A chassis strain lacking bglS gene and its application

Abstract

The present invention belongs to the field of genetic engineering and enzyme engineering, and relates to a chassis strain lacking the bglS gene and its application. The present invention provides a genetically engineered chassis strain of Bacillus subtilis, wherein the chassis strain is constructed by deleting the glucanase encoding gene on the Bacillus subtilis host genome. bglS The described chassis bacteria can efficiently express alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase. This method is also suitable for increasing the expression of other exocrine proteins, laying the foundation for efficient expression of heterologous proteins and promoting the efficient expression and industrial production of alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase.

Description

Technical Field

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

[0002] Alkaline proteases, also known as serine proteases, are enzymes that catalyze the hydrolysis of peptide bonds. Their active centers contain serine, and they are therefore known as serine proteases. These enzymes hydrolyze protein peptide bonds within the alkaline pH range, not only hydrolyzing peptide bonds but also amide and ester bonds, as well as performing transesterification and transpeptidation. Alkaline proteases are widely used in the food, laundry, and leather industries. Because microbial proteases are all extracellular, they offer advantages over plant and animal proteases, such as relatively simple downstream processing, low cost, wide availability, ease of bacterial culture, and high yield. They possess all the properties of plant and animal proteases, but possess greater hydrolytic and alkali resistance than neutral proteases, are heat-resistant, and possess a certain degree of esterase activity, making them amenable to industrial production.

[0003] Aminopeptidases are a class of exoproteases that specifically recognize amino acid residues at the N-termini of proteins and polypeptide chains, hydrolyzing them to release free amino acids. Synergistic hydrolysis with alkaline proteases and carboxypeptidases can effectively reduce the bitterness of protein hydrolysates and produce a variety of bioactive peptides, which have broad application prospects in the food and medical industries. However, current aminopeptidases suffer from low production, poor enzyme activity, and susceptibility to inactivation at high temperatures. Therefore, increasing the expression level of aminopeptidases is of great value in improving their effectiveness and expanding their applications.

[0004] α-Amylase, also known as 1,4-α-D-glucan glucanohydrolase, is a type of endonuclease that can hydrolyze α-1,4-D glucosidic bonds within starch, resulting in partial depolymerization. Based on their thermal stability and optimal reaction temperature, α-amylases are generally classified into high-temperature α-amylases, medium-temperature thermostable α-amylases, non-thermostable α-amylases, and low-temperature α-amylases. Amylases suitable for applications in maltose syrup, food baking, and textile desizing have been isolated from plants and microorganisms of various types and functions. Multiple amylase genes have also been isolated, and a variety of amylase products are now commercially produced.

[0005] sThis gene encodes an endo-β-1,3-1,4-glucanase that specifically targets the 1,3- and 1,4-glycosidic bonds of β-glucans. It can be used to produce bioethanol by saccharifying lignocellulosic biomass, enhance flavor in wine and juice production, and improve cellulose digestion in farm animals by adding it to feed. bglS also promotes cellulose digestion and plant hormone activation and participates in the hydrolysis of mammalian glucosylceramides. No studies have been reported to affect protein expression through gene editing of bglS.

[0006] Bacillus subtilis has a relatively clear genetic metabolic pathway, is non-pathogenic, has no codon bias, has a short fermentation cycle, and is suitable for high cell density culture. Furthermore, Bacillus subtilis has a strong ability to secrete proteins directly outside the cell. Therefore, Bacillus subtilis is a commonly used food-safe strain. However, the Bacillus subtilis high-efficiency expression system still limits the expression of many proteins and is currently limited to the production of a small number of industrial enzymes. Therefore, achieving efficient extracellular expression of aminopeptidases, alkaline proteases, thermostable α-amylases, and mesophilic α-amylases in Bacillus subtilis is of great significance for their industrial production. Summary of the Invention

[0007] In view of the above problems, the purpose of the present invention is to provide a Bacillus subtilis chassis strain and its application that can produce high levels of alkaline protease, aminopeptidase, high-temperature resistant α-amylase and medium-temperature α-amylase through genetic engineering modification of the host.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a genetically engineered Bacillus subtilis substrate, wherein the substrate is missing the glucanase encoding gene on the Bacillus subtilis host genome. s expression;

[0010] Furthermore, the Bacillus subtilis host is Bacillus subtilis WB600;

[0011] Furthermore, the amino acid sequence of the glucanase is shown in SEQ ID NO.1;

[0012] Furthermore, the coding gene s The nucleotide sequence is shown in SEQ ID NO.2;

[0013] Preferably, the base bacteria is a knockout of the glucanase encoding gene on Bacillus subtilis WB600 s Income.

[0014] In a second aspect, the present invention provides the use of the genetically engineered Bacillus subtilis substrate described in the first aspect in the expression of proteases;

[0015] The protease includes but is not limited to alkaline protease, aminopeptidase, high-temperature resistant α-amylase, medium-temperature α-amylase and the like.

[0016] 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;

[0017] Furthermore, the encoding gene of the alkaline protease is shown in SEQ ID NO. 3, and is derived from alkaliphilic Bacillus.

[0018] 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;

[0019] Furthermore, the aminopeptidase encoding gene is shown in SEQ ID NO. 4, which is derived from Bacillus PS11 (2022).

[0020] In a fifth aspect, the present invention provides a genetically engineered bacterium for producing a mesophilic α-amylase, which is obtained by heterologously overexpressing a mesophilic α-amylase on the basis of the genetically engineered base bacterium described in the first aspect;

[0021] Furthermore, the gene encoding the mesophilic α-amylase is shown in SEQ ID NO. 5, and is derived from Bacillus subtilis BF7658.

[0022] 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 base bacterium described in the first aspect;

[0023] Furthermore, the gene encoding the thermostable α-amylase is shown in SEQ ID NO.6, which is derived from Bacillus subtilis.

[0024] In a seventh aspect, the present invention provides applications 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.

[0025] In an eighth aspect, the present invention provides a method for producing an alkaline protease, an aminopeptidase, a mesophilic α-amylase, or a thermostable α-amylase, the method comprising: culturing any one of the genetically engineered bacteria described in aspects 3 to 6 in a culture medium to produce the protease; and collecting the protease from the genetically engineered bacteria and / or the culture medium. The protease is selected from an alkaline protease, an aminopeptidase, a mesophilic α-amylase, or a thermostable α-amylase.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention is based on the genome of Bacillus subtilis s By knocking out the gene, an industrial chassis strain that can efficiently express alkaline protease, aminopeptidase, medium-temperature α-amylase, and high-temperature resistant α-amylase was obtained. This 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 resistant α-amylase.

[0028] The present invention provides a Bacillus subtilis chassis strain capable of high production of alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase. When heterologously overexpressing alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase, after 60 h of shake flask fermentation, the alkaline protease activity of the fermentation broth reached a maximum of 28763.56 U / mL after 48 h, an increase of 20.28% compared with the control bacteria; the aminopeptidase activity of the fermentation broth reached a maximum of 9894.99 U / mL after 36 h, an increase of 15.00% compared with the control; the mesophilic α-amylase activity of the fermentation broth reached a maximum of 283.76 U / mL after 48 h, an increase of 25.76% compared with the control bacteria; and the thermostable α-amylase activity of the fermentation broth reached a maximum of 958.53 U / mL after 48 h, an increase of 20.45% compared with the control bacteria. This indicates that the glucanase encoding gene has been knocked out. s 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

[0029] Figure 1 This is the construction process of the temperature-sensitive knockout vector pWH-T2-ΔbglS in the embodiment;

[0030] Figure 2 This is the single-crossover verification of the bglS gene knockout in the example; wherein M is a marker; lane 1 is the knockout bacteria.

[0031] Figure 3 This is the verification of bglS gene knockout in the example; wherein M is a marker; lane 1 is WB600, and lane 2 is WB600ΔbglS.

[0032] Figure 4 The enzymatic activity of alkaline protease expressed by the genetically engineered bacterium WB600ΔbglS-AprE in the examples is shown.

[0033] Figure 5 The enzymatic activity of the aminopeptidase expressed by the genetically engineered bacterium WB600ΔbglS-YwaD in the examples is shown.

[0034] Figure 6The enzymatic activity of the mesophilic α-amylase expressed by the genetically engineered bacterium WB600ΔbglS-AmyE in the examples is shown.

[0035] Figure 7 The enzymatic activity of the thermostable α-amylase expressed by the genetically engineered bacterium WB600ΔbglS-AmyD in the examples is shown. DETAILED DESCRIPTION

[0036] The present invention will be 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 appropriate ones may be selected from the known means and materials that can solve the corresponding technical problems. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications made to the material composition and dosage in these embodiments also fall within the scope of protection of the present invention.

[0037] The present invention provides a genetically engineered Bacillus subtilis strain that expresses a deletion of the bglS gene on the Bacillus subtilis genome. The nucleotide sequence of the bglS gene is shown in SEQ ID NO. 2. According to the present invention, the deletion of the bglS gene can be achieved by conventional methods in the art, for example, by inactivating or knocking out the gene.

[0038] The present invention provides a genetically engineered bacterium expressing alkaline protease, which is obtained by expressing alkaline protease in the above-mentioned genetically engineered Bacillus subtilis chassis bacteria. The nucleotide sequence of the alkaline protease encoding gene AprE is shown in SEQ ID NO.3.

[0039] The present invention provides a genetically engineered bacterium expressing aminopeptidase, which is obtained by expressing aminopeptidase in the above-mentioned genetically engineered Bacillus subtilis chassis bacteria. The nucleotide sequence of the aminopeptidase encoding gene ywaD is shown in SEQ ID NO.4.

[0040] 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 genetically engineered Bacillus subtilis chassis bacteria. The nucleotide sequence of the gene AmyE encoding the mesophilic α-amylase is shown in SEQ ID NO.5.

[0041] 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. The nucleotide sequence of the thermostable α-amylase encoding gene AmyD is shown in SEQ ID NO.6.

[0042] According to a preferred embodiment of the present invention, the bglS gene on the Bacillus subtilis genome can be knocked out by conventional means in the art, for example, by constructing a knockout vector by homologous recombination and electrotransforming it into Bacillus subtilis, and knocking out the above gene 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 selection marker.

[0043] 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 the 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 the alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase genes is transferred into Bacillus subtilis by conventional means in the art.

[0044] 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.

[0045] The present invention provides a method for constructing strains of the above-mentioned 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 and overexpressing the alkaline protease encoding gene AprE, the aminopeptidase encoding gene YwaD, the mesophilic α-amylase encoding gene AmyE, or the thermostable α-amylase encoding gene AmyD into Bacillus subtilis.

[0046] According to a specific embodiment of the present invention, the construction method includes: knocking out the bglS gene in the starting strain Bacillus subtilis WB600 to obtain the strain WB600ΔbglS.

[0047] Furthermore, the expression vector containing the alkaline protease encoding gene AprE expression cassette was respectively transformed into strains WB600 and WB600ΔbglS, and finally alkaline protease-producing Bacillus subtilis WB600-AprE and WB600ΔbglS-AprE were obtained.

[0048] Furthermore, the expression vector containing the aminopeptidase encoding gene YwaD expression cassette was respectively transformed into strains WB600 and WB600ΔbglS, and finally aminopeptidase-producing Bacillus subtilis WB600-YwaD and WB600ΔbglS-YwaD were obtained.

[0049] Furthermore, the expression vector containing the expression cassette of the mesophilic α-amylase encoding gene AmyE was respectively transformed into strains WB600 and WB600ΔbglS, and finally Bacillus subtilis WB600-AmyE and WB600ΔbglS-AmyE producing mesophilic α-amylase were obtained.

[0050] Furthermore, the expression vector containing the thermostable α-amylase encoding gene AmyD expression cassette was respectively transformed into strains WB600 and WB600ΔbglS, and finally Bacillus subtilis WB600-AmyD and WB600ΔbglS-AmyD with high thermostable α-amylase production were obtained.

[0051] According to a more preferred embodiment of the present invention, the construction method comprises the following steps:

[0052] (1) Knock out the target gene and obtain knockout strains

[0053] The target gene bglS gene was knocked out according to the following steps:

[0054] 1) Obtain the UP and DOWN ends of the homologous sequences on both sides of the target gene through PCR amplification;

[0055] 2) Obtain the linear knockout plasmid vector by double enzyme digestion and agarose gel electrophoresis;

[0056] 3) Connect the homologous sequence with the linear vector through seamless cloning technology to obtain the knockout plasmid;

[0057] 4) The knockout plasmid is methylated by methylation induction;

[0058] 5) Electroporate the methylation-modified knockout plasmid into Bacillus subtilis WB600 competent cells;

[0059] 6) Knockout strains were screened by single and double crossover and sequence verified by sequencing to obtain the WB600ΔbglS chassis strain;

[0060] 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.

[0061] (2) Construction of overexpression of alkaline protease gene, aminopeptidase gene, mesophilic α-amylase gene, and thermostable α-amylase gene

[0062] Recombinant plasmids carrying expression cassettes for the alkaline protease gene AprE, the aminopeptidase gene YwaD, the mesophilic α-amylase gene AmyE, and the thermostable α-amylase gene AmyD were electroporated into the knockout strain WB600ΔbglS, respectively. For comparison, they were also transformed into the starting strain WB600. The resulting alkaline protease-producing engineered strains were named WB600ΔbglS-AprE and WB600-AprE; the aminopeptidase-producing engineered strains were named WB600ΔbglS-YwaD and WB600-YwaD; the mesophilic α-amylase-producing engineered strains were named WB600ΔbglS-AmyE and WB600-AmyE; and the thermostable α-amylase-producing engineered strains were named WB600ΔbglS-AmyD and WB600-AmyD.

[0063] The strain produces alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase through shake flask fermentation.

[0064] Alkaline protease activity was determined using the Folin-phenol method described in Appendix B of GB / T 23527.1-2023. One unit of enzyme activity (U / mL) is 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. Three replicates were performed for each sample, and the results were averaged.

[0065] Aminopeptidase activity was determined using the LNA method. One unit of enzyme activity (U / mL) is defined as the amount of enzyme required to hydrolyze leucine-p-nitroaniline to produce 1 μg of p-NA per minute per mL of enzyme solution at 50°C and pH 9.0. The assay consists of 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. The solution was preheated at 50°C for 5 minutes, followed by the addition of 0.2 mL of 50 mmol / L leucine solution. The reaction was then incubated at 50°C for 10 minutes, followed by the addition of 1 mL of 40% acetic acid, followed by shaking. The solution was allowed to stand for 10 minutes, and the absorbance was measured at 405 nm. Three replicates were performed for each sample, and the results were averaged. References Y. Fundoiano-Hershcovitz, L. Rabinovitch, S. Shulami, V. Reiland, G. Shoham, and Y. Shoham (2005). The ywad gene from Bacillus subtilisencodes a double-zinc aminopeptidase.FEMS MicrobiolLett,243,157-163.

[0066] The mesophilic α-amylase activity assay follows the method described in GB / T 24401-2009: a dilute iodine solution, a soluble starch solution at a final concentration of 20 g / L, a pH 6.0 phosphate buffer, and a 0.1 mol / L hydrochloric acid solution. 20 mL of soluble starch solution (20 g / L) and 5 mL of phosphate buffer (pH 6.0) were mixed and preheated at 60°C for 8 minutes. 1 mL of the appropriately diluted enzyme solution was then added. The mixture was reacted at 60°C for 5 minutes and then added to a mixture of 5 mL of dilute iodine solution and 500 μL of 0.1 mol / L hydrochloric acid for color development. The absorbance was measured at 660 nm using 500 μL of 0.1 mol / L hydrochloric acid and 5 mL of dilute iodine solution as a blank. Three replicates were performed for each sample, and the results were averaged.

[0067] The thermostable α-amylase activity was determined according to the method described in GB / T 24401-2009: a dilute iodine solution, a soluble starch solution at a final concentration of 20 g / L, a pH 6.0 phosphate buffer, and a 0.1 mol / L hydrochloric acid solution. 20 mL of the soluble starch solution (20 g / L) and 5 mL of the phosphate buffer (pH 6.0) were mixed and preheated at 70°C for 8 minutes. 1 mL of the appropriately diluted enzyme solution was then added. The mixture was reacted at 70°C for 5 minutes and then added to a mixture of 5 mL of dilute iodine solution and 500 μL of 0.1 mol / L hydrochloric acid for color development. The absorbance was measured at 660 nm using 500 μL of 0.1 mol / L hydrochloric acid and 5 mL of dilute iodine solution as a blank. Three replicates were performed for each sample, and the results were averaged.

[0068] The present invention provides a method for efficiently producing alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase. The method comprises culturing the above-mentioned strains expressing 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.

[0069] 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: corn flour 64 g / L, soybean cake powder 40 g / L, disodium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 0.3 g / L, high temperature amylase 0.7 g / L, and the rest is water.

[0070] Unless otherwise specified, some of the culture media used in the examples are as follows:

[0071] Seed culture medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, and the rest is water;

[0072] Fermentation medium: corn flour 64 g / L, soybean meal 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.

[0073] Preparation of competent culture medium for Bacillus sp.

[0074] 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;

[0075] 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.

[0076] The strains and plasmids involved in the examples are shown in Table 1:

[0077] Table 1

[0078]

[0079] Note: pWH-T2 and pWB980 are commercial plasmids.

[0080] The primer information involved in the embodiment is shown in Table 2:

[0081] Table 2

[0082]

[0083] Note: The underlined part is the restriction endonuclease.

[0084] The present invention will be described in more detail below through specific examples.

[0085] Example 1: Construction of genetically engineered chassis strain WB600ΔbglS

[0086] (1) Knockout of target gene

[0087] 1) Amplify the homologous sequence of the target gene

[0088] According to Bacillus subtilis sThe 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 and bglS-UP-R, and bglS-DOWN-F and bglS-DOWN-R, were used (see Primer Table 2) to perform PCR amplification using the Bacillus subtilis WB600 genome as a template. The amplification reaction system was as follows:

[0089]

[0090] 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.

[0091] The PCR product was subjected to agarose gel electrophoresis. 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.

[0092] 2) Linearization of expression vector

[0093] The pWH-T2 plasmid was extracted according to the kit's manual. After double digestion with XbaI and SmaI, the product was subjected to agarose gel electrophoresis and recovered using a DNA gel recovery kit to obtain the linearized vector sequence.

[0094] The double enzyme digestion system is as follows:

[0095]

[0096] After mixing, enzyme digestion was performed in a 37°C water bath for 2 h. After the reaction was completed, the enzyme digestion product was subjected to agarose gel electrophoresis, and the size was 4260 bp. The enzyme digestion product was then recovered using a small amount of DNA recovery kit: linear pWH-T2 plasmid.

[0097] 3) Construction of knockout vector

[0098] 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.

[0099] The seamless cloning enzyme reaction system is as follows:

[0100]

[0101] After mixing evenly, react in a 50 °C water bath for 15 min.

[0102] 4) Methylation modification of knockout vector and transformation into Bacillus subtilis WB600 competent cells

[0103] The knockout vector pWH-T2-ΔbglS was transformed into EC135.P.Bam. competent cells by chemical transformation. When the culture medium OD 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.

[0104] The methylated knockout vector was transformed into Bacillus subtilis WB600 competent cells using the transfection method, and the knockout vector verification primers SmaI-F and XbaI-R were used for colony PCR verification.

[0105] 5) Single exchange verification

[0106] After selecting the successfully transformed single clones and culturing them at 45°C for 2-3 generations, they were diluted and spread, and single colonies were picked for colony PCR verification. Since single crossover may occur in upstream or downstream homologous sequences during single crossover, primers SmaI-F and DJH-DOWN-R were used for verification (the results are shown in Figure 2). Figure 2 shown).

[0107] 6) Dual exchange verification

[0108] The single clones that have been successfully exchanged were selected and cultured at 37°C for 3-4 generations. After dilution and plating, single colonies were selected for colony PCR verification. The primers used were SJH-F and SJH-R (the results are shown in Figure 2). Figure 3 The strain after single and double crossover verification was named WB600ΔbglS.

[0109] Example 2: Construction of alkaline protease producing strain

[0110] The alkaline protease gene AprE was introduced into the above-mentioned genetically engineered bacteria WB600ΔbglS.

[0111] (1) The nucleotide sequence containing the pLY-2 promoter and amyE signal peptide (SEQ ID NO. 7), and the alkaline protease encoding gene (SEQ ID NO. 3) were synthesized by a biological company;

[0112] (2) The pWB980 plasmid was extracted using a kit and double digested with EcoRI and BamHI using the same enzyme digestion system as in Example 1;

[0113] (3) 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 using seamless cloning enzyme to obtain the recombinant plasmid Ply-2 -SP amyE -AprE-pWB980; the connection system is as follows:

[0114]

[0115] After mixing the system, react at 50°C for 15 min;

[0116] (4) The recombinant plasmid obtained in step (3) was transformed into Bacillus subtilis WB600 and WB600ΔbglS, respectively, as described below;

[0117] ① Pick newly activated single colonies of Bacillus subtilis WB600 and WB600ΔbglS respectively and culture them in 5 mL LB liquid medium at 37°C and 220 rpm overnight;

[0118] ② Transfer 100 μL of culture medium to 5 mL of SPI medium and culture at 37°C and 220 rpm until the end of logarithmic growth OD 600 =1.2 (about 3-4 h);

[0119] ③ Take 200 μL of culture medium grown to the end of the logarithmic phase and add it to 2 mL of SPII medium. Incubate at 37°C and 100 rpm for 1.5 h.

[0120] ④ Add 20 μL of 10 mmol / L EGTA to the cells in the above SPII medium and culture at 37°C, 100 rpm for 10 min;

[0121] ⑤Add ligation product: recombinant plasmid P ly-2 -SP amyE -AprE-pWB980, 37°C, 100 rpm, 30 min;

[0122] ⑥ Adjust the speed to 220 rpm and continue incubation for 1.5 hours. Plate the bacterial solution onto LB selection plates containing 100 μg / mL kanamycin and incubate at 37°C for 12 hours. Screen for positive transformants for verification. The resulting engineered strains producing alkaline protease were named WB600-AprE and WB600ΔbglS-AprE.

[0123] Example 3: Construction of an aminopeptidase-producing strain

[0124] The same method as in Example 2 was used to construct the aminopeptidase encoding gene expression plasmid P 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.

[0125] 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 in Example 2).

[0126] Example 4: Construction of a mesophilic α-amylase producing strain

[0127] The same method as in Example 2 was used to construct the medium-temperature α-amylase encoding gene expression plasmid P ly-2 -SP amyE - AmyE-pWB980, only the alkaline protease encoding gene is replaced with the mesophilic α-amylase encoding gene AmyE shown in SEQ ID NO.5.

[0128] 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, named WB600-AmyE and WB600ΔbglS-AmyE, respectively (the construction process was basically the same as that in Example 2).

[0129] Example 5: Construction of a thermostable α-amylase producing strain

[0130] The same method as in Example 2 was used to construct the expression plasmid P encoding the gene for thermostable α-amylase. 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.

[0131] 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, named WB600-AmyD and WB600ΔbglS-AmyD, respectively (the construction process was basically the same as that in Example 2).

[0132] Example 6: Production of alkaline protease, aminopeptidase, mesophilic α-amylase and thermostable α-amylase using genetically engineered bacteria

[0133] 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 streaked on LB plates, inverted and cultured overnight at 37°C. Activated single colonies were picked and placed in 5 mL of LB medium, shaken and cultured at 37°C, 220 r / min for 12 h, and then transferred to 50 mL of LB liquid medium at a 2% inoculum size until the OD reached 0. 600 When the concentration reaches 0.8-1.0, the inoculum volume is transferred to a baffled bottle containing 100 mL of fermentation medium at a 2% inoculum volume and cultured at 37°C and 220 r / min for 60 h.

[0134] Samples were taken at fixed points at 12, 24, 36, 48, and 60 hours, centrifuged at 4°C and 12,000 r / min for 2 minutes, and the fermentation supernatant was taken. After appropriate dilution, the alkaline protease activity was determined according to the national standard method, the aminopeptidase activity was determined according to the LNA method, and the medium-temperature α-amylase and high-temperature α-amylase activities were determined according to the national standard method. The results are as follows Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 shown.

[0135] Among them, the highest alkaline protease activity in the fermentation broth after 48 h reached 28763.56 U / mL, an increase of 20.28% compared with the control bacteria; the highest aminopeptidase activity in the fermentation broth after 36 h reached 9894.99 U / mL, an increase of 15.00% compared with the control bacteria; the highest mesophilic α-amylase activity in the fermentation broth after 48 h reached 283.76 U / mL, an increase of 25.76% compared with the control bacteria; the highest thermostable α-amylase activity in the fermentation broth after 48 h reached 958.53 U / mL, an increase of 20.45% compared with the control bacteria (Table 3);

[0136] It can be seen that the present invention knocks out the glucanase encoding gene in Bacillus subtilis, and the resulting bacteria can efficiently heterologously express multiple enzymes such as alkaline protease, aminopeptidase, mesophilic α-amylase, and thermostable α-amylase, and provides an innovative idea for constructing a chassis strain with high production of multiple enzymes.

[0137] Table 3 Enzyme production experiment

[0138]

[0139] The present invention increases the yield of industrial enzymes by enhancing 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 levels of other Bacillus-related genes for culturing bacteria, producing compounds, or expressing other proteins.

[0140] Although the present invention has been disclosed 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 use of a genetically engineered Bacillus subtilis substrate in the expression of alkaline protease, aminopeptidase, thermostable α-amylase or mesophilic α-amylase, characterized in that: The base bacteria is obtained by deleting the glucanase encoding gene on the host genome of Bacillus subtilis WB600. s The expression is obtained; The gene encoding the alkaline protease is shown in SEQ ID NO.3; the gene encoding the aminopeptidase is shown in SEQ ID NO.4; the gene encoding the mesophilic α-amylase is shown in SEQ ID NO.5; and the gene encoding the thermostable α-amylase is shown in SEQ ID NO.

6.

2. The use according to claim 1, wherein The amino acid sequence of the glucanase is shown in SEQ ID NO.

1.

3. A genetically engineered bacterium for producing alkaline protease, aminopeptidase, mesophilic α-amylase or thermostable α-amylase, characterized in that: It is obtained by heterologously overexpressing alkaline protease, aminopeptidase, mesophilic α-amylase or thermostable α-amylase on the basis of genetically engineered bacteria. The base bacteria is obtained by deleting the glucanase encoding gene on the host genome of Bacillus subtilis WB600. s The expression is obtained; The gene encoding the alkaline protease is shown in SEQ ID NO.3; the gene encoding the aminopeptidase is shown in SEQ ID NO.4; the gene encoding the mesophilic α-amylase is shown in SEQ ID NO.5; and the gene encoding the thermostable α-amylase is shown in SEQ ID NO.6.

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