CN113151270A - Promoter for efficiently expressing alkaline protease and application thereof - Google Patents

Abstract

The invention provides a novel promoter P64 for improving the activity of alkaline protease, wherein the promoter is derived from bacillus amyloliquefaciens and is obtained by a genome transcriptome data analysis and enzyme activity detection method. Under the regulation and control of a novel promoter P64, the alkaline protease gene is efficiently expressed in a recombinant engineering strain Bacillus amyloliquefaciens P64-pLF/11018, the enzyme activity reaches 12751U/mL, is 13.6 times of that of wild type Bacillus amyloliquefaciens (938U/mL), and has great industrial application potential. The invention not only is applied to industrial production, but also lays a foundation for the research of the high-efficiency expression of the exogenous gene in the bacillus amyloliquefaciens, and has important significance for promoting the further development of the gene technology and the microbial metabolic engineering.

Description

Promoter for efficiently expressing alkaline protease and application thereof

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a promoter for efficiently expressing an alkaline protease gene and application thereof.

Background art:

bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a bacterium with high affinity with Bacillus subtilis and can inhibit the growth of various plant pathogenic bacteria. In its own growth process, a series of metabolites can be produced, which enable bacillus amyloliquefaciens to have broad fungal and bacterial inhibitory activity. Meanwhile, the plant growth promoting agent has the function of promoting plant growth, and can enhance the autoimmune system by inducing plants to improve the disease resistance. Research shows that the bacillus amyloliquefaciens has stronger protein secretion capacity and has outstanding expression in the aspect of heterologous gene expression hosts.

The protease used as an industrial enzyme preparation can catalyze the hydrolysis of protein and polypeptide and has important application value in cheese production, meat tenderization and vegetable protein modification. Compared with the protease from animals and plants, the protease from microorganisms has the characteristics of relatively simple downstream technical treatment, low price, easy culture of thalli and high yield. In recent years, researchers at home and abroad make a great deal of work on alkaline protease gene engineering, and great progress is made. Tang et al constructed recombinant expression plasmids of alkaline protease genes using Bacillus shuttle expression vectors, and carried out high expression studies in Bacillus subtilis. The protease gene is obtained by cloning from the bacillus pumilus, and the like, and the protease gene is expressed in escherichia coli and the bacillus pumilus respectively, so that the enzyme activity is improved by two times. Bacillus amyloliquefaciens is an important industrial microorganism and is a production host of various industrial enzymes (protease, alpha-amylase and beta-glucanase), so the research on the Bacillus amyloliquefaciens alkaline protease becomes a hotspot. The royal jelly and the like take bacillus amyloliquefaciens as a host, and the activity of the alkaline protease is improved by 25 percent by optimizing an expression element.

Promoters are essential regulatory elements for gene expression in bacteria, and determine the strength and timing of gene expression. The expression of the gene can be changed by inserting or deleting the promoter, and the research on the growth and development of the thalli and the metabolic regulation and control can be realized. The promoter is also the basis for constructing various expression systems and realizing the expression of heterologous genes. At present, the research on the efficient expression of protease by using a promoter derived from bacillus amyloliquefaciens is not much, and the number of promoters which can be perfectly used for constructing industrial strains is extremely limited in terms of the existing promoters, so that the starting capacity and the regulation and control mode have many problems. Therefore, the method is very important for obtaining more promoters with high transcription strength and has great significance for researching the functions of the bacillus amyloliquefaciens genome.

The invention content is as follows:

for the above reasons, the present invention aims to provide a promoter for efficiently expressing an alkaline protease gene, and applications of the promoter.

The technical scheme of the invention is as follows:

a promoter P64 for efficiently expressing alkaline protease gene has the nucleotide sequence shown in SEQ ID NO. 1.

Meanwhile, the invention also provides a recombinant vector containing the promoter P64.

Accordingly, the present invention also provides a host cell containing the above recombinant vector.

In addition, the invention also provides application of the promoter P64 in expression of alkaline protease genes.

In addition, the invention provides primers P64-F and P64-R capable of amplifying the promoter P64, and the nucleotide sequences are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3.

Preferably, the recombinant vector containing the promoter P64 is P64-pLF-AprE, and is constructed by the following method: 1) taking an escherichia coli-bacillus shuttle vector as a template, and performing reverse PCR amplification to obtain a linear vector fragment; 2) connecting a linear vector fragment with the promoter P64, transforming the connected product into a host cell for culture and screening positive clones; 3) and collecting the screened positive transformants, extracting plasmids for sequencing, and obtaining the P64-pLF-AprE with correct sequencing. Wherein, the primers used for the reverse PCR amplification in the step 1) are pLF-F and pLF-R, and the nucleotide sequences are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5.

Preferably, the invention provides a host cell containing P64-pLF-AprE, wherein the host cell is Escherichia coli or Bacillus amyloliquefaciens.

The novel strong promoter P64 is obtained by screening from a genome of amylolytic bacillus amyloliquefaciens, can efficiently express alkaline protease genes in the amylolytic bacillus amyloliquefaciens, has the corresponding enzyme activity after expression 13.6 times of that of a wild type, and has good application potential in the field of industrial enzyme preparations. In addition, the invention also constructs a recombinant vector and an engineering strain containing the promoter P64. The invention can be applied to industrial production and also provides a powerful tool for the research of the high-efficiency expression of the exogenous gene in the bacillus amyloliquefaciens.

Description of the drawings:

FIG. 1 shows the result of electrophoresis of the promoter fragment; wherein, the lane M is a nucleic acid molecular weight standard, and the 240bp bands in the lanes 1 and 2 are promoter amplification fragments.

FIG. 2 electrophoresis results of the linear vector fragment; wherein, Lane M is a nucleic acid molecular weight standard, and the band at 6.9kb in Lane 1-4 is an amplified fragment of the linear vector.

FIG. 3 shows the results of enzyme digestion verification of the recombinant vector; wherein, lane M is a nucleic acid molecular weight standard, and lanes 1-4 are bands at 1.2kb and 5.9kb, which are cleavage product bands.

FIG. 4 general flow chart of recombinant vector construction.

FIG. 5 shows the restriction enzyme digestion verification result of the Bacillus amyloliquefaciens positive transformant; wherein, lane M is a nucleic acid molecular weight standard; the bands at 1.2kb and 5.9kb in lanes 1 and 2 are the bands of the cleavage products.

FIG. 6 shows the results of enzyme activity assays of wild type Bacillus amyloliquefaciens and constructed engineering bacteria P64-pLF/11018 and PA-pLF/11018.

The specific implementation mode is as follows:

the process of the invention is described below by means of specific embodiments. Unless otherwise specified, all technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims.

The strains used were: escherichia coli JM109, Escherichia coli EC135, and Bacillus amyloliquefaciens 11018 (culture Collection of microorganisms of Tianjin science and technology university).

Enzymes and reagents used: restriction enzymes SacI, KpnI, plasmid extraction kit and GoldstarDNA polymerase were purchased from Takara, and ligase BiscMIX was purchased from Quanyujin. Trichloroacetic acid, folin phenol, glycerol were purchased from Tianjin Bai Oxetai.

The culture medium used:

LB medium (g/L): peptone 10, NaCl 10 and yeast powder 5.

Fermentation medium (g/L): corn flour 64, bean cake powder 40, high-temperature amylase 0.7, potassium dihydrogen phosphate 0.4 and disodium hydrogen phosphate 4.

Example 1 promoter screening

Performing transcriptome sequencing (sequencing is completed by Jinzhi Biotechnology, Inc. of Suzhou) on the bacillus amyloliquefaciens to obtain transcriptome sequencing data; the expression level is measured by the TPM value of the gene expression quantity in the transcriptome data, the TPM value is sequenced, and the promoter sequence with higher gene transcription level in each period including P64 is selected.

Example 2 extraction of Bacillus amyloliquefaciens genome

(1) Collecting the bacterial liquid cultured in the culture medium overnight, centrifuging for 10min at 4000r/min, discarding the supernatant, adding 100 mu l of TE Buffer, and then oscillating for resuspension;

(2) adding 15 μ L (50mg/mL) of lysozyme, and carrying out water bath at 37 ℃ for 10 min;

(3) adding 20 mu L of proteinase K and 60 mu L of 10% SDS, and carrying out water bath for 2-3h until the mixture is clear;

(4) adding phenol-like substance with the same volume, shaking, mixing, centrifuging at 12000r/min for 5min, and collecting supernatant;

(5) adding 60-80% isopropanol, turning upside down, mixing, standing at-70 deg.C for 5min, centrifuging at 12000r/min for 5min, and removing supernatant;

(6) washing with 500 μ L ethanol twice, centrifuging at 12000rmp for 5min, removing supernatant, and air drying for 5-10 min;

(7) the obtained genome was dissolved in 50. mu.L of sterile water.

Example 3 cloning of promoter and vector fragments

The upstream and downstream primers P64-F/P64-R were designed and synthesized based on the sequence of the promoter P64 (primer synthesis was performed by Jinzhi Biotech, Suzhou). The nucleotide sequences of the primers P64-F and P64-R are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

Taking the bacillus amyloliquefaciens genome as a template and taking P64-F/P64-R as a primer to carry out PCR amplification reaction. The PCR reaction system and the reaction procedure are shown in Table 1 and Table 2, respectively. After the reaction, a fragment of about 240bp was obtained, and the product was recovered by cutting gel and verified by agarose gel electrophoresis, the results of which are shown in FIG. 1.

Table 1 PCR amplification reaction System for promoters

GoldStar: the kit comprises DNA polymerase, PCR Buffer and Mg²⁺dNTPs, etc. in a pre-mixing system

TABLE 2 PCR amplification reaction procedure for promoters

An Escherichia coli-bacillus shuttle vector (PA-pLF-AprE, containing Escherichia coli and bacillus replicon, chloramphenicol resistance genes, kanamycin resistance genes and alkaline protease gene AprE) is used as a template, and primers pLF-F/pLF-R are designed and synthesized by utilizing SnapGene software. The nucleotide sequences of pLF-F and pLF-R are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively. Reverse PCR was performed using the primers pLF-F/pLF-R, and the reaction system and procedure are shown in Table 3 and Table 4, respectively. After completion of the amplification, a linear vector fragment pLF-AprE of about 6.9kbp was obtained, and the product was recovered by cutting the gel and verified by agarose gel electrophoresis, the result of which is shown in FIG. 2.

TABLE 3 PCR amplification reaction System for Linear vectors

Primertar star: the prepared premixed system in the kit comprises polymerase, buffer solution and Mg²⁺dNTPs, etc

TABLE 4 PCR amplification reaction procedure for Linear vectors

Example 4 construction of recombinant vector

The PCR product of the linear vector pLF-AprE is recovered by cutting gel, then is connected with a purified and recovered promoter P64, and the connected product is transformed into Escherichia coli JM 109. The transformation process is as follows: (1) adding appropriate amount of ligation product into the competence, gently mixing, and standing on ice for 30 min; (2) putting the mixture into 42 ℃ water bath for 90s, carrying out ice bath for 2min, adding a resuscitation solution, and carrying out shaking culture at 37 ℃ and 220r/min for 40 min; (3) centrifuging at 4000r/min for 5min, spreading a proper volume of bacterial liquid on a chloramphenicol resistant plate, and culturing at 37 ℃ overnight.

And (4) selecting positive transformants, transferring the positive transformants to a liquid LB culture medium, culturing for a period of time, and extracting plasmids for enzyme digestion verification analysis. The results of agarose gel electrophoresis after digestion are shown in FIG. 3: after being cut by KpnI/SmaI enzyme, the fragment of about 5900bp and 1200bp is obtained. After enzyme digestion verification, the promoter sequence is sent to a sequencing mechanism (Suzhou Jinzhi Biotechnology, Inc.) for sequencing. The plasmid with correct sequencing is the successfully constructed recombinant vector P64-pLF-AprE. FIG. 4 is a general flow chart of the construction of the recombinant vector of the present invention.

Example 5 construction of engineered Strain

1. And transferring the successfully constructed vector to escherichia coli for methylation modification. The specific process is as follows: (1) adding appropriate amount of recombinant vector P64-pLF-AprE into the competence of Escherichia coli EC135, mixing gently, and standing on ice for 30 min; water bath at 42 deg.c for 90 sec and ice bath for 2 min; (2) adding a resuscitation solution after ice bath, and carrying out shaking culture at 37 ℃ for 220r/min for a period of time; (3) centrifuging at 4000r/min for 5min, coating a proper volume of bacterial liquid on a resistant plate, and culturing at 37 ℃ overnight; (4) after 12h, selecting a transformant single colony, culturing the transformant single colony in a 5mL LB test tube until the OD of the thallus reaches 0.2, and adding an arabinose inducer; (5) after further overnight incubation, plasmids were extracted.

2. The extracted recombinant plasmid is electrically transferred to bacillus amyloliquefaciens. The specific process is as follows: (1) adding 5 mu L of recombinant plasmid into 80 mu L of bacillus amyloliquefaciens 11018 competent cell suspension, mixing and transferring into an electric rotating cup; (2) standing for 2min, performing electric shock at 2500V, immediately adding resuscitation solution after electric shock, mixing, and resuscitating in a shaking table at 37 deg.C for 3 hr; (3) coating the bacterial suspension on a Kan resistant LB plate, culturing at 37 ℃ for a period of time, and screening positive transformants; (4) the positive transformants were selected and inoculated in LB liquid medium and cultured overnight at 37 ℃ at 220 r/min. The transformant was identified by digestion with recombinant plasmid, and the results of agarose gel electrophoresis after digestion are shown in FIG. 5: after the enzyme digestion is carried out by KpnI/SmaI, fragments of about 5900bp and 1200bp are obtained, which shows that the construction of the engineering strain of the bacillus amyloliquefaciens P64-pLF/11018 is successful.

The same construction method of the engineering strain is adopted to construct the engineering bacterium bacillus amyloliquefaciens PA-pLF/11018 containing the shuttle vector (PA-pLF-AprE) of the escherichia coli-bacillus.

Example 6 expression and analysis of engineered strains

The activity of the promoter for expressing alkaline protease in Bacillus amyloliquefaciens was evaluated and compared.

The constructed engineering bacteria P64-pLF/11018 and PA-pLF/11018 and the wild type bacillus amyloliquefaciens 11018 are selected for fermentation culture, and the specific operation is as follows: respectively inoculating the 3 strains into LB liquid culture medium, performing shaking culture at 37 ℃ at 220r/min for 12h, then respectively transferring the strains into triangular flasks containing 100mL of fermentation culture medium at 2% of inoculum size, performing shaking culture at 37 ℃ at 220r/min for 48 h; taking the fermentation liquor, centrifuging for 3min at 12000r/min, and taking the supernatant for enzyme activity determination.

The enzyme activity determination method is carried out according to a GB/T23527-2009 appendix B Folin phenol method. 1 enzyme activity unit (U/mL) is defined as the amount of enzyme required for hydrolyzing casein to produce 1. mu.g of tyrosine in 1mL of enzyme solution at 40 ℃ and pH of 10.5 for 1 min.

The results of enzyme activity measurements of the three bacteria are shown in Table 5 and FIG. 6. As can be seen from the figure and the table, the enzyme activity of the alkaline protease expressed by the promoter P64 in the engineering bacteria P64-PLF/11018 is 13.6 times of that of the wild type, and is also obviously higher than that of the protease expressed by the PA promoter of the bacillus subtilis.

TABLE 5 determination of the alkaline protease enzyme activity of the three bacteria

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Claims (7)

1. The nucleotide sequence of a promoter P64 of an alkaline protease gene is shown as SEQ ID NO. 1.

2. A recombinant vector comprising the promoter P64 of claim 1.

3. A host cell comprising the recombinant vector of claim 2.

4. The use of the promoter P64 of claim 1 for the expression of alkaline protease gene.

5. Primers P64-F and P64-R for amplifying the promoter P64 of claim 1, wherein: the nucleotide sequences of the primers P64-F and P64-R are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3.

6. The recombinant vector according to claim 2, wherein: the recombinant vector is P64-pLF-AprE and is constructed by the following method: 1) taking an escherichia coli-bacillus shuttle vector as a template, and performing reverse PCR amplification to obtain a linear vector fragment; 2) connecting a linear vector fragment with the promoter P64, transforming the connected product into a host cell for culture and screening positive clones; 3) collecting the screened positive transformants, extracting plasmids for sequencing, wherein the correctly sequenced positive transformants are P64-pLF-AprE;

wherein, the primers used for the reverse PCR amplification in the step 1) are pLF-F and pLF-R, and the nucleotide sequences are respectively shown as SEQ ID NO. 4 and SEQ ID NO. 5.

7. A host cell comprising the recombinant vector of claim 6, wherein: the host cell is escherichia coli or bacillus amyloliquefaciens.

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