium chassis and heterologously overexpresses the alkaline protease gene aprE (GenBank: ACR24262.1).

[0035] The present invention provides a genetically engineered bacterium for producing keratinase, which is based on the above-mentioned Bacillus amyloliquefaciens genetic engineering bacterium chassis and heterologously overexpresses the keratinase gene kerK (GenBank: AAS86761.1).

[0036] According to the present invention, the heterologous overexpression of the above genes can be carried out by conventional means in the art. For example, the gene is made by conventional means in the artywaD , aprE or kerK The expression cassette is inserted into the genome of Bacillus amyloliquefaciens, or an expression vector containing the above gene expression cassette is transferred into Bacillus amyloliquefaciens by conventional means in the art.

[0037] According to the present invention, the overexpression means that the amount of the gene expression product is significantly higher than the original level.

[0038] The present invention provides a method for producing protease using the above-mentioned genetically engineered bacterium, including: culturing the genetically engineered bacterium in a medium to produce protease; and collecting the protease from the genetically engineered bacterium and / or the medium. Among them, the protease is selected from aminopeptidase, alkaline protease or keratinase.

[0039] The present invention provides a method for constructing the above-mentioned genetically engineered bacterium, including: in Bacillus amyloliquefaciens, point-mutating the deoxyribonucleic acid regulatory protein rsbX gene, and optionally introducing an alkaline protease gene aprE , an aminopeptidase gene ywaD or a protease gene kerK into Bacillus amyloliquefaciens and overexpressing it.

[0040] According to a preferred embodiment of the present invention, the construction method further includes the following steps: (1) Knock out and point-mutate the target gene to obtain a chassis strain The method for knocking out and point-mutating the target gene to obtain a chassis strain refers to the construction process of Bacillus amyloliquefaciens obtained in previous studies (see Patent CN116218750A). It should be noted that the order of the above gene knockouts does not affect the achievement of the object of the present invention, as long as the result of all the above target genes being knocked out can be finally achieved. The target gene is the deoxyribonucleic acid regulatory protein coding gene rsbX The point mutation is carried out according to the following steps: 1) Obtain the upstream and downstream homologous sequences of the target gene by PCR amplification; 2) Obtain a linear point mutation plasmid vector by double digestion and agarose nucleic acid gel electrophoresis; 3) Connect the homologous sequences and the linear vector by seamless cloning technology to obtain a genomic point mutation plasmid; 4) Induce methylation modification of the point mutation plasmid by methylation; 5) Electrotransform the methylated modified point mutation plasmid into the competent cells of Bacillus amyloliquefaciens; 6) Screen out the point mutation strain by single and double exchanges and verify the sequence by sequencing to obtain the chassis strain; 7) The growth performance was determined by shake flask culture.

[0041] The specific operation methods of the above steps can all be realized according to technical manuals, textbooks or literature reports that are easily accessible to those skilled in the art.

[0042] (2) Heterologous overexpression of protease genes The recombinant plasmids carrying the alkaline protease gene aprE , aminopeptidase gene ywaD or protease gene kerK expression cassettes were respectively electrotransformed into the above knockout strains and genome point mutation strains, and control bacteria were set up to obtain alkaline protease-producing bacteria, aminopeptidase-producing bacteria, and keratinase-producing bacteria respectively. The above strains produced alkaline protease, aminopeptidase, and keratinase respectively through shake flask culture.

[0043] According to a preferred embodiment of the present invention, the protease gene ywaD , aprE or kerK were transferred into the strains through the recombinant plasmids Ply-2-SPamyE- ywaD -pWB980, Ply-2-SPamyE- aprE -pWB980, Ply-2 -SPamyE- kerK -pWB980.

[0044] Unless otherwise specified, in the following examples: Seed medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and the rest is water; Fermentation medium: 64 g / L corn starch, 40 g / L soybean cake powder, 4 g / L disodium hydrogen phosphate, 0.3 g / L potassium dihydrogen phosphate, 0.7 g / L thermostable amylase.

[0045] Medium for preparing competent cells of Bacillus amyloliquefaciens: LBS medium: 5 g / L yeast extract, 10 g / L peptone, 5 g / L sodium chloride, 9.1085 g / L sorbitol; Recovery medium: 5 g / L yeast extract, 10 g / L peptone, 5 g / L sodium chloride, 9.1085 g / L sorbitol, 6.92246 g / L mannitol.

[0046] The method for determining the enzyme activity of aminopeptidase refers to the LNA method, that is, 1 enzyme activity unit (U / mL) is defined as the amount of enzyme required for 1 mL of enzyme solution to hydrolyze leucine p-nitroanilide to produce 1 μg of p-nitroaniline in 1 min at 60 °C and pH 9. Each sample was set with three replicates, and the results were averaged.

[0047] The alkaline protease activity determination method was carried out according to the Folin phenol method in Appendix B of GB / T 23527-2009, that is, 1 enzyme activity unit (U / mL) was defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine in 1 mL of enzyme solution at 40°C and pH 10.5 for 1 min. Each sample was repeated three times and the results were averaged.

[0048] The method for determining the activity of keratinase is that one unit of enzyme activity (U / mL) is defined as the amount of enzyme required to hydrolyze keratin to produce 1 μg of tyrosine in 1 mL of enzyme solution at 40°C and pH 10 for 1 min. Each sample was repeated three times and the results were averaged.

[0049] The plasmids involved in the following examples are shown in Table 1: Table 1: Plasmids

[0050] Note: pWH-T2 and pWB980 are commercial plasmids.

[0051] The primers involved in the following examples are shown in Table 2: Table 2: Primers

[0052] The present invention will be described in more detail below through specific examples.

[0053] Example 1: Construction of Bacillus amyloliquefaciens chassis strain In this example, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Based on CGMCC No. 11218, six extracellular protease genes were knocked out aprE, bpr, vpr, mpr, nprE, epr , exopolysaccharide gene cluster eps , polyglutamate gene cluster pgs ,and sacB The strain obtained by the gene is the host (the strain and its construction method have been disclosed in the patent application CN116218750A specification 0094-0116 paragraphs, and is named Δ acfi strains), and then proceed rsbX Gene point mutation, the steps are as follows: (1) Amplify the homologous sequence of the target gene Bacillus amyloliquefaciens rsbX Gene sequence (SEQ ID NO: 2) using primers rsbX -up-F / R and primers rsbX -down-F / R was obtained by PCR amplification rsbX The homology arm sequences at both ends of the gene sequence are up and down. The homology arm amplification primers are shown in Table 2.acfi Using the genome as a template, the amplification reaction system is as follows:

[0054] The amplification program is set as follows: pre-denaturation: 95°C for 5 min; denaturation: 95°C for 30 s; annealing: 58°C for 45 s; extension: 72°C for 10 s; 30 cycles; extension: 72°C for 10 min.

[0055] The PCR products are subjected to agarose gel electrophoresis. The sizes of the electrophoresis bands at the up and down ends are between 500 bp and 1000 bp. Then, the PCR products are recovered using a small amount of DNA recovery kit, and the rsbX upstream and downstream homologous arm fragments of the gene are obtained. (2) Linearization of the expression vector The pWH-T2 plasmid is extracted using a plasmid miniprep kit. After XbaI and SmaI double digestion, agarose gel electrophoresis is performed, and then the products are recovered using a DNA gel recovery kit to obtain the linearized vector fragment.

[0056] The double digestion system is as follows:

[0057] After mixing, digest with enzymes in a water bath at 37°C for 2 h. After the reaction is completed, the digested products are subjected to agarose gel electrophoresis. The target band is 4260 bp, and then the digested products, the linear pWH-T2 plasmid, are recovered using a small amount of DNA recovery kit.

[0058] (3) Construction of the point mutation plasmid The linear pWH-T2 plasmid fragment obtained by double digestion and the upstream and downstream homologous arms of the rsbX gene (carrying the mutation site) are ligated by seamless cloning to form the recombinant plasmid pWH-T2-rsbX * (see the construction schematic diagram in Figure 1 , and this plasmid contains the rsbX * gene shown in SEQ ID NO.4).

[0059] The seamless cloning enzyme reaction system is as follows:

[0060] After mixing evenly, react in a water bath at 50°C for 15 min.

[0061] (4) Methylation modification of the point mutation vector and electrotransformation into competent cells of Bacillus amyloliquefaciens The constructed point mutation vector pWH-T2- rsbX *Transform it into EC135.P.Bam. competent cells by chemical transformation. When the OD600 value of the culture medium is 0.2, add 80 μL of 50 mg / mL arabinose aqueous solution for methylation induction, and culture it overnight in a shaker at 30 °C.

[0062] Use the chemical transformation method to transform the methylated plasmid into Bacillus amyloliquefaciens Δ acfi competent cells, and use the point mutation vector verification primers SmaI -F, XbaI -R for colony PCR verification.

[0063] (5) Single crossover verification After picking the successfully electrotransformed monoclonal colonies and culturing them for 3 - 4 generations at 45 °C, dilute and spread them, and pick single colonies for colony PCR verification. Since single crossovers may occur in either the upstream or downstream homologous sequences during the single crossover process, two sets of primers SmaI -F, DJH-R and DJH-F, XbaI -R are used for verification. Among them, the group of primers SmaI -F, DJH-R is successfully verified, as Figure 2 shown.

[0064] (6) Double crossover verification Pick the successfully single-crossover monoclonal colonies and culture them for 6 - 9 generations at 37 °C, dilute and spread them, pick single colonies for colony PCR verification. The primers used are SJH-F, SJH-R, and the results are as Figure 3 shown. The strain after correct single and double crossover verification is named: Δ acfi - rsbX * . The monoclonal colonies with single crossover are passaged and cultured for 6 - 9 generations at 37 °C in a tube of LB without antibiotics, then the bacterial solution is diluted and spread. The transformants obtained by dilution coating are transferred to a Kan-resistant plate and a non-resistant plate by the replica plating method, and the colonies that do not grow on the Kan-resistant plate but grow on the non-resistant plate are selected for PCR verification. The primers used are SJH-F, SJH-R, and the results are as Figure 3 shown. The correct band is 2000 bp. Select the monoclonal colonies with correct bands and streak them in three zones on the Kan-resistant plate for re-verification, and send the strains that are still correct after re-verification for sequencing verification. The strains with correct sequencing verification are the strains with successful point mutation. The strain after correct single and double crossover verification is named: Δ acfi - rsbX * .

[0065] It should be noted that if other Bacillus amyloliquefaciens (such as CGMCC No. 11218) is used as the starting strain, the above steps can also be used to point-mutate the genersbX The primers are selected according to the target gene, and the operation methods and conditions are basically the same.

[0066] Growth of the genetically engineered bacterium Δ acfi-rsbX * : Growth of the genetically engineered bacterium Δ acfi - rsbX * and the control bacterium Δ acfi were cultured in the seed medium for 72 h respectively, and the changes in the bacterial concentration and the viable cell count were measured at 24 h, 48 h and 72 h. 100 μL of the bacterial liquid was diluted with normal saline by different multiples, 1 mL of the bacterial suspension with different concentrations was placed in a sterilized petri dish, and 15 - 20 mL of the medium cooled to about 50 °C was poured into the petri dish and gently mixed, and then cultured and counted. The strain Δ acfirsbX * and the control strain Δ acfi were cultured simultaneously. It can be seen from Figure 4 that at the same bacterial liquid concentration at 24 h and 72 h, the number of viable strains contained in Δ acfi - rsbX * is more, showing higher growth advantage and growth vitality, indicating that rsbX the gene point mutation has no effect on the growth of the strain and can also improve the survival ability of the strain.

[0067] Example 2: Construction of aminopeptidase-producing bacteria The aminopeptidase gene acfi - rsbX * was introduced into the above-mentioned genetically engineered bacteria Δ acfi and Δ ywaD .

[0068] (1) The nucleotide sequences of the pLY-2 promoter and amyE the signal peptide (SEQ ID NO: 8), and the aminopeptidase gene ywaD (SEQ ID NO: 6) were synthesized by a biological company; (2) The pWB980 plasmid was extracted using a kit and digested with EcoRI and BamHI. The digestion system was the same as in Example 1; (3) The pLY-2 promoter, the signal peptide of Bacillus amyloliquefaciens, the recovered fragment of the aminopeptidase gene amyE and the linearized vector pWB980 obtained by digestion were ligated using seamless cloning enzyme to obtain the recombinant plasmid Ply-2-SPamyE- ywaD pWB980; the ligation system is as follows: ywaD- ​

[0069] After mixing the system, react at 50 °C for 15 min; (4) Electroporate the recombinant plasmids obtained in step (3) into Bacillus amyloliquefaciens Δ acfi - rsbX * 、Δ acfi as described below; ① Wash the electroporation cuvette with 75% alcohol, irradiate it under ultraviolet light for more than 20 min, and pre-cool it on ice; ② Mix 100 μL of competent cells and the ligation product DNA and add them to the electroporation cuvette, and place it on ice for 2 min; ③ Electroporate at 2500 V, and the electroporation time is generally 4 - qs; ④ Immediately after electroporation, add 1 ml of recovery medium, incubate at 37 °C for 3 h, take the bacterial solution and spread it on an LB screening plate containing 100 μg / mL kanamycin, and culture it at 37 °C for 12 h to screen for positive transformants for verification. The genetically engineered strains producing aminopeptidase were named Δ acfi - rsbX * - ywaD 、Δ acfi - ywaD respectively.

[0070] Example 3: Construction of alkaline protease-producing bacteria Introduce the alkaline protease gene acfi - rsbX * 、Δ acfi into the above-mentioned genetically engineered bacteria Δ aprE (SEQ ID NO: 5). Among them, the recombinant plasmid Ply-2-SPamy E- aprE -pWB980, and the construction process is basically the same as that in Example 2. The genetically engineered strains producing alkaline protease were named Δ acfi - rsbX * - aprE 、Δ acfi - aprE respectively.

[0071] Example 4: Construction of keratinase-producing bacteria Introduce the keratinase gene acfi-rsbX * 、Δ acfi into the above-mentioned genetically engineered bacteria Δ kerK (SEQ ID NO: 7). Among them, the recombinant plasmid Ply-2-SPamyE- kerK-pWB980, the construction process is basically the same as that in Example 2. The obtained genetically engineered strains producing keratinase are respectively named Δ acfi - rsbX * - kerK 、Δ acfi - kerK 。

[0072] Example 5: Production of aminopeptidase, alkaline protease and keratinase using genetically engineered bacteria Flask fermentation: Respectively streak the three groups of genetically engineered bacteria Δ acfi - YwaD and Δ acfi - rsbX * - YwaD 、Δ acfi - aprE and Δ acfi - rsbX * - aprE 、Δ acfi - kerK and Δ acfi - rsbX * - kerK on an LB plate for three-zone streaking, incubate inverted at 37 °C overnight, pick the activated single colonies into 5 mL of LB medium, shake culture at 37 °C and 220 r / min for 12 h, transfer with an inoculum of 2% to 50 mL of LB liquid medium until the OD 600 reaches about 1.0, and then transfer with an inoculum of 2% to a baffled flask containing 100 mL of fermentation medium, shake culture at 37 °C and 220 r / min for 48 h. Sampling is carried out at regular intervals, centrifuge at 4 °C and 12,000 r / min for 2 min, and take the supernatant of the fermentation broth. After appropriate dilution, the activities of aminopeptidase, alkaline protease and keratinase are measured.

[0073] The results are shown in Table 3. After measurement, the recombinant aminopeptidase activity in the fermentation supernatant with Δ acfi-rsbX * as the chassis bacterium is 138% of the expression activity of the control bacterium, and the recombinant alkaline protease activity in the fermentation supernatant with Δ acfi-rsbX * as the chassis bacterium is 109% of the expression activity of the control bacterium; the recombinant keratinase activity in the fermentation supernatant with Δ acfirsbX * as the chassis bacterium is 122% of the expression activity of the control bacterium.

[0074] Table 3: Enzyme production experiment

[0075] The present invention improves the production of industrial enzymes by enhancing the activity of strains during the fermentation process. The selected Bacillus amyloliquefaciens chassis is an example, and the selected protease is also an example, rather than a limitation. Those skilled in the art can also use this method to knock out or weaken the expression levels of other related Bacillus genes for culturing cells, producing compounds, or expressing other proteins.

[0076] Although the present invention has been disclosed above with 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 the forms and details of 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 deoxyribonucleic acid regulatory protein RsbX mutant, characterized in that, The mutant was obtained by introducing a T165I mutation into the wild-type RsbX shown in SEQ ID NO.1; the amino acid sequence of the RsbX mutant is shown in SEQ ID NO.

3.

2. The coding gene of the RsbX mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

4.

4. The application of the RsbX mutant according to claim 1 in the high-efficient expression of heterologous proteases.

5. A Bacillus amyloliquefaciens genetic engineering chassis bacterium, characterized in that The deoxyribonucleic acid regulatory protein RsbX expressed by the chassis bacterium has a T165I mutation.

6. The Bacillus amyloliquefaciens genetic engineering chassis bacterium according to claim 5, wherein The chassis bacterium does not express the following genes on the genome of the Bacillus amyloliquefaciens host: six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr , the extracellular polysaccharide gene cluster eps , the polyglutamic acid gene cluster pgs , and sacB genes.

7. The application of the chassis bacterium according to claim 5 in the expression of heterologous proteins.

8. The application according to claim 7, characterized in that The heterologous proteins include, but are not limited to: alkaline protease, aminopeptidase, keratinase.

9. A genetically engineered bacterium for producing protease, characterized in that, It is obtained by heterologously overexpressing alkaline protease, aminopeptidase or keratinase on the basis of the genetically engineered chassis bacterium according to claim 5.

10. The genetically engineered bacterium according to claim 9, characterized in that, The coding gene of the alkaline protease is shown in SEQ ID NO.5; the coding gene of the aminopeptidase is shown in SEQ ID NO.6; the coding gene of the aminopeptidase is shown in SEQ ID NO.7.

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