Chassis strain with deletion of bglS gene and application thereof

Abstract

The invention belongs to the field of gene engineering and enzyme engineering, and relates to a bglS gene-deleted chassis strain and application thereof. The invention provides a bacillus subtilis genetic engineering chassis bacterium. The chassis bacterium is obtained by deleting the expression of a glucanase coding gene bglS on a bacillus subtilis host genome. The chassis bacterium can efficiently express alkaline protease, aminopeptidase, medium-temperature alpha-amylase and high-temperature-resistant alpha-amylase, and the method is also suitable for improving the expression quantity of other exocrine proteins and lays a foundation for efficiently expressing heterologous proteins. High-efficiency expression and industrial production of alkaline protease, aminopeptidase, medium-temperature alpha-amylase and high-temperature-resistant alpha-amylase are promoted.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and enzyme engineering, and relates to a chassis strain lacking a bglS gene and application thereof. Background Art

[0002] Alkaline protease is a type of enzyme that can catalyze the hydrolysis of peptide bonds. Its active center contains serine, and it is also called serine protease. Enzymes that hydrolyze protein peptide bonds in the alkaline pH range can not only hydrolyze peptide bonds, but also have the functions of hydrolyzing amide bonds, ester bonds, and transesterification and transpeptidation. Alkaline protease has a wide range of uses in the food, washing and leather industries. Since microbial proteases are all extracellular enzymes, compared with animal and plant proteases, they have the advantages of relatively simple downstream technical processing, low price, wide source, easy cultivation of bacteria, and high yield. They have all the characteristics of animal and plant proteases, and have stronger hydrolysis ability and alkali resistance than neutral proteases, strong heat resistance and certain esterase activity, and are easy to realize industrial production.

[0003] Aminopeptidase is a type of exoprotease that can specifically recognize the amino acid residues at the N-terminus of proteins and polypeptide chains, and hydrolyze them to release free amino acids. Synergistic hydrolysis with alkaline proteases, carboxypeptidases, etc. can effectively reduce the bitterness in protein hydrolysates and prepare a variety of bioactive peptides, which have broad application prospects in the food and medical industries. However, the current aminopeptidase production is low, the enzyme activity is poor, and it is easy to be inactivated under high temperature conditions. Therefore, increasing the expression of aminopeptidase is of great value in improving the application effect of aminopeptidase and broadening its application field.

[0004] α-Amylase, also known as 1,4-α-D-glucan glucanohydrolase, can hydrolyze α-1,4-D glucosidic bonds from the inside of starch, resulting in partial depolymerization of starch. It is a type of endonuclease. According to the thermal stability and optimal reaction temperature of α-amylase, α-amylase is usually divided into high-temperature α-amylase, medium-temperature heat-resistant α-amylase, non-heat-resistant α-amylase and low-temperature α-amylase. Amylase suitable for maltose syrup, food baking, textile desizing industry, etc. A large number of different types and functions of amylase have been isolated from plants and microorganisms from different sources, and a variety of amylase genes have been isolated. A variety of amylase products are now industrially produced.

[0005] ikBThe gene encodes an endo-β-1,3-1,4-glucanase that specifically acts on the 1,3 and 1,4 glycosidic bonds of β-glucans. It can produce bioethanol by saccharifying lignocellulosic biomass, can enhance flavor in wine and juice production, and can be added to feed to improve cellulose digestion by farm animals. In addition, bglS promotes cellulose digestion and plant hormone activation, and participates in the hydrolysis of mammalian glucosylceramide. There is no related research in the prior art on affecting protein expression by gene editing of bglS.

[0006] Bacillus subtilis has a clear genetic metabolic pathway, is non-pathogenic, has no codon preference, has a short fermentation cycle, and is suitable for high cell density culture. In addition, Bacillus subtilis has a strong ability to secrete proteins and can be directly secreted outside the cell. Therefore, Bacillus subtilis is a commonly used food safety strain. However, the Bacillus subtilis efficient expression system still limits the expression of many proteins and is currently limited to the production of a small amount of industrial enzymes. Therefore, achieving efficient expression of aminopeptidases, alkaline proteases, thermostable α-amylases, medium-temperature α-amylases, etc. outside the cell of Bacillus subtilis is of great significance for its industrial production. Summary of the invention

[0007] In view of the above problems, the object of the present invention is to provide a Bacillus subtilis chassis strain capable of high production of alkaline protease, aminopeptidase, thermostable α-amylase and mesophilic α-amylase by genetic engineering to transform the host and its application.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: In a first aspect, the present invention provides a genetically engineered Bacillus subtilis substrate, wherein the substrate is missing a gene encoding a glucanase on the Bacillus subtilis host genome. ikB Expression; Further, the Bacillus subtilis host is Bacillus subtilis WB600; Furthermore, the amino acid sequence of the glucanase is shown in SEQ ID NO.1; Furthermore, the coding gene ikB The nucleotide sequence is shown in SEQ ID NO.2; Preferably, the base bacteria is a knockout of the glucanase encoding gene on Bacillus subtilis WB600 ikB Income.

[0009] In a second aspect, the present invention provides the use of the genetically engineered Bacillus subtilis substrate bacteria described in the first aspect in the expression of proteases; The protease includes, but is not limited to, alkaline protease, aminopeptidase, thermostable α-amylase, mesophilic α-amylase, and the like.

[0010] In a third aspect, the present invention provides a genetically engineered bacterium for producing alkaline protease, which is obtained by heterologously overexpressing alkaline protease on the basis of the genetically engineered chassis bacterium described in the first aspect; Furthermore, the coding gene of the alkaline protease is shown in SEQ ID NO.3, and is derived from alkaliphilic Bacillus.

[0011] In a fourth aspect, the present invention provides a genetically engineered bacterium for producing aminopeptidase, which is obtained by heterologously overexpressing aminopeptidase on the basis of the genetically engineered chassis bacterium described in the first aspect; Furthermore, the aminopeptidase encoding gene is shown in SEQ ID NO.4, which is derived from Bacillus PS11 (2022).

[0012] In a fifth aspect, the present invention provides a genetically engineered bacterium for producing mesophilic α-amylase, which is obtained by heterologously overexpressing mesophilic α-amylase on the basis of the genetically engineered chassis bacterium described in the first aspect; Furthermore, the gene encoding the mesophilic α-amylase is shown in SEQ ID NO.5, and is derived from Bacillus subtilis BF7658.

[0013] In a sixth aspect, the present invention provides a genetically engineered bacterium for producing a thermostable α-amylase, which is obtained by heterologously overexpressing a thermostable α-amylase on the basis of the genetically engineered chassis bacterium described in the first aspect; Furthermore, the gene encoding the thermostable α-amylase is shown in SEQ ID NO.6, and is derived from Bacillus subtilis.

[0014] In a seventh aspect, the present invention provides the use of the genetically engineered bacteria as described in the third to sixth aspects above, particularly in the production of alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase, respectively.

[0015] In an eighth aspect, the present invention provides a method for producing alkaline protease, aminopeptidase, mesophilic α-amylase or thermostable α-amylase, the method comprising: culturing any one of the genetically engineered bacteria described in the third aspect to the sixth aspect in a culture medium to produce a protease; and collecting the protease from the genetically engineered bacteria and / or the culture medium. The protease is selected from alkaline protease, aminopeptidase, mesophilic α-amylase or thermostable α-amylase.

[0016] The beneficial effects of the present invention are as follows: The present invention is based on the genome of Bacillus subtilis ikBBy knocking out the gene, an industrial chassis strain that can efficiently express alkaline protease, aminopeptidase, medium-temperature α-amylase and high-temperature α-amylase was obtained, which can lay the foundation for the efficient expression of heterologous proteins and promote the efficient expression and industrial production of alkaline protease, aminopeptidase, medium-temperature α-amylase and high-temperature α-amylase.

[0017] The present invention provides a Bacillus subtilis chassis strain capable of high production of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase. In the case of heterologous overexpression of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase, after 60 h of shake flask fermentation, the alkaline protease activity of the fermentation liquid in 48 h reaches a maximum of 28763.56 U / mL, which is 20.28% higher than that of the control bacteria, the aminopeptidase activity of the fermentation liquid in 36 h reaches a maximum of 9894.99 U / mL, which is 15.00% higher than that of the control, the mesophilic α-amylase activity of the fermentation liquid in 48 h reaches a maximum of 283.76 U / mL, which is 25.76% higher than that of the control bacteria, and the thermostable α-amylase activity of the fermentation liquid in 48 h reaches a maximum of 958.53 U / mL, which is 20.45% higher than that of the control bacteria. It is indicated that the glucanase encoding gene is knocked out. ikB It can effectively increase the enzymatic activity of multiple enzymes, and this method is also suitable for increasing the expression level of other exocrine proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The construction process of the temperature-sensitive knockout vector pWH-T2-ΔbglS in the embodiment; Figure 2 This is the single exchange verification of bglS gene knockout in the example; wherein M is: marker; lane 1 is: knockout bacteria.

[0019] Figure 3 This is the verification of bglS gene knockout in the example; wherein M is: marker; lane 1 is: WB600, and lane 2 is: WB600ΔbglS.

[0020] Figure 4 The enzymatic activity of alkaline protease expressed by the genetically engineered bacterium WB600ΔbglS-AprE in the examples.

[0021] Figure 5 The enzymatic activity of the aminopeptidase expressed by the genetically engineered bacterium WB600ΔbglS-YwaD in the examples.

[0022] Figure 6 The enzymatic activity of the mesophilic α-amylase expressed by the genetically engineered bacterium WB600ΔbglS-AmyE in the examples.

[0023] Figure 7The enzymatic activity of the thermostable α-amylase expressed by the genetically engineered bacterium WB600ΔbglS-AmyD in the examples. DETAILED DESCRIPTION

[0024] The present invention is further described below by specific embodiments. Unless otherwise specified, the technical means, materials, etc. involved in the following embodiments may be well known to those skilled in the art, and suitable ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications of the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0025] The present invention provides a genetically engineered Bacillus subtilis substrate, which performs deletion expression of the bglS gene on the Bacillus subtilis genome, wherein the nucleotide sequence of the bglS gene is shown in SEQ ID NO. 2. According to the present invention, the deletion expression of the bglS gene can be performed by conventional means in the art, for example, the gene is inactivated or knocked out by conventional means in the art.

[0026] The present invention provides a genetically engineered bacterium expressing alkaline protease, which is obtained by expressing alkaline protease in the above-mentioned Bacillus subtilis genetically engineered chassis bacteria. The nucleotide sequence of the alkaline protease encoding gene AprE is shown in SEQ ID NO.3.

[0027] The present invention provides a genetically engineered bacterium expressing aminopeptidase, which is obtained by expressing aminopeptidase in the above-mentioned genetically engineered bacterium of Bacillus subtilis. The nucleotide sequence of the aminopeptidase encoding gene ywaD is shown in SEQ ID NO.4.

[0028] The present invention provides a genetically engineered bacterium expressing a mesophilic α-amylase, which is obtained by expressing the mesophilic α-amylase in the above-mentioned Bacillus subtilis genetically engineered chassis bacteria. The nucleotide sequence of the mesophilic α-amylase encoding gene AmyE is shown in SEQ ID NO.5.

[0029] The present invention provides a genetically engineered bacterium expressing a thermostable α-amylase, which is obtained by expressing the thermostable α-amylase in the above-mentioned Bacillus subtilis genetically engineered chassis bacteria, and the nucleotide sequence of the thermostable α-amylase encoding gene AmyD is shown in SEQ ID NO.6.

[0030] According to a preferred embodiment of the present invention, the knockout of the bglS gene on the Bacillus subtilis genome can be carried out by conventional means in the art, for example, by homologous recombination, a knockout vector is constructed and electrotransformed into Bacillus subtilis, and the gene is knocked out from the genome through single-double exchange. Preferably, the knockout vector is a pWH-T2 plasmid containing a Kana resistance gene and a 5-Fu reverse screening marker.

[0031] According to the present invention, the method of heterologous overexpression of the above-mentioned genes can adopt conventional means in the art, for example, the expression cassettes of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase genes are inserted into the genome of Bacillus subtilis by conventional means in the art, or the expression vector containing the expression cassettes of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase genes is transferred into Bacillus subtilis by conventional means in the art.

[0032] According to a preferred embodiment of the present invention, the heterologous overexpression of the alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase genes is achieved by electroporating expression plasmids (pWB980-AprE, pWB980-YwaD, pWB980-AmyE and pWB980-AmyD) containing expression cassettes of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase into Bacillus subtilis, respectively.

[0033] The present invention provides a method for constructing strains of the alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase, comprising: inactivating or knocking out the bglS gene shown in the nucleotide sequence SEQ ID NO.2 in Bacillus subtilis WB600, and optionally introducing an alkaline protease encoding gene AprE, an aminopeptidase encoding gene YwaD, a mesophilic α-amylase encoding gene AmyE or a thermostable α-amylase encoding gene AmyD into Bacillus subtilis and overexpressing them.

[0034] According to a specific embodiment of the present invention, the construction method comprises: knocking out the bglS gene in the starting strain Bacillus subtilis WB600 to obtain the strain WB600ΔbglS.

[0035] Furthermore, the expression vector containing the expression cassette of the alkaline protease encoding gene AprE was respectively transferred into the strains WB600 and WB600ΔbglS, and finally the alkaline protease-producing Bacillus subtilis WB600-AprE and WB600ΔbglS-AprE were obtained.

[0036] Furthermore, the expression vector containing the expression cassette of the aminopeptidase encoding gene YwaD was respectively transferred into the strains WB600 and WB600ΔbglS, and finally the aminopeptidase-producing Bacillus subtilis WB600-YwaD and WB600ΔbglS-YwaD were obtained.

[0037] Furthermore, the expression vector containing the expression cassette of the mesophilic α-amylase encoding gene AmyE was respectively transferred into strains WB600 and WB600ΔbglS, and finally Bacillus subtilis WB600-AmyE and WB600ΔbglS-AmyE producing mesophilic α-amylase were obtained.

[0038] Furthermore, the expression vector containing the expression cassette of the thermostable α-amylase encoding gene AmyD was respectively transferred into the strains WB600 and WB600ΔbglS, and finally the Bacillus subtilis WB600-AmyD and WB600ΔbglS-AmyD with high thermostable α-amylase production were obtained.

[0039] According to a more preferred embodiment of the present invention, the construction method comprises the following steps: (1) Knock out the target gene and obtain knockout strains The target gene bglS gene is knocked out according to the following steps: 1) Obtain the UP and DOWN ends of the homologous sequences on both sides of the target gene through PCR amplification; 2) Obtain the linear knockout plasmid vector by double enzyme digestion and agarose nucleic acid gel electrophoresis; 3) Connect the homologous sequence with the linear vector through seamless cloning technology to obtain the knockout plasmid; 4) The knockout plasmid is methylated by methylation induction; 5) The methylation-modified knockout plasmid was electroporated into Bacillus subtilis WB600 competent cells; 6) The knockout strains were screened by single and double crossover, and the sequences were verified by sequencing to obtain the WB600ΔbglS chassis strain; The specific operation methods of the above steps can be implemented according to technical manuals, textbooks or literature reports that are easily available to those skilled in the art.

[0040] (2) Construction of overexpression of alkaline protease gene, aminopeptidase gene, medium-temperature α-amylase gene, and high-temperature resistant α-amylase gene The recombinant plasmids carrying the expression cassettes of the alkaline protease gene AprE, the aminopeptidase gene YwaD, the mesophilic α-amylase gene AmyE, and the thermostable α-amylase gene AmyD were electro-transformed into the above knockout strain WB600ΔbglS, respectively. As a comparison, they were also transformed into the starting strain WB600. The engineered strains producing alkaline protease were named WB600ΔbglS-AprE and WB600-AprE; the engineered strains producing aminopeptidase were named WB600ΔbglS-YwaD and WB600-YwaD; the engineered strains producing mesophilic α-amylase were named WB600ΔbglS-AmyE and WB600-AmyE; the engineered strains producing thermostable α-amylase were named WB600ΔbglS-AmyD and WB600-AmyD.

[0041] The strain produces alkaline protease, aminopeptidase, medium-temperature alpha-amylase and high-temperature resistant alpha-amylase through shake flask fermentation.

[0042] The enzyme activity of alkaline protease was determined according to the Folin-phenol method in Appendix B of GB / T 23527.1-2023, i.e., 1 unit of enzyme activity (U / mL) is defined as the amount of enzyme required for 1 mL of enzyme solution to hydrolyze casein to produce 1 μg of tyrosine at 40 °C and pH 10.5 for 1 min. Each sample was repeated three times and the results were averaged.

[0043] The method for determining the enzyme activity of aminopeptidase was carried out according to the LNA method, that is, 1 unit of enzyme activity (U / mL) was defined as the amount of enzyme required for 1 mL of enzyme solution to hydrolyze leucine p-nitroaniline to generate 1 μgρ-NA per minute at 50 °C and pH 9.0. It includes Tris-HCl buffer, 50 mmol / L leucine solution, and 40% acetic acid solution. 0.2 mL of appropriately diluted enzyme solution was mixed with 2.6 mL of Tris-HCl buffer, preheated at 50 °C for 5 min, 0.2 mL of leucine solution (50 mmol / L) was added, reacted at 50 °C for 10 min, and 1 mL of 40% acetic acid was added and shaken, allowed to stand for 10 min, and the absorbance was measured at 405 nm. Each sample was repeated three times, and the results were averaged. ReferencesY.Fundoiano-Hershcovitz, L.Rabinovitch,S.Shulami, V.Reiland, G.Shoham, and Y.Shoham(2005).The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase.FEMS MicrobiolLett,243,157-163.

[0044] The method for determining the activity of mesophilic α-amylase is based on GB / T 24401-2009: including dilute iodine solution, soluble starch solution with a final concentration of 20 g / L, phosphate buffer with a pH of 6.0, and hydrochloric acid solution with a concentration of 0.1 mol / L. 20 mL of soluble starch solution (20 g / L) and 5 mL of phosphate buffer (pH 6.0) were mixed, preheated at 60 °C for 8 min, and then 1 mL of appropriately diluted enzyme solution was added. After reacting at 60 °C for 5 minutes, it was added to a mixed solution of 5 mL of dilute iodine solution and 500 μL of hydrochloric acid (0.1 mol / L) for color development, and 500 μL of hydrochloric acid (0.1 mol / L) and 5 mL of dilute iodine solution were used as blanks, and the absorbance was measured at 660 nm. Each sample was repeated three times, and the results were averaged.

[0045] The method for determining the activity of thermostable α-amylase is in accordance with GB / T 24401-2009: including dilute iodine solution, soluble starch solution with a final concentration of 20 g / L, phosphate buffer with a pH of 6.0, and hydrochloric acid solution with a concentration of 0.1 mol / L. 20 mL of soluble starch solution (20 g / L) and 5 mL of phosphate buffer (pH 6.0) were mixed, preheated at 70 °C for 8 min, and then 1 mL of appropriately diluted enzyme solution was added. After reacting at 70 °C for 5 minutes, it was added to a mixed solution of 5 mL of dilute iodine solution and 500 μL of hydrochloric acid (0.1 mol / L) for color development, and 500 μL of hydrochloric acid (0.1 mol / L) and 5 mL of dilute iodine solution were used as blanks, and the absorbance was measured at 660 nm. Each sample was repeated three times, and the results were averaged.

[0046] The present invention provides a method for efficiently producing alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase. The method comprises culturing the strains expressing the alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase under suitable conditions, and collecting the alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase from the culture.

[0047] According to a preferred embodiment of the present invention, the suitable conditions refer to a culture temperature of 35-37°C, a rotation speed of 200-220 r / min and the following fermentation medium composition: 64 g / L corn flour, 40 g / L soybean cake powder, 4 g / L disodium hydrogen phosphate, 0.3 g / L potassium dihydrogen phosphate, 0.7 g / L high temperature amylase, and the rest is water.

[0048] Unless otherwise specified, some of the culture media used in the examples are as follows: Seed culture medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest is water; Fermentation medium: corn flour 64 g / L, soybean cake powder 40 g / L, disodium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 0.3 g / L, thermostable amylase 0.7 g / L, and the rest is water.

[0049] Preparation medium for Bacillus competent cells: LBS medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, sorbitol 9.1085 g / L, and the rest is water; Recovery medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, sorbitol 9.1085 g / L, mannitol 6.92246 g / L, and the rest is water.

[0050] The strains and plasmids involved in the examples are shown in Table 1: Table 1

[0051] Note: pWH-T2 and pWB980 are commercial plasmids.

[0052] The primer information involved in the embodiment is shown in Table 2: Table 2

[0053] Note: The underlined part is the restriction endonuclease.

[0054] The present invention will be described in more detail below through specific examples.

[0055] Example 1: Construction of genetically engineered chassis strain WB600ΔbglS (1) Knockout of target gene 1) Amplify the homologous sequence of the target gene Bacillus subtilis ikB The gene sequence (SEQ ID NO.2) was designed and amplified by PCR to obtain the homology arm sequences UP and DOWN at both ends of the target gene sequence. Two sets of primers, bglS-UP-F, bglS-UP-R and bglS-DOWN-F, bglS-DOWN-R (see Primer Table 2), were used to perform PCR amplification using the genome of Bacillus subtilis WB600 as a template; the amplification reaction system was as follows:

[0056] The amplification program was set as follows: pre-denaturation: 95 °C for 5 min; denaturation: 95 °C for 30 s; annealing: 56 °C for 45 s; extension: 72 °C for 25 s; reaction for 30 cycles; extension: 72 °C for 10 min.

[0057] The PCR product was subjected to agarose gel electrophoresis, and the size of the electrophoresis bands at the UP and DOWN ends was between 400 bp and 600 bp. The PCR product, i.e., the upstream and downstream homology arm fragments of the target gene, was then recovered using a small amount of DNA recovery kit.

[0058] 2) Linearization of expression vector The pWH-T2 plasmid was extracted, and the extraction process was referred to the operating manual of the kit. After double digestion with XbaI and SmaI, agarose gel electrophoresis was performed, and the product was recovered by a DNA gel recovery kit to obtain a linearized vector sequence.

[0059] The double enzyme digestion system is as follows:

[0060] After mixing, the enzyme digestion was carried out in a 37 ℃ water bath for 2 h. After the reaction was completed, the digestion product was subjected to agarose gel electrophoresis, and the size was 4260 bp. The digestion product was then recovered using a small amount of DNA recovery kit: linear pWH-T2 plasmid.

[0061] 3) Construction of knockout vector The linear pWH-T2 vector fragment obtained by enzyme digestion and the upstream and downstream homology arm UP end and DOWN end fragments of the target gene were connected by seamless cloning to form the recombinant plasmid pWH-T2-ΔbglS.

[0062] The seamless cloning enzyme reaction system is as follows:

[0063] After mixing well, react in a 50 ℃ water bath for 15 min.

[0064] 4) Methylation modification of knockout vector and transformation into Bacillus subtilis WB600 competent cells The knockout vector pWH-T2-ΔbglS was transformed into EC135.P.Bam. competent cells by chemical transformation. 600 When the value was 0.2, 80 μL of 50 mg / mL arabinose aqueous solution was added for methylation induction, and the cells were cultured in a shaking incubator at 30 °C overnight.

[0065] The methylated knockout vector was transformed into Bacillus subtilis WB600 competent cells using the transformation method, and the knockout vector verification primers SmaI-F and XbaI-R were used for colony PCR verification.

[0066] 5) Single exchange verification The successfully transformed single clones were selected and cultured at 45°C for 2-3 generations. After dilution and plating, single colonies were picked for colony PCR verification. Since single exchange may occur in the upstream or downstream homologous sequences during the single exchange process, primers SmaI-F and DJH-DOWN-R were used for verification (the results are shown in Figure 2). Figure 2 as shown).

[0067] 6) Dual exchange verification The single clones that have been successfully exchanged were selected and cultured at 37°C for 3-4 generations. After dilution and plating, a single colony was selected for colony PCR verification. The primers used were SJH-F and SJH-R (the results are shown in Figure 3 The strain after single and double exchange verification was named WB600ΔbglS.

[0068] Example 2: Construction of alkaline protease producing strain The alkaline protease gene AprE was introduced into the above-mentioned genetically engineered bacteria WB600ΔbglS. (1) A nucleotide sequence containing the pLY-2 promoter and amyE signal peptide (SEQ ID NO.7), and an alkaline protease encoding gene (SEQ ID NO.3) were synthesized by a biological company; (2) Extracting the pWB980 plasmid using a kit and performing double digestion with EcoRI and BamHI, the digestion system was the same as in Example 1; (3) Using seamless cloning enzyme, the pLY-2 promoter obtained above was connected with the amyE signal peptide of Bacillus amyloliquefaciens, the recovered fragment of the alkaline protease gene AprE, and the linearized vector pWB980 obtained by enzyme digestion to obtain the recombinant plasmid P ly-2 -SP amyE -AprE-pWB980; the connection system is as follows:

[0069] After mixing the system, react at 50°C for 15 min; (4) The recombinant plasmid obtained in step (3) was transformed into Bacillus subtilis WB600 and WB600ΔbglS respectively, as described below; ① Pick newly activated single colonies of Bacillus subtilis WB600 and WB600ΔbglS respectively and place them in 5 mL LB liquid medium, culture them at 37°C, 220 r / min, overnight; ②Take 100 μL of culture medium and transfer it to 5 mL of SPI medium. Cultivate at 37°C and 220 r / min until the end of logarithmic growth OD 600 =1.2 (about 3-4 h); ③ Take 200 μL of the culture medium grown to the end of the logarithmic phase and add it to 2 mL of SPII medium. Culture at 37°C and 100 r / min for 1.5 h. ④ Add 20 μL 10 mmol / L EGTA to the bacteria in the above SPII medium and culture at 37°C and 100 r / min for 10 min; ⑤Add ligation product: recombinant plasmid P ly-2 -SP amyE -AprE-pWB980, 37°C, 100 rpm, 30 min; ⑥ Adjust the speed to 220 r / min, continue to culture for 1.5 h, take the bacterial solution and spread it on the LB screening plate containing 100 μg / mL kanamycin, culture at 37℃ for 12 h, and screen the positive transformants for verification. The genetically engineered strains producing alkaline protease were named WB600-AprE and WB600ΔbglS-AprE.

[0070] Example 3: Construction of aminopeptidase producing strain The aminopeptidase encoding gene expression plasmid P was constructed in the same manner as in Example 2. ly-2 -SP amyE -YwaD-pWB980, only the alkaline protease encoding gene is replaced with the aminopeptidase encoding gene YwaD shown in SEQ ID NO.4.

[0071] The recombinant plasmid P ly-2 -SP amyE -YwaD-pWB980 was transformed into Bacillus subtilis WB600 and WB600ΔbglS, respectively, to obtain aminopeptidase-producing genetically engineered strains named WB600-YwaD and WB600ΔbglS-YwaD, respectively (the construction process was basically the same as that in Example 2).

[0072] Example 4: Construction of mesophilic α-amylase producing strain The medium-temperature α-amylase encoding gene expression plasmid P was constructed in the same manner as in Example 2. ly-2 -SP amyE - AmyE-pWB980, only the alkaline protease encoding gene is replaced with the medium-temperature α-amylase encoding gene AmyE shown in SEQ ID NO.5.

[0073] The recombinant plasmid P ly-2 -SP amyE-AmyE-pWB980 was transformed into Bacillus subtilis WB600 and WB600ΔbglS, respectively, to obtain genetically engineered strains producing mesophilic α-amylase, which were named WB600-AmyE and WB600ΔbglS-AmyE, respectively (the construction process was basically the same as that in Example 2).

[0074] Example 5: Construction of a thermostable α-amylase producing strain The same method as in Example 2 was used to construct a high temperature resistant α-amylase encoding gene expression plasmid P ly-2 -SP amyE -AmyD-pWB980, only the alkaline protease encoding gene is replaced with the thermostable α-amylase encoding gene AmyD shown in SEQ ID NO.6.

[0075] The recombinant plasmid P ly-2 -SP amyE -AmyD-pWB980 was transformed into Bacillus subtilis WB600 and WB600ΔbglS, respectively, to obtain genetically engineered strains producing thermostable α-amylase, which were named WB600-AmyD and WB600ΔbglS-AmyD, respectively (the construction process was basically the same as that in Example 2).

[0076] Example 6: Production of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase using genetically engineered bacteria Shake flask fermentation: The four groups of genetically engineered bacteria WB600-AprE, WB600ΔbglS-AprE, WB600-YwaD, WB600ΔbglS-YwaD, WB600-AmyE, WB600ΔbglS-AmyE, WB600-AmyD, and WB600ΔbglS-AmyD constructed in Examples 2-5 were respectively streaked on LB plates, inverted and cultured overnight at 37°C, and activated single colonies were picked and placed in 5 mL LB medium, shaken and cultured at 37°C, 220 r / min for 12 h, and transferred to 50 mL LB liquid medium at an inoculum size of 2% until the OD reached 0. 600 When the concentration reaches 0.8-1.0, transfer the inoculum to a baffled bottle containing 100 mL of fermentation medium at a 2% inoculum volume and culture at 37°C and 220 r / min for 60 h.

[0077] Samples were taken at fixed points at 12, 24, 36, 48, and 60 hours, respectively, and centrifuged at 4 °C, 12000 r / min for 2 min. The fermentation supernatant was taken and diluted appropriately to determine the activity of alkaline protease according to the national standard method, the activity of aminopeptidase according to the LNA method, and the activity of medium-temperature α-amylase and thermostable α-amylase according to the national standard method. The results are as follows Figure 4 , Figure 5, Figure 6 , Figure 7 shown.

[0078] Among them, the highest alkaline protease activity in the 48 h fermentation broth reached 28763.56 U / mL, which was 20.28% higher than that of the control bacteria; the highest aminopeptidase activity in the 36 h fermentation broth reached 9894.99 U / mL, which was 15.00% higher than that of the control bacteria; the highest mesophilic α-amylase activity in the 48 h fermentation broth reached 283.76 U / mL, which was 25.76% higher than that of the control bacteria; the highest thermostable α-amylase activity in the 48 h fermentation broth reached 958.53 U / mL, which was 20.45% higher than that of the control bacteria (Table 3); It can be seen that the present invention knocks out the glucanase encoding gene in Bacillus subtilis, and the bacteria finally obtained can efficiently heterologously express multiple enzymes such as alkaline protease, aminopeptidase, mesophilic α-amylase, thermostable α-amylase, etc., and provides an innovative idea for constructing a chassis strain with high production of multiple enzymes.

[0079] Table 3 Enzyme production experiment

[0080] The present invention increases the yield of industrial enzymes by increasing the activity of the strain during the fermentation process. The selected Bacillus subtilis starting strain is only an example, and the selected protease is also an example, not a limitation. Those skilled in the art can also use this method to knock out or weaken the expression level of other Bacillus-related genes for culturing bacteria, producing compounds, or expressing other proteins.

[0081] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details 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 substrate, characterized in that: The base bacteria deletes the glucanase encoding gene on the Bacillus subtilis host genome ikB expression obtained.

2. The genetically engineered Bacillus subtilis substrate bacteria according to claim 1, characterized in that: The Bacillus subtilis host is Bacillus subtilis WB600.

3. The genetically engineered Bacillus subtilis substrate according to claim 1, characterized in that: The amino acid sequence of the glucanase is shown in SEQ ID NO.

1.

4. The use of a genetically engineered Bacillus subtilis substrate bacteria in the expression of protease according to claim 1, characterized in that: The protease is alkaline protease, aminopeptidase, high temperature resistant α-amylase or medium temperature α-amylase.

5. The use according to claim 4, characterized in that The coding gene of the alkaline protease is shown in SEQ ID NO.3; the coding gene of the aminopeptidase is shown in SEQ ID NO.4; the coding gene of the medium-temperature α-amylase is shown in SEQ ID NO.5; and the coding gene of the high-temperature resistant α-amylase is shown in SEQ ID NO.

6.

6. A genetically engineered bacterium for producing protease, characterized in that: The invention is obtained by heterologously overexpressing alkaline protease, aminopeptidase, medium-temperature α-amylase or high-temperature resistant α-amylase on the basis of the genetically engineered chassis bacteria described in claim 1.

7. Use of the genetically engineered bacteria according to claim 6 in the production of alkaline protease, characterized in that: The genetically engineered bacteria is obtained by heterologously overexpressing alkaline protease on the basis of the genetically engineered chassis bacteria described in claim 1.

8. The use of the genetically engineered bacteria according to claim 6 in producing aminopeptidase, characterized in that: The genetically engineered bacteria is obtained by heterologously overexpressing aminopeptidase on the basis of the genetically engineered chassis bacteria described in claim 1.

9. Use of the genetically engineered bacteria according to claim 6 in the production of mesophilic α-amylase, characterized in that: The genetically engineered bacteria are obtained by heterologously overexpressing mesophilic α-amylase on the basis of the genetically engineered chassis bacteria described in claim 1.

10. Use of the genetically engineered bacteria according to claim 6 in producing thermostable α-amylase, characterized in that: The genetically engineered bacteria are obtained by heterologously overexpressing a high-temperature resistant α-amylase on the basis of the genetically engineered chassis bacteria described in claim 1.

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