CN112522173A - Engineering bacterium for producing heterologous alkaline protease and construction method thereof - Google Patents

Abstract

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

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 particularly relates to an engineering bacterium for producing heterologous alkaline protease and a construction method thereof.

Background

Alkaline proteases (Alkaline proteases), a class of enzymes that catalyze the hydrolysis of peptide bonds, whose active center contains serine, also known as serine proteases, enzymes that hydrolyze protein peptide bonds in the Alkaline pH range, which not only hydrolyze peptide bonds but also hydrolyze amide bonds, ester bonds and transesterification and transpeptidation. The enzyme is widely present in animal pancreas, bacteria and mould, and the enzyme activity can be specifically inhibited by diisopropyl phosphoryl fluoride (DFP), benzyl sulfonyl fluoride (PMSF) and Potato Inhibitor (PI). The alkaline protease has wide application in the industries of food, washing, leather making and the like. Because the protease secreted by the microorganism is extracellular enzyme, compared with the protease from animals and plants, the protease has the characteristics of relatively simple downstream technical treatment, low price, wide source, easy culture of thalli, high yield and the like; and the high-yield strain is simple and rapid in breeding, has stronger hydrolysis capacity and alkali resistance capacity, has higher heat resistance and certain esterase activity compared with neutral protease, and is easy to realize industrial production.

The species used industrially for the production of proteases are mainly of the genus bacillus, including: the bacillus licheniformis, the bacillus subtilis and the bacillus amyloliquefaciens have the following advantages that the bacillus amyloliquefaciens has strong secretion capacity and becomes the most potential heterologous protein expression host: (1) is an internationally recognized GRAS organism, is nonpathogenic, does not produce exotoxin and endotoxin, and has no pollution to the environment; (2) the cell wall has simple composition, is convenient for protein secretion, and does not contain heat-source lipopolysaccharide; (3) many phages and plasmids used in molecular biological experiments can be used as the transformation tools, and recombinant DNA is easy to transfer; (4) the protein is directly secreted into an extracellular culture medium without accumulation, thereby being beneficial to downstream recovery and purification of the protein and reducing the operation cost of the whole production chain; (5) the bacillus is a unicellular organism, can reach very high cell density in the fermentation process, and the culture medium is relatively simple, low in cost and high in yield, and meets the requirements of industrial production. At present, the method is widely applied to industrial production of food and enzyme preparations, such as large-scale production of alpha-amylase, protease, feed additive, beta-glucanase and the like. However, the bacillus amyloliquefaciens still has the problems of unstable expression system and low expression quantity in the aspect of heterologous protein expression, and because the bacillus amyloliquefaciens can secrete a plurality of extracellular proteases which can strongly degrade heterologous proteins, the reduction of the degradation of the proteases of the bacteria is the 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 by directionally modifying a genetically engineered host. And the viscosity of the engineering bacteria is greatly reduced in the fermentation process, which is beneficial to the separation and purification of the protease product.

The technical scheme of the invention is summarized as follows:

a genetic engineering bacterium, regard Bacillus amyloliquefaciens as the host, 8 kinds of extracellular protease aprE, epr, mpr, vpr, nprE, bpr, wprA, aprX of the host are lacked at first, get the deficient strain of extracellular protease; the host alpha-amylase and exopolysaccharide gene cluster eps is also deleted; and introducing the high copy plasmid expressing the heterologous alkaline protease into a host to obtain a genetic engineering 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 of Bacillus alkalophilus origin.

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

The invention also provides application of the genetic engineering bacteria in fermentation production of the alkaline protease, and the enzyme activity of the alkaline protease in the fermentation liquor of the genetic engineering bacteria can reach 19524U/mL or 64328U/mL to the maximum.

The invention also provides a construction method of the gene engineering bacteria, in a specific implementation mode, 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide coding gene clusters on a host genome are subjected to serial knockout mainly by using a temperature-sensitive plasmid mediated homologous recombination method, and an expression plasmid containing a heterologous alkaline protease gene is introduced.

The present invention also provides a method for efficiently producing a heterologous alkaline protease by culturing the genetically engineered bacterium under suitable conditions and collecting the alkaline protease from the culture.

The invention has the beneficial effects that:

according to the invention, 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide gene clusters eps on a bacillus amyloliquefaciens genome are knocked out in series, a strain with main extracellular enzyme deletion is constructed, meanwhile, a high-copy plasmid for expressing alkaline protease from bacillus alcalophilus is introduced into the strain to obtain a genetic engineering bacterium, and the activity of the recombinant alkaline protease in fermentation liquor after 48h fermentation culture is 19524U/mL at the highest; in a 5L fermentation tank, the activity of the recombinant alkaline protease reaches 64328U/mL, and the gene engineering bacteria of the invention obviously improve the expression quantity of heterologous protease. According to the invention, the main extracellular enzymes secreted by the bacillus amyloliquefaciens are knocked out, so that the influence of the protease on the expression of the exogenous alkaline protease is reduced. The method is simple and easy to implement, is suitable for a bacillus amyloliquefaciens system, lays a foundation for mediating the high-efficiency expression of the heterologous protease gene in the bacillus amyloliquefaciens expression system, promotes the industrial large-scale production of the heterologous protease, and can be applied to the high-efficiency expression of other heterologous genes.

Protease secreted by the thallus is divided into an intracellular protease and an extracellular protease, wherein the extracellular protease needs to enter a secretion channel under the guidance of a signal peptide and can exert various physiological functions by being secreted outside the cell. In bacillus amyloliquefaciens, there are mainly 4 protein secretion pathways: the Sec secretory pathway, the Tat secretory pathway, the Com secretory pathway and the ABC secretory pathway, wherein most proteins are secreted by means of the Sec pathway and a signal peptide starting with 2 arginines is secreted by means of the Tat pathway. Analysis of the Signal peptides of alkaline protease and a-amylase by Signal P software was that of the Sec secretory pathway, and both were secreted by the Sec pathway. The highest expression quantity of a-amylase in extracellular products expressed by the bacillus amyloliquefaciens CGMCC No.11218 is obtained by combining the analysis result of mass spectrum detection on the fermentation liquid (figure 4 and figure 5), the secretion of the a-amylase is presumed to occupy the secretion channel of the alkaline protease, so that the a-amylase encoding gene and the extracellular polysaccharide encoding gene cluster eps are knocked out, the viscosity of the fermentation liquid 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, and the separation and purification of the products are simplified while the expression quantity of target proteins is improved.

Drawings

FIG. 1: constructing a temperature-sensitive knockout vector T2;

FIG. 2: homologous recombination knockout principle.

FIG. 3: and (3) carrying out nucleic acid electrophoresis verification on the extracellular protease knockout strain (wherein delta represents a gene knockout strain, and CK is a control taking an undeleted strain as a template).

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

FIG. 5: and (5) analyzing the mass spectrum of the fermentation liquor.

FIG. 6: and (4) verifying the amyE gene knockout strain by nucleic acid electrophoresis.

FIG. 7: schematic structure of eps gene cluster.

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

FIG. 9: and (3) carrying out nucleic acid electrophoresis verification on the recombinant strains J1, J2 and J3.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are 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. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The following embodiments use the following media and enzyme activity determination methods:

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

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

Coli competence preparation medium:

LB culture medium: 5g/L of yeast powder, 10g/L of peptone and 5g/L of sodium chloride; CaCl₂Solution: 0.1 mol/L.

Preparing a culture medium by bacillus amyloliquefaciens in a competent manner:

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

recovering the culture 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 is carried out according to a Folin phenol method in GB/T23527-2009 appendix B, namely 1 enzyme activity unit (U/mL) is defined as the enzyme quantity required by 1mL of enzyme solution to hydrolyze casein for 1min to generate 1 mu g of tyrosine under the conditions of 40 ℃ and pH 10.5.

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

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

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

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

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

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

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

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

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

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

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

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

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

1) according to the aprE, epr, mpr, vprE, bpr, wprA, aprX, amyE genes and nucleotide sequences of extracellular polysaccharide coding gene clusters eps on a bacillus amyloliquefaciens CGMCC No.11218 genome, respective upper and lower homologous arm primers are respectively designed by using software SnapGene, the length of the homologous arms 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 homologous arms, and performing overlapped PCR amplification by using the recovered product as a template to obtain overlapped fragments of the upper and lower homologous arms;

3) purifying and recovering the overlapped fragments, performing double enzyme digestion on the recovered product by using Sac1 and Sma1, and connecting the purified product with a T2 temperature-sensitive type vector cut by the same enzyme to obtain 10 different temperature-sensitive type knockout vectors;

4) transforming the ligation product into a competent cell of EC135, carrying out methylation modification, electrically transforming the modified plasmid into Bacillus amyloliquefaciens CGMCC No.11218, carrying out subculture on a positive transformant in an LB test tube with 45 ℃ and kanamycin resistance, wherein under the condition, a replicon of the knockout vector cannot be normally replicated, and then screening out a single-crossover strain in which the knockout vector is integrated on a host genome; subculturing the strain subjected to single exchange in a non-resistance LB test tube at 37 ℃ for about 5 generations, performing a check plate experiment, preliminarily considering that the colony which does not grow on a kanamycin-resistant plate but grows on the non-resistance plate is successfully knocked out, and performing colony PCR verification and sequencing to obtain a single gene knocked-out strain;

5) electrically transferring the second knockout plasmid to competent cells of the single-gene knockout strain in the step 4), screening out the knockout strain with the second gene deletion through single exchange and double exchange for 2 passages, and sequencing and verifying; continuously repeating the operation of the step 4) to transfer into a third knockout plasmid and a fourth knockout plasmid … on the basis, and finally obtaining 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide gene cluster eps serial knockout strains;

6) electrically transferring the expression plasmid containing the heterologous alkaline protease gene to an original host CGMCC No.11218 by an electric shock conversion mode, and screening to obtain a positive transformant, which is marked as J1; simultaneously, electrically transforming the expression plasmid into 8 strains with knocked-out extracellular protease genes in the 5) process, and marking the strains as J2; simultaneously transferring the expression plasmid to 8 extracellular protease genes, alpha-amylase genes and extracellular polysaccharide gene cluster eps serial knockout strains finally obtained in the step 5), and verifying that a correct transformant is the final engineering bacterium and is recorded as J3;

7) and carrying out shake flask fermentation on the recombinant strains J1, J2 and J3, and measuring the alkaline protease activity of the three recombinant strains after 48 hours.

It should be noted that the terms first and second used in the detailed description of the embodiments only indicate that one operation object is distinguished from another operation object, and do not limit or imply that an actual sequential relationship exists between the operation objects.

The following examples further describe the specific operations of the various steps and the results obtained.

Example 1:

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

1. Obtaining the overlapping segment of homologous arm of the knockout gene (taking epr as an example).

According to the sequence information of the whole genome of the bacillus amyloliquefaciens, taking an epr gene as an example, an upstream and a downstream homologous arm primers of epr are designed through SnapGene, a target fragment is amplified through PCR, the amplified upstream and downstream homologous arm fragments are purified and recovered, and overlapping PCR is carried out by taking a recovered product as a template, wherein the sequences of the primers and the enzyme cutting sites are shown in Table 1.

The reaction system used for amplification of the Epr homology arms was 50. mu.L, as follows:

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

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

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

2. and (5) constructing a knockout vector.

And purifying the overlapped PCR product, performing double digestion by using Sma1-Sac1 restriction endonuclease, recovering a double digestion product, and connecting the double digestion product with a T2 vector cut by the same enzyme overnight to obtain the knockout vector.

The enzyme digestion system is as follows:

the ligation conditions were 16 ℃, 6h or overnight ligation, and the ligation system was as follows:

4.5. mu.L of the fragment of interest

Linear T2 fragment 0.5. mu.L

Solution I 5.0μL；

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

(1) taking out the competence in a refrigerator at minus 80 ℃, and immediately placing on ice for 3-5 min;

(2) sucking 8 mu L of the ligation product into the competent cells, gently mixing the ligation product and the competent cells uniformly, and placing the mixture on ice for 20 min;

(3) after ice bath, the mixture is transferred into a 42 ℃ water bath kettle, is subjected to heat shock for 90s, is immediately subjected to ice bath for 3min, and is added with 750 mu L of LB recovery solution;

(4) resuscitating in a shaker at 37 deg.C and 220r/min for about 1h, spreading on double-resistant plate containing 100. mu.g/mL kanamycin and 100. mu.g/mL spectinomycin, and culturing in incubator at 37 deg.C for 12 h.

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

4. And (3) electrically transferring the knockout plasmid successfully subjected to methylation modification into the bacillus amyloliquefaciens, wherein the electric transfer method comprises the following steps:

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

(2) 100 μ L of competent and 10ng plasmid DNA were mixed and added to an electric rotor and left on ice for 3 min.

(3) The 2500V shock is generally 4-6 ms.

Immediately after electric shock, 1ml of recovery medium was added, and recovery was carried out at 37 ℃ for 3 hours. Kanamycin plates were plated, incubated at 37 ℃ for 12h, and transformants were selected for validation.

Selecting a positive transformant which is verified to be correct, placing the positive transformant in 5mL of LB liquid culture medium added with kanamycin resistance, carrying out shaking culture at 45 ℃ at 180r/min for 12h, taking 10 mu L of bacterial liquid, transferring the bacterial liquid into fresh 5mL of liquid LB culture medium added with kanamycin, and marking as a second generation; when the plasmid is continuously transmitted to the 3 rd generation, the plasmid is diluted and coated on a kanamycin plate, and after the kanamycin plate is cultured for 12 hours at 45 ℃, colony PCR (polymerase chain reaction) is carried out to verify whether the knockout plasmid is integrated at the corresponding position of a genome.

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

5. Knocking out aprE, mpr, vpr, nprE, bpr, aprX, wprA, amyE and eps gene clusters of the host according to the steps 1-4, wherein the homologous arm amplification primers are shown in the table 1, and the amplification system and the amplification program are the same as the step 1. Obtaining a knockout strain.

TABLE 1 amplification primers for knock-out gene homology arms

6. The expression plasmid containing the heterologous alkaline protease gene is introduced into the 3 constructed genetic engineering bacteria by an electric shock transformation method, and the specific process is as follows:

(1) the nucleotide sequences of the pLY-2 promoter and the signal peptide of Bacillus subtilis amyE (shown as SEQ ID NO: 1) were synthesized by Kingzhi Biotechnology, Inc., Suzhou;

(2) the PCR technology is used for amplifying alkaline protease gene (the sequence is shown as GenBank: FJ 940727.1) by taking the basophilic bacillus genome as a template, the alkaline protease gene is purified and recovered, 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 signal peptide fragment of bacillus subtilis amyE, an alkaline protease gene recovery fragment from bacillus alcalophilus and a linearization vector pWB980 by using a seamless cloning enzyme purchased from Beijing Quanji biotechnology limited; the linking system is as follows:

(4) reacting the reaction system in the step (3) at 50 ℃ for 15min, and transferring the reaction system into bacillus subtilis WB600 by the following method;

firstly, selecting a newly activated Bacillus subtilis WB600 single colony to be cultured in 5mL LB liquid culture medium at 37 ℃ and 220r/min overnight;

② transferring 100 mul of culture solution into 5mL of SPI culture medium, culturing at 37 ℃ and 220r/min until OD600 is 1.2 (about 3-4 h) at the end of logarithmic growth;

③ putting 200 mu L of culture solution which grows to the end of logarithmic phase into 2mL of SPII culture medium, culturing for 1.5h at 37 ℃ and 100 r/min;

fourthly, 20 mu L of 10mmol/L EGTA is added into the thalli of the SPII culture medium, and the thalli are cultured for 10min at 37 ℃ and 100 r/min;

adding the ligation product, and culturing at 37 ℃ and 100r/min for 30 min;

sixthly, adjusting the rotating speed to 220r/min, continuously culturing for 1.5h, taking bacterial liquid, coating the bacterial liquid on an LB screening plate containing 100 mu g/mL kanamycin, culturing for 12h at 37 ℃, and screening positive transformants for verification.

Extracting a plasmid of a transformant which is verified to be correct, electrically transferring the plasmid into an original host CGMCC No.11218, and verifying a positive transformant which is marked as J1; simultaneously, electrically transforming the plasmids into 8 strains with the knocked-out extracellular protease genes in the step 5), and marking the strains as J2; simultaneously transferring the plasmid to the knockout strain finally obtained in the step 5), and verifying a positive transformant, wherein the positive transformant is marked as J3; the verification results are shown in fig. 9.

Example 2:

expression and analysis of heterologous alkaline protease gene engineering bacteria.

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

The enzyme activity of the alkaline protease in the fermentation supernatant of the recombinant strain is determined according to the national standard GB/T23527-2009 appendix B Folin phenol method, the enzyme activity of the alkaline protease in the fermentation supernatant of 3 recombinant strains is determined to be the highest within 48h, and the activity of the recombinant alkaline protease expressed by the recombinant strain J3 is the highest within 19524U/mL, which is 1.40 times that of the recombinant strain J1 (the enzyme activity is 13985U/mL), and is 1.15 times that of the recombinant strain J2 (the enzyme activity is 17042U/mL).

Example 3:

amplification experiment of recombinant alkaline protease gene engineering bacteria.

A single colony of the recombinant bacteria on the plate is picked up and inoculated into 100mL LB liquid culture medium containing 50 mu g/mL kanamycin at 37 ℃ at 220 r/min. After culturing for 4h, the cells were inoculated into 100mL of fermentation medium at 37 ℃ at 220r/min in an inoculum size of 2%, and after culturing for 8h, the cells were inoculated into a fermentor containing 3L of liquid and having a total volume of 5L in an inoculum size of 2% for amplification experiments. Wherein the dissolved oxygen in the fermentation tank is controlled to be more than 40 percent in the fermentation process, the stirring speed is 600-700rpm, the temperature is 37 ℃, the feeding is started when the pH is 7 (the feeding culture medium: the cottonseed protein is 50g/L, and the dextrin is 300g/L), the initial feeding amount is 100g/h, and the DE value is controlled to be 15mg/mL-18mg/mL by feeding in the whole fermentation period. The defoaming agent is properly added in the early stage of fermentation to control the generation of foam in the fermentation process. Sampling every two hours, and determining the activity of the alkaline protease by a Folin phenol method. The measured alkaline protease enzyme activity of the extracellular protease recombinant strain reaches 64328U/mL at most after fermentation for 58h, and the viscosity of the fermentation liquid is greatly reduced, so that the dissolved oxygen is effectively improved, and the production cost is reduced.

Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> Tianjin science and technology university, Shandong Longkote enzyme preparations Co., Ltd

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Claims (6)

1. A genetic engineering bacterium, regard Bacillus amyloliquefaciens as the host, 8 kinds of extracellular protease aprE, epr, mpr, vpr, nprE, bpr, wprA, aprX of the host are lacked at first, get the deficient strain of extracellular protease; the host alpha-amylase and exopolysaccharide gene cluster eps is also deleted; and introducing the high copy plasmid expressing the heterologous alkaline protease into a host to obtain a genetic engineering bacterium for efficiently producing the heterologous alkaline protease.

2. The genetically engineered bacterium of claim 1, wherein the bacillus amyloliquefaciens has a preservation number of CGMCC No. 11218.

3. The genetically engineered bacterium of claim 1, wherein the heterologous alkaline protease is an alkaline protease derived from Bacillus alkalophilus.

4. The genetically engineered bacterium of claim 1, wherein the high copy plasmid expressing the heterologous alkaline protease comprises a nucleotide sequence as set forth in SEQ ID NO: 1 with the signal peptide amyE.

5. Use of the genetically engineered bacterium of any one of claims 1 to 4 for the fermentative production of alkaline proteases.

6. A method for efficiently producing a heterologous alkaline protease, which comprises culturing the genetically engineered bacterium according to any one of claims 1 to 4 under suitable conditions and collecting the alkaline protease from the culture.

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