CN112522173B - Engineering bacterium for producing heterologous alkaline protease and construction method thereof - Google Patents

Abstract

The invention aims to provide a genetically engineered bacterium capable of efficiently and stably producing heterologous protease by directionally modifying a genetically engineered host, wherein bacillus amyloliquefaciens is used as the host, and 8 extracellular proteases aprE, epr, mpr, vpr, nprE, bpr, wprA, aprX of the host are deleted to obtain an extracellular protease defective strain; the host alpha-amylase and extracellular polysaccharide gene cluster eps are also deleted; and introducing the high-copy plasmid for expressing the heterologous alkaline protease into a host to obtain a genetically engineered bacterium for efficiently producing the heterologous alkaline protease. In addition, the viscosity of the engineering bacteria is greatly reduced in the fermentation process, and the separation and purification of the product protease are facilitated.

Description

Engineering bacterium for producing heterologous alkaline protease and construction method thereof

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and in particular relates to engineering bacteria for producing heterologous alkaline protease and a construction method thereof.

Background

Alkaline protease (Alkaline protease), an enzyme capable of catalyzing and hydrolyzing peptide bonds, the active center of which contains serine, also known as serine protease, is an enzyme capable of hydrolyzing protein peptide bonds in the pH value alkalescence range, and has the functions of hydrolyzing peptide bonds, hydrolyzing amide bonds, ester bonds, and transesterifying and transpeptiding. Such enzymes are widely found in animal pancreas, bacteria, mold, and the enzyme activity can be specifically inhibited by Diisopropylphosphoryl Fluoride (DFP), phenylmethylsulfonyl fluoride (PMSF), potato Inhibitor (PI), and the like. Alkaline proteases find wide use in the food, washing, and tanning industries. As the protease secreted by the microorganism is extracellular enzyme, compared with the protease of animal and plant sources, the protease has the characteristics of relatively simple downstream technical treatment, low cost, wide source, easy culture of thalli, high yield and the like; and the high-yield strain is simple and quick to breed, has stronger hydrolysis capability and alkali resistance capability compared with neutral protease, has larger heat resistance and certain esterase activity, and is easy to realize industrial production.

The species used industrially for the production of proteases are mainly bacillus species, including: bacillus licheniformis, bacillus subtilis and bacillus amyloliquefaciens, wherein, bacillus amyloliquefaciens has strong secretion ability to make it the most potential heterologous protein expression host, in addition, bacillus amyloliquefaciens has the following advantages: (1) Is internationally accepted GRAS organism, is non-pathogenic, does not produce exotoxin and endotoxin, and has no pollution to the environment; (2) The cell wall has simple composition, is convenient for secretion of protein, and does not contain pyrogenic lipopolysaccharide; (3) Many phages and plasmids used in molecular biology experiments can be used as tools for transformation, and recombinant DNA is easy to transfer; (4) The protein is directly secreted into extracellular culture medium, so that the protein cannot accumulate, the downstream recovery and purification of the protein are facilitated, and the operation cost of the whole production chain is reduced; (5) The bacillus is a single-cell organism, can reach very high cell density in the fermentation process, has relatively simple culture medium, low cost and high yield, and meets the requirements of industrial production. The method is widely applied to the industrial production of foods and enzyme preparations at present, such as the large-scale production of alpha-amylase, protease, feed additives, beta-glucanase and the like. However, in the aspect of the expression of heterologous proteins, the bacillus amyloliquefaciens still has the problems of unstable expression system and low expression quantity, and as the bacillus amyloliquefaciens can secrete a plurality of extracellular proteases, the enzymes can have strong degradation effect on the heterologous proteins, so that the reduction of the degradation effect of the protease of the bacteria is a key for improving the expression of the exogenous proteases.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a genetically engineered bacterium capable of efficiently and stably producing heterologous protease through directional transformation of a genetically engineered host. In addition, the viscosity of the engineering bacteria is greatly reduced in the fermentation process, and the separation and purification of the product protease are facilitated.

The technical scheme of the invention is summarized as follows:

a genetically engineered bacterium takes bacillus amyloliquefaciens as a host, 8 extracellular proteases aprE, epr, mpr, vpr, nprE, bpr, wprA, aprX of the host are deleted to obtain an extracellular protease defective strain; the host alpha-amylase and extracellular polysaccharide gene cluster eps are also deleted; and introducing the high-copy plasmid for expressing the heterologous alkaline protease into a host to obtain a genetically engineered bacterium for efficiently producing the heterologous alkaline protease.

Preferably, the preservation number of the bacillus amyloliquefaciens is CGMCC No.11218 (biological preservation information is disclosed in patent CN 105087448B).

Preferably, the heterologous alkaline protease is an alkaline protease from Bacillus alcalophilus.

Preferably, the high copy plasmid expressing the heterologous alkaline protease contains a nucleotide sequence as set forth in SEQ ID NO:1 and a signal peptide amyE.

The invention also provides application of the genetically engineered bacterium in fermentation production of alkaline protease, wherein the enzyme activity of the alkaline protease in fermentation broth of the genetically engineered bacterium reaches 19524U/mL or 64328U/mL at most.

The invention also provides a construction method of the genetically engineered bacterium, in a specific implementation mode, 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide coding gene clusters on a host genome are knocked out in series mainly by using a temperature-sensitive plasmid-mediated homologous recombination method, and an expression plasmid containing heterologous alkaline protease genes is introduced.

The invention also provides a method for efficiently producing the heterologous alkaline protease, which comprises the steps of culturing the genetically engineered bacterium under proper conditions and collecting the alkaline protease from the culture.

The invention has the beneficial effects that:

the invention constructs a strain with main extracellular enzyme deletion by knocking out 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide gene clusters eps on a bacillus amyloliquefaciens genome in series, simultaneously introduces high-copy plasmid expressing alkaline protease from bacillus alcalophilus into the strain to obtain genetic engineering bacteria, and the activity of recombinant alkaline protease in fermentation broth is 19524U/mL at the highest after 48h fermentation culture; in a 5L fermentation tank, the activity of the recombinant alkaline protease reaches 64328U/mL, and the genetically engineered bacterium provided by the invention obviously improves the expression quantity of the heterologous protease. The invention reduces the influence of self protease on the expression of the exogenous alkaline protease by knocking out the main extracellular enzyme secreted by the bacillus amyloliquefaciens. The method is simple and easy to implement, is suitable for a bacillus amyloliquefaciens system, lays a foundation for mediating the efficient expression of the heterologous protease gene in the bacillus amyloliquefaciens expression system, promotes the industrialized mass production of the heterologous protease, and can be applied to the efficient expression of other heterologous genes.

Proteases secreted by cells are classified into intracellular and extracellular proteases, and the extracellular proteases are required to enter a secretion channel under the guidance of a signal peptide and to exert various physiological functions by secretion to the outside of the cell. In bacillus amyloliquefaciens, there are mainly 4 protein secretion pathways: the Sec, tat, com and ABC secretion pathways, most of which rely on the Sec pathway for secretion, and the signal peptide starting with 2 arginines is secreted via the Tat pathway. Analysis by Signal P software of alkaline protease and a-amylase Signal peptide is a Sec secretory pathway Signal peptide, both are through the Sec pathway secretion. And combining the analysis result of mass spectrometry detection on the fermentation broth to obtain an extracellular product expressed by bacillus amyloliquefaciens CGMCC No.11218, wherein the expression quantity of a-amylase is highest (shown in figures 4 and 5), and supposing that the secretion of the a-amylase occupies a secretion channel of alkaline protease, so that the encoding gene of the a-amylase and the extracellular polysaccharide encoding gene cluster eps are knocked out, and the viscosity of the fermentation broth is reduced by deleting the main extracellular enzyme secreted by the bacillus amyloliquefaciens and the eps gene cluster for blocking the synthesis of extracellular polysaccharide, so that the separation and purification of the product are simplified while the expression quantity of target protein is improved.

Drawings

Fig. 1: a construction process of a temperature-sensitive knockout carrier T2;

fig. 2: principle of homologous recombination knockout.

Fig. 3: extracellular protease knockout strain nucleic acid electrophoresis verification (wherein Δ represents a gene knockout strain, CK is a control with a non-knockout strain as a template).

Fig. 4: SDS-PAGE of recombinant strain J1 fermentation broth.

Fig. 5: and (5) mass spectrometry analysis results of fermentation liquor.

Fig. 6: and (5) performing nucleic acid electrophoresis verification on the amyE gene knockout strain.

Fig. 7: schematic diagram of eps gene cluster structure.

Fig. 8: and (5) performing nucleic acid electrophoresis verification on the eps gene cluster knockout strain.

Fig. 9: recombinant strains J1, J2 and J3 were verified by nucleic acid electrophoresis.

Detailed Description

The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

The culture medium and the enzyme activity measurement method used in the following embodiments are as follows:

seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 5g/L of sodium chloride;

fermentation medium: 64g/L corn flour, 40g/L bean cake powder, 4g/L disodium hydrogen phosphate, 0.3g/L potassium dihydrogen phosphate and 0.7g/L high temperature amylase.

E.coli competent preparation medium:

LB medium: 5g/L of yeast powder, 10g/L of peptone and 5g/L of sodium chloride; caCl (CaCl) ₂ Solution: 0.1mol/L.

Bacillus amyloliquefaciens competent preparation medium:

LBS medium: 5g/L of yeast powder, 10g/L of peptone, 5g/L of sodium chloride and 9.1085g/L of sorbitol;

resuscitating medium: 5g/L of yeast powder, 10g/L of peptone, 5g/L of sodium chloride, 9.1085g/L of sorbitol and 6.92246g/L of mannitol.

The method for measuring the enzyme activity of the alkaline protease used in the invention is carried out according to the GB/T23527-2009 annex B Fu Lin Fen method, namely 1 enzyme activity unit (U/mL) is defined as the amount of enzyme required by 1mL of enzyme solution to react for 1min at 40 ℃ and pH of 10.5 to hydrolyze casein to generate 1 mug of tyrosine.

The nucleotide sequences of the genes involved in the following embodiments are as follows (NCBI accession numbers):

aprE: GU825966.1 (594 bp to 1742 bp);

epr: CP054415.1 (genome-wide 3751547bp to 3753295 bp);

mpr: CP054415.1 (genome-wide 886397bp to 887311 bp);

vpr: CP002634.1 (genome-wide 3706130bp to 3708541 bp);

nprE: k02497.1 (254 bp to 1819 bp);

bpr: CP054415.1 (genome-wide 1624354bp to 1628643 bp);

wprA: CP018902.1 (genome-wide 310740bp to 313415 bp);

aprX: CP018902.1 (genome-wide 983721bp to 985049 bp);

amyE: MT590601.1 (564 bp to 2108 bp);

eps: CP018902.1 (genome-wide 2456935bp to 2472647 bp).

The technical scheme of the invention is further detailed as follows:

the invention constructs a genetic engineering strain for efficiently producing heterologous proteinase, which mainly uses a temperature-sensitive plasmid-mediated homologous recombination method to perform tandem knockout on 8 extracellular proteinase genes, alpha-amylase genes and extracellular polysaccharide coding gene clusters on a host genome, and introduces an expression plasmid containing the heterologous alkaline proteinase genes, and comprises the following steps:

1) According to the nucleotide sequences of aprE, epr, mpr, vpr, nprE, bpr, wprA, aprX, amyE genes and extracellular polysaccharide encoding gene clusters eps on the genome of bacillus amyloliquefaciens CGMCC No.11218, the software Snapge is utilized to respectively design respective upper and lower homologous arm primers, the length of the homologous arm is controlled to be about 400-500 bp, and the homologous arm mediated knockout efficiency of the length is higher;

2) Purifying and recovering the amplified fragments of the upper and lower homology arms, and carrying out overlapped PCR (polymerase chain reaction) amplification by taking the recovered products as templates to obtain overlapped fragments of the upper and lower homology arms;

3) Purifying and recovering the overlapped fragments, carrying out double enzyme digestion on the recovered products by Sac1 and Sma1, and connecting the purified products with T2 temperature-sensitive type vectors cut by the same enzyme to obtain 10 different temperature-sensitive knockout vectors;

4) Transferring the connection product into competent cells of EC135 for methylation modification, transferring the modified plasmid into bacillus amyloliquefaciens CGMCC No.11218, subculturing the positive transformant in LB test tubes with 45 ℃ and kanamycin resistance, and under the condition, the replicon of the knockout vector cannot normally replicate, thereby screening out a single exchange strain of the knockout vector integrated on a host genome; subculturing the strain subjected to single exchange in an antibiotic-free LB test tube at 37 ℃ for about 5 generations, performing a spot plate test, preliminarily considering that the colony growing on the antibiotic-free plate is successful in knockout due to the fact that the colony is not long on the kanamycin-resistant plate, and obtaining a single gene knockout strain through colony PCR verification and sequencing;

5) Electrotransferring the second knockout plasmid to competent cells of the single-gene knockout strain in the step 4), performing single-exchange and double-exchange for 2 passages, screening out the knockout strain with the second gene deletion, and sequencing and verifying; continuing to repeat the operation of the step 4) on the basis, transferring into a third knockout plasmid and a fourth knockout plasmid …, and finally obtaining 8 strains with extracellular protease genes, alpha-amylase genes and extracellular polysaccharide gene cluster eps which are knocked out in series;

6) The expression plasmid containing the heterologous alkaline protease gene is electrically transferred into a primary host CGMCC No.11218 by utilizing an electric shock conversion mode, and a positive transformant is obtained by screening and is marked as J1; meanwhile, the expression plasmid is electrically transformed into the strain with 8 extracellular protease genes knocked out in the process of 5), and the strain is marked as J2; simultaneously, the expression plasmid is electrically transferred to the strain with 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide gene cluster eps which are finally obtained in the step 5) and knocked out in series, and the correct transformant is verified to be the final engineering bacterium, and is marked as J3;

7) Shake flask fermentation was performed on recombinant strains J1, J2, J3, and alkaline protease activities of the three recombinant strains were determined after 48h.

It should be noted that the terms first and second used in the detailed description of the solution merely denote distinguishing one operation object from another operation object, and do not limit or imply that there is a real sequential relationship between these operation objects.

The following examples further describe the specific operation of each step and the results obtained.

Example 1:

a genetically engineered bacterium for producing heterologous alkaline protease and a construction method thereof.

1. Obtaining overlapping fragments of homologous arms of the knocked-out gene (epr is taken as an example).

According to the whole genome sequence information of bacillus amyloliquefaciens, using an epr gene as an example, designing an upstream and downstream homology arm primer of the epr through Snapge, amplifying a target fragment by utilizing PCR, purifying and recovering the amplified upstream and downstream homology arm fragments, and carrying out overlapping PCR by taking the recovered product as a template, wherein the sequence of the primer and the enzyme cutting site are shown in a table 1.

The reaction system used in amplifying the Epr homology arms was 50. Mu.L, as follows:

the annealing temperature of Epr was 56℃and the extension time corresponds to the length of the gene, the reaction procedure being as follows:

the reaction system used for overlap PCR was 50. Mu.L, as follows:

the annealing temperature of the overlap PCR was 56℃and the extension time corresponds to the length of the gene, the reaction procedure was as follows:

2. construction of knockout vectors.

And (3) double-enzyme digestion is carried out on the overlapped PCR products after purification by using Sma1-Sac1 restriction endonuclease, then the double-enzyme digestion products are recovered and then are connected with a T2 vector which is subjected to the same enzyme digestion overnight, so as to obtain the knockout vector.

The enzyme digestion system is as follows:

the connection conditions are 16 ℃,6 hours or overnight connection, and the connection system is as follows:

4.5. Mu.L of fragment of interest

Linear T2 fragment 0.5. Mu.L

Solution I 5.0μL；

3. The ligation products were transformed into competent cells of EC135 for methylation modification as follows:

(1) Taking out competence in a refrigerator at the temperature of minus 80 ℃ and immediately placing the competence on ice for 3 to 5 minutes;

(2) Sucking 8 mu L of the connection product into competent cells, gently mixing, and standing on ice for 20min;

(3) After ice bath, transferring the mixture into a water bath kettle at 42 ℃, performing heat shock for 90 seconds, immediately performing ice bath for 3 minutes, and adding 750 mu L of LB recovery liquid;

(4) Resuscitates at 37deg.C with 220r/min shaker for about 1 hr, spreads on double antibody plate containing 100 μg/mL kanamycin and 100 μg/mL Qimycin, and cultures at 37deg.C in incubator for 12 hr.

Transferring the positive transformant into liquid LB culture medium containing kanamycin and Qcomycin resistance for culturing at 37 ℃, adding arabinose for induction culture at 30 ℃ for about 12 hours when the OD600 reaches about 0.2, extracting plasmids by using the Omega small-amount rapid extraction kit, and carrying out the operation steps according to the instructions attached to the kit.

4. The knockout plasmid with successful methylation modification is electrotransformed into bacillus amyloliquefaciens, and the electrotransformation method is as follows:

(1) Cleaning the electric rotating cup with 75% alcohol, irradiating for more than 20min under an ultraviolet lamp, and pre-cooling on ice.

(2) mu.L of competent and 10ng of plasmid DNA were mixed and added to an electrocuvette and left on ice for 3min.

(3) 2500V shock, typically 4-6ms shock time.

Immediately after the electric shock, 1ml of resuscitation medium was added, and resuscitated for 3 hours at 37 ℃. Kanamycin plates were spread and incubated in an incubator at 37℃for 12h, and transformants were selected for verification.

Selecting positive transformants which are verified to be correct, putting the positive transformants into 5mL of LB liquid medium added with kanamycin resistance, shaking and culturing for 12 hours at 45 ℃ and 180r/min, transferring 10 mu L of bacterial liquid into fresh 5mL of liquid LB medium added with kanamycin, and marking the liquid LB medium as a second generation; on successive passage 3, the colonies were diluted and plated on kanamycin plates, incubated at 45℃for 12h, and colony PCR verified whether the knockdown plasmid was integrated at the corresponding location in the genome.

Transferring the colony subjected to single exchange into 5mL of non-antibiotic LB liquid medium, performing shake culture at 37 ℃ and 220r/min, recording as a generation every 12h, continuously carrying out passage for 3-4 times, diluting and coating bacterial liquid on a non-antibiotic LB agar plate, allowing the grown single colony to stand on a non-antibiotic and kanamycin-resistant agar plate, screening out the colony which does not grow on the kanamycin-resistant plate and grows on the non-antibiotic plate, and performing colony PCR verification and sequencing.

5. Then the aprE, mpr, vpr, nprE, bpr, aprX, wprA, amyE and eps gene clusters of the host are knocked out according to the steps 1-4, the homology arm amplification primers are shown in table 1, and the amplification system and the amplification procedure are the same as those in the step 1. Obtaining the knocked-out strain.

Table 1 knockout Gene homology arm amplification primers

6. The electric shock transformation method is utilized to introduce the expression plasmid containing the heterologous alkaline protease gene into the constructed 3 strains of genetic engineering bacteria, and the specific process is as follows:

(1) The nucleotide sequences of the pLY-2 promoter and the Bacillus subtilis amyE signal peptide (shown in SEQ ID NO: 1) were synthesized by Suzhou Jin Weizhi Biotechnology Co., ltd;

(2) The alkaline protease gene (the sequence is shown as GenBank: FJ 940727.1) is amplified by using the PCR technology and using the alkalophilic bacillus genome as a template, and is purified and recovered, and the primers are shown in Table 1, and the reaction system and the reaction conditions are as follows:

(3) Connecting a pLY-2 promoter with a bacillus subtilis amyE signal peptide fragment, an alkaline protease gene recovery fragment derived from bacillus alcaligenes and a linearization vector pWB980 by using a seamless cloning enzyme purchased from Beijing full gold biotechnology Co-Ltd; the connection system is as follows:

(4) The reaction system in the step (3) is reacted for 15min at 50 ℃ and then is transformed into bacillus subtilis WB600, and the method is as follows;

(1) selecting a single colony of the newly activated bacillus subtilis WB600, and culturing the single colony in 5mL of LB liquid medium at 37 ℃ for 220r/min overnight;

(2) transfer 100 μl of culture solution into 5mL SPI culture medium, culturing at 37deg.C and 220r/min until OD600 = 1.2 (about 3-4 h) at the end of logarithmic growth;

(3) 200 mu L of culture solution grown to the end of a log phase is taken to be placed in 2mL of SPII culture medium, and is cultured for 1.5h at 37 ℃ and 100 r/min;

(4) 20 mu L of 10mmol/L EGTA is added into the thallus of the SPII culture medium, and the mixture is cultured for 10min at 37 ℃ and 100 r/min;

(5) adding the connection product, and culturing at 37deg.C and 100r/min for 30min;

(6) the rotation speed is regulated to 220r/min, the culture is continued for 1.5 hours, bacterial liquid is coated on an LB screening plate containing 100 mug/mL kanamycin, the culture is carried out for 12 hours at 37 ℃, and positive transformants are screened for verification.

Extracting plasmids of the correct transformants, electrically transferring the plasmids to an original host CGMCC No.11218, verifying positive transformants, and marking the positive transformants as J1; meanwhile, electrically converting the plasmid into strains with 8 extracellular protease genes knocked out in the step 5), and marking the strains as J2; simultaneously, electrically transferring the plasmid to the knocked-out strain finally obtained in the step 5), and verifying a positive transformant and marking the positive transformant as J3; the verification result is shown in fig. 9.

Example 2:

and (5) expressing and analyzing the heterologous alkaline protease genetic engineering bacteria.

Single colonies of recombinant strains J1, J2 and J3 on fresh LB plates were inoculated into 50mL kanamycin sulfate resistant seed medium, respectively, cultured with shaking at 37℃for 12h at 220r/min, inoculated into kanamycin resistant fermentation medium at 2% inoculum size, and cultured for 48h at 37℃at 220r/min.

The enzyme activity of alkaline protease in the fermentation supernatant of the recombinant strain is measured according to the national standard GB/T23527-2009 annex B Fu Lin Fenfa, the activity of alkaline protease in the fermentation supernatant of 3 recombinant strains reaches the highest value in 48h, and the activity of recombinant alkaline protease expressed by recombinant strain J3 reaches 19524U/mL which is 1.40 times that of recombinant strain J1 (enzyme activity is 13985U/mL) and 1.15 times that of recombinant strain J2 (enzyme activity is 17042U/mL).

Example 3:

amplification experiment of recombinant alkaline protease genetically engineered bacteria.

The recombinant bacteria single colony on the plate is picked and inoculated into 100mL of LB liquid medium containing 50 mug/mL kanamycin, and the temperature is 37 ℃ and 220r/min. After 4 hours of cultivation, the culture medium was transferred to 100mL of fermentation medium at 37℃and 220r/min, and after 8 hours of cultivation, the culture medium was transferred to a fermentation tank with a total volume of 5L and a liquid loading amount of 3L at 2% for amplification experiments. Wherein the dissolved oxygen amount in the fermentation tank is controlled to be more than 40%, the stirring speed is 600-700rpm, the temperature is 37 ℃, the feeding is started when the pH=7 (the feeding culture medium is 50g/L of cottonseed protein and 300g/L of dextrin), the initial feeding amount is 100g/h, and the DE value is controlled to be 15mg/mL-18mg/mL through feeding during the whole fermentation period. Foam generation during fermentation is controlled by properly adding an antifoaming agent in the early stage of fermentation. Samples were taken every two hours, and Fu Lin Fenfa was assayed for alkaline protease activity. The measured alkaline protease activity of the recombinant strain of extracellular protease reaches a maximum of 64328U/mL after 58h of fermentation, the viscosity of fermentation liquid is greatly reduced, the dissolved oxygen is effectively improved, and the production cost is reduced.

Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

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Claims (3)

1. A genetically engineered bacterium takes bacillus amyloliquefaciens CGMCC No.11218 as a host, 8 extracellular proteases aprE, epr, mpr, vpr, nprE, bpr, wprA, aprX of the host are deleted to obtain an extracellular protease defective strain; host alpha-amylase and exopolysaccharide gene clustersepsA deletion; introducing high-copy plasmid expressing heterologous alkaline protease into a host to obtain a genetically engineered bacterium for efficiently producing the heterologous alkaline protease; the high copy plasmid for expressing the heterologous alkaline protease contains a nucleotide sequence shown in SEQ ID NO:1 and a signal peptide amyE;

the aprE is NCBI: 594bp to 1742bp in GU 825966.1; the epr is NCBI: 3751547bp to 3753295bp in CP 054415.1; the mpr is NCBI: 886397bp to 887311bp in CP 054415.1; the vpr is NCBI: 3706130bp to 3708541bp in CP 002634.1; the nprE is NCBI: 254bp to 1819bp in K02497.1; the bpr is NCBI: 1624354bp to 1628643bp in CP 054415.1; the wprA is NCBI: 310740bp to 313415bp in CP 018902.1; the aprX is NCBI: 983721bp to 985049bp in CP 018902.1; the amyE is NCBI: 564bp to 2108bp in MT 590601.1; the eps is NCBI: 2456935bp to 2472647bp in CP 018902.1; the heterologous alkaline protease is alkaline protease derived from bacillus alcalophilus.

2. Use of the genetically engineered bacterium of claim 1 for the fermentative production of alkaline protease.

3. A method for efficiently producing a heterologous alkaline protease, which comprises culturing the genetically engineered bacterium of claim 1 under suitable conditions and collecting the alkaline protease from the culture.

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