CN116218750A

CN116218750A - Bacillus amyloliquefaciens chassis fungus and construction method and application thereof

Info

Publication number: CN116218750A
Application number: CN202211257312.1A
Authority: CN
Inventors: 李玉; 路福平; 刘逸寒; 李庆刚; 宋广超; 史超硕
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-10-14
Filing date: 2022-10-14
Publication date: 2023-06-06

Abstract

The invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and particularly relates to a chassis knockout strain and application thereof. The bacillus amyloliquefaciens genetic engineering strain does not express six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs and sacB genes on the genome of the bacillus amyloliquefaciens to construct chassis bacteria, and on the basis, the keratinase genes kerk, the aminopeptidase genes ywaD and the alkaline protease genes aprE are expressed in a heterologous manner, so that the bacillus amyloliquefaciens chassis strain and a production strain capable of producing keratinase, aminopeptidase and alkaline protease in a high yield are finally obtained, and a new idea is provided for constructing the protease high-yield strain.

Description

Bacillus amyloliquefaciens chassis fungus and construction method and application thereof

Technical field:

the invention belongs to the technical field of bioengineering, relates to breeding of industrial microorganisms, and in particular relates to a chassis strain, a construction method and application thereof.

The background technology is as follows:

keratinase (e.c. 3.4.21/24/99.11) is a class of proteases capable of specifically degrading insoluble keratins into soluble proteins, polypeptides or amino acids. Bacterial keratinase is of great interest in practical use. The method for treating the feathers by using the microbial fermentation method is a method which is developed faster at present, and the method utilizes an enzyme production system in the microbial fermentation process to degrade the feathers, has mild reaction conditions and can reserve nutrient elements to the greatest extent; the degradation products contain amino acid components contained in feathers, and the content of essential amino acids for animal bodies is obviously improved; the microbial fermentation method has low energy consumption in feather treatment process and is environment-friendly. It has wide application prospect in leather making.

Aminopeptidases (APs for short, EC 3.4.11) are a class of exoproteases that selectively degrade polypeptide chains and protein N-terminal amino acid residues, releasing free amino acids. The aim of reducing the bitter taste can be achieved by catalyzing and degrading hydrophobic amino acid residues at the N end of the bitter peptide. Therefore, the aminopeptidase can significantly improve the hydrolysis degree of casein and soy protein hydrolysate and reduce bitterness. The aminopeptidase enzyme method has high debitterizing hydrolysis efficiency, mild conditions and easy operation, and is widely applied to the modern industries such as food and the like. However, the existing aminopeptidase generally has the problems of low yield, poor enzyme activity, easy inactivation under high temperature conditions and the like. Aminopeptidases of high catalytic activity and stability are lacking in the fields of food processing and the like.

Alkaline protease, which is an enzyme capable of catalyzing and hydrolyzing peptide bonds, the active center of the alkaline protease contains serine, is also called serine protease, and 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. It has wide application in the industries of food, washing, leather making, etc. As the microbial protease is extracellular enzyme, compared with animal and plant source protease, the microbial protease has the advantages of relatively simple downstream technical treatment, low cost, wide source, easy culture of thalli, high yield, simple and quick breeding of high-yield strains, stronger hydrolysis capability and alkali resistance capability compared with neutral protease, higher heat resistance, certain esterase activity and easy realization of industrial production.

Accordingly, there is a need in the art to further dig keratinase, aminopeptidase and alkaline protease having industrial application properties, more ways to obtain keratinase, aminopeptidase and alkaline protease, and more convenient industrial production using keratinase, aminopeptidase and alkaline protease.

The invention comprises the following steps:

in view of the above problems, an object of the present invention is to provide a Bacillus amyloliquefaciens chassis strain capable of highly producing keratinase, aminopeptidase and alkaline protease by genetic engineering of a host.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect, the present invention provides a Bacillus amyloliquefaciens genetically engineered strain that does not express six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs, and sacB genes on the Bacillus amyloliquefaciens host genome;

further, the bacillus amyloliquefaciens host is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.11218;

the sacB gene has the sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

the extracellular protease gene aprE has a sequence shown in SEQ ID NO:2, a nucleotide sequence shown in seq id no;

the extracellular protease gene bpr has the sequence shown in SEQ ID NO:3, a nucleotide sequence shown in figure 3;

the extracellular protease gene vpr has the sequence shown in SEQ ID NO:4, a nucleotide sequence shown in seq id no;

the extracellular protease gene mpr has the sequence shown in SEQ ID NO:5, a nucleotide sequence shown in seq id no;

the extracellular protease gene nprE has a sequence shown in SEQ ID NO:6, a nucleotide sequence shown in seq id no;

the extracellular protease gene epr has the sequence shown in SEQ ID NO: 7;

the extracellular polysaccharide gene cluster eps has a nucleotide sequence shown as 2456935bp to 2472647bp on GenBank: CP 018902.1;

the polyglutamic acid gene cluster pgs has a sequence shown in SEQ ID NO:8, a nucleotide sequence shown in seq id no;

preferably, the Bacillus amyloliquefaciens genetically engineered strain knocks out extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs, and sacB genes on a host genome.

In a second aspect, the invention provides a genetically engineered bacterium for producing keratinase, which is obtained by heterologously expressing keratinase on the basis of the genetically engineered bacillus amyloliquefaciens strain;

further, the keratinase encoding gene kerk has the sequence of SEQ ID NO:9 is derived from Bacillus licheniformis.

In a third aspect, the invention provides a genetically engineered bacterium for producing aminopeptidase, which is obtained by heterologously expressing an aminopeptidase gene on the basis of the genetically engineered bacillus amyloliquefaciens strain;

further, the coding gene ywaD of the aminopeptidase has the sequence of SEQ ID NO:10, derived from Bacillus subtilis.

In a fourth aspect, the invention provides a genetically engineered bacterium for producing alkaline protease, which is obtained by heterologously expressing alkaline protease on the basis of the genetically engineered bacillus amyloliquefaciens strain;

further, the coding gene AprE of the alkaline protease has a sequence shown in SEQ ID NO:11, derived from Bacillus clausii.

In a fifth aspect, the present invention provides the use of a genetically engineered bacterium as described above for producing a keratinase, aminopeptidase or alkaline protease, in particular for producing a keratinase, aminopeptidase or alkaline protease, respectively.

In a sixth aspect, the present invention provides a method for producing keratinase, aminopeptidase or alkaline protease efficiently, comprising culturing the genetically engineered bacterium for producing keratinase, aminopeptidase or alkaline protease under appropriate conditions, respectively, and collecting keratinase, aminopeptidase and alkaline protease from the culture, respectively.

The beneficial effects of the invention are as follows:

the invention provides a bacillus amyloliquefaciens chassis strain capable of producing keratinase, aminopeptidase and alkaline protease at high yield, and the knockout of sacB genes in a host is beneficial to the expression of the keratinase, aminopeptidase and alkaline protease by the chassis strain. Under the condition of carrying keratinase, aminopeptidase and alkaline protease expression cassettes respectively, fermenting the keratinase, aminopeptidase and alkaline protease expression cassettes respectively by shaking bottles for 48 hours, wherein the activity of the keratinase in the fermentation liquor is as high as 3333.7U/mL, and the activity of the keratinase is improved by about 0.55 times by taking a starting strain as a host; the enzyme activity of the liquid ammonia peptidase is up to 9357U/mL, and the starting strain is taken as a host to improve the enzyme activity by about 0.53 times; the maximum enzyme activity of alkaline protease in the fermentation broth reaches 20046.2U/mL, and the initial strain is taken as a host to improve the enzyme activity by about 0.47 times.

Description of the drawings:

fig. 1: the construction process of the knockout carrier;

fig. 2: verifying sacB gene knockout;

wherein M: maker 1: knock-out strain 2: an original strain;

fig. 3: enzyme activity comparison of the knocked-out strain expressed keratinase;

fig. 4: enzyme activity comparison of aminopeptidase expressed by the knocked-out strain;

fig. 5: comparison of enzyme activities of the knockout strain expressing alkaline protease.

The specific embodiment is as follows:

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.

In a first aspect, the present invention provides a genetically engineered strain of bacillus amyloliquefaciens that does not express the six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs, and sacB genes on the bacillus amyloliquefaciens host genome; preferably, the sacB gene and six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene cluster eps, polyglutamic acid gene cluster pgs have the amino acid sequence of SEQ ID NO: 1-9; preferably the bacillus amyloliquefaciens host is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.11218.

At the same time SEQ ID NO:1-9 are identical to published genetic information on NCBI as follows:

the sacB gene has the sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification; namely 3067621bp to 3069039bp on GenBank:CP 018902.1.

The extracellular protease gene aprE has a sequence shown in SEQ ID NO:2, a nucleotide sequence shown in seq id no; i.e., 594bp to 1742bp on GenBank, GU 825966.1.

The extracellular protease gene bpr has the sequence shown in SEQ ID NO:3, a nucleotide sequence shown in figure 3; namely 1624354bp to 1628643bp on GenBank:CP 054415.1.

The extracellular protease gene vpr has the sequence shown in SEQ ID NO:4, a nucleotide sequence shown in seq id no; namely 3706130bp to 3708541bp on GenBank:CP 002634.1.

The extracellular protease gene mpr has the sequence shown in SEQ ID NO:5, a nucleotide sequence shown in seq id no; namely 886397bp to 887311bp on GenBank:CP 054415.1.

The extracellular protease gene nprE has a sequence shown in SEQ ID NO:6, a nucleotide sequence shown in seq id no; namely 668734bp to 670299bp on GenBank:CP 018902.1.

The extracellular protease gene epr has the sequence shown in SEQ ID NO: 7; namely 3751547bp to 3753295bp on GenBank:CP 054415.1.

The extracellular polysaccharide gene cluster eps has a nucleotide sequence shown as 2456935bp to 2472647bp on GenBank: CP 018902.1.

The polyglutamic acid gene cluster pgs has a sequence shown in SEQ ID NO:8, a nucleotide sequence shown in seq id no; namely 2616648bp to 2617796bp on GenBank:CP 018902.1.

According to the present invention, the means for not expressing the above-mentioned gene may be conventional means in the art, for example, inactivation of the gene or knocking out the gene by conventional means in the art, and a preferred means is gene knocking out. The mode of knockout can be conventional in the art, for example, by homologous recombination, constructing a knockout vector, which is preferably a pQ-T2 plasmid (i.e., T2 (2) -ori plasmid), and electrically transforming the vector into Bacillus amyloliquefaciens, and knocking out the above gene from the genome by single or double exchange.

In a second aspect, the present invention provides an application of the bacillus amyloliquefaciens genetic engineering strain in the use of the bacillus amyloliquefaciens genetic engineering strain as a keratinase, aminopeptidase or alkaline protease expression chassis fungus, wherein the bacillus amyloliquefaciens genetic engineering strain is used as the chassis fungus to heterologously express a keratinase gene kerk, an aminopeptidase gene ywaD or an alkaline protease gene AprE to obtain a genetic engineering fungus for producing keratinase, aminopeptidase or alkaline protease; preferably, the nucleotide sequence of the keratinase gene kerk is shown in SEQ ID NO:9, genBank: AY590140.1; the nucleotide sequence of the aminopeptidase gene ywaD is shown in SEQ ID NO:10, namely 4049025-4050392bp on GenBank:CP 053102.1; the nucleotide sequence of the alkaline protease gene AprE is shown in SEQ ID NO:11, genBank: 17bp to 1159bp on FJ 940727.1.

According to the present invention, the heterologous expression of the kerk, ywaD, or AprE gene may be performed by conventional means in the art, for example, by inserting an expression cassette of the keratinase gene kerk, aminopeptidase gene ywaD, or alkaline protease gene AprE into the genome of bacillus amyloliquefaciens, or by transferring an expression vector comprising the expression cassette of the keratinase gene kerk, aminopeptidase gene ywaD, or alkaline protease gene AprE into bacillus amyloliquefaciens by conventional means in the art.

In the invention, the original strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.11218, and the construction method of the chassis strain comprises the following steps of: in the original strain CGMCC No.11218, the epr, vpr, aprE, bpr, mpr, nprE, eps and pgs are knocked out to obtain the strain BA delta 6 delta eps delta pgs, and then the sacB gene is knocked out to obtain the strain BA delta 6 delta eps delta pgs delta sacB.

Further, expression cassettes containing a keratinase gene kerk, an aminopeptidase gene ywaD, or an alkaline protease gene AprE were inserted into the genomes of the strains CGMCC No.11218, BA Δ6 Δeps Δpgs Δsacb, respectively, to give high-yield strains named as follows:

according to a more preferred embodiment of the invention, the construction method comprises the steps of:

(1) Knocking out target genes and obtaining each knocked-out strain

The target genes comprise six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs and sacB of bacillus amyloliquefaciens, and are knocked out according to the following steps, and it is noted that the sequence of gene knockdown does not affect the achievement of the target of the invention, so long as the result of all knockdown of the genes can be finally achieved, and the knockdown sequence adopted according to the specific embodiment of the invention is only used for obtaining knockdown strains of all steps so as to facilitate comparison of knockdown effects:

1) Obtaining up ends and down ends of homologous sequences on two sides of a target gene through PCR amplification;

2) Obtaining a linear knockout plasmid vector through double digestion and agarose nucleic acid gel electrophoresis;

3) Connecting homologous sequences with a linear vector through a seamless cloning technology to obtain a knockout plasmid;

4) Methylation-induced modification of the knockout plasmid;

5) Electrotransformation of the methylation-modified knockout plasmid into bacillus amyloliquefaciens competent cells;

6) Screening out the knocked-out strain through single and double exchange, and verifying the sequence through sequencing;

the specific methods of operation of the steps described above can be carried out according to technical manuals, textbooks or literature reports readily available to the person skilled in the art.

(2) Heterologous expression of keratinase genes

The recombinant plasmid carrying the keratinase gene kerk expression cassette is electrically transformed into the above knockout strain BA delta 6 eps delta pgs delta SacB, and is also electrically transformed into the above knockout strain BA delta 6 eps delta pgs and a starting strain CGMCC No.11218 simultaneously, so that each genetic engineering strain BA delta 6 eps delta pgs delta SacB-K, BA-K and WT-K for producing keratinase is obtained, and the strains produce keratinase through shake flask fermentation.

Recombinant strains expressing aminopeptidase genes and alkaline protease genes are constructed by a similar method, and the strains respectively produce aminopeptidase and alkaline protease by shake flask fermentation.

Method for measuring enzyme activity of keratinase: the method is characterized in that 1% of soluble keratin is used as a substrate, and a Fulin phenol color development method is adopted for measurement. Taking 350 mu L of enzyme solution (fermentation supernatant) with proper dilution, adding the same volume of substrate, reacting for 20min at 40 ℃, adding 700 mu L0.4M trichloroacetic acid to stop the reaction, centrifuging at 15000rpm for 5min, taking 200 mu L of supernatant, and sequentially adding 200 mu L of Fu Lin Fen and 1mL of 0.4M Na ₂ CO ₃ After fully and uniformly mixing, preserving the temperature at 40 ℃ for 20min, and measuring the absorption of a color development system under 660nm wavelengthLight value. The control group was charged with trichloroacetic acid and substrate in reverse order relative to the experimental group, and the rest was the same. Definition of enzyme activity: in the above reaction system, the absorbance was 1 enzyme activity unit (U) per 0.01 increase. 3 replicates were measured for each sample.

The enzyme activity measurement method of aminopeptidase refers to the BpNA method. Definition of enzyme activity: hydrolysis of leucine paranitroaniline at 60℃and pH9.0 results in 1. Mu. g p-NA per minute, defined as a unit of enzyme activity, expressed in U/ml.

The method for measuring the enzyme activity of alkaline protease 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 enzyme amount required by 1mL of enzyme solution to hydrolyze casein at 40 ℃ for 1min under the condition of pH 10.5 to generate 1 mug of tyrosine.

According to a preferred embodiment of the invention, the keratinase gene kerk, the aminopeptidase gene ywaD and the alkaline protease gene AprE are each produced by recombinant plasmid P _ly-2 -SPamyE-KerK-pWB980、P _ly-2 -SPamyE-ywaD-pWB980、P _ly-2 The SPamyE-AprE-pWB980 was transferred into a chassis strain, in which the promoter P _ly-2 The nucleotide sequence of the peptide SPamyE is shown as SEQ ID NO: shown at 12.

The invention provides application of the genetically engineered bacterium for producing keratinase, aminopeptidase or alkaline protease in high-yield keratinase, aminopeptidase or alkaline protease.

According to a preferred embodiment of the invention, the use of the genetically engineered strain BA delta 6 eps delta pgs delta SacB-K for fermenting and producing keratinase is that the enzyme activity of the keratinase in the fermentation broth of the genetically engineered strain is up to 3333.7 +/-53.8U/mL, and the enzyme activity of the genetically engineered strain BA-K is improved by about 0.25 times.

The use of the genetic engineering strain BA delta 6 eps delta pgs delta SacB-Y for producing aminopeptidase by fermentation is that the enzyme activity of the aminopeptidase in fermentation broth of the genetic engineering strain reaches 9357U/mL at most, and the BA-Y strain is taken as a comparison enzyme activity to be improved by about 0.19 times.

The genetically engineered strain BA Δ six Δ EPS Δ Pgs Δ SacB-A is used for fermentation and production of keratinase. The highest enzyme activity of keratinase in the fermentation broth of the genetically engineered strain reached 20046.2U/mL, which increased by about 0.18 times compared to the BA-A strain.

The invention provides a method for efficiently producing keratinase, aminopeptidase and alkaline protease, which comprises the steps of respectively culturing genetically engineered strains for producing keratinase, aminopeptidase or alkaline protease under proper conditions, and respectively collecting keratinase, aminopeptidase and alkaline protease from the culture.

According to a preferred embodiment of the invention, the suitable conditions are a culture temperature of 37℃and a fermentation medium composition of: 64g/L corn flour, 40g/L bean cake powder, 4g/L disodium hydrogen phosphate, 0.3g/L monopotassium phosphate, 0.7g/L high temperature amylase and the balance of water.

The present invention will be described in more detail with reference to specific examples. In the following examples, unless otherwise specified:

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

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

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 strains and plasmids referred to in the examples are shown in tables 1 and 2:

TABLE 1

Strain	Knocked out genes	Fragment Length kb
			BAΔ6ΔepsΔpgs	epr,vpr,aprE,bpr,mpr,nprE,eps,pgs	15.928+15.714+10.471
BAΔ6ΔepsΔpgsΔSacB	epr,vpr,aprE,bpr,mpr,nprE，eps，pgs，sacB	42.113+1.419

Note that: wherein 6 represents six extracellular protease genes of epr, vpr, aprE, bpr, mpr, nprE, eps gene encodes eps synthetic protease, pgs gene encodes polyglutamic acid synthesis-related enzyme, sacB gene encodes levan sucrose transferase.

TABLE 2

Plasmid(s)	Use of the same	Resistance to
			pQ-T2 (T2 (2) -ori plasmid)	Knock-out vector	Kana
P _ly-2 -SPamyE-kerk-pWB980	Expression cassette carrying kerk	Kana
			P _ly-2 -SPamyE-ywaD-pWB980	Expression cassette carrying ywaD	Kana
P _ly-2 -SPamyE-AprE-pWB980	AprE-carrying expression cassette	Kana

Note that: t2 (2) -ori plasmid (pQ-T2 plasmid is adopted as a substitution name in the application), and is disclosed in CN201810898060.8, namely a malR-knocked bacillus licheniformis strain, a construction method and application; the pWB980 plasmid is a commercial plasmid.

Primer information relating to the examples is shown in Table 3:

TABLE 3 Table 3

The invention is further illustrated by the following detailed description.

Example 1: construction of genetically engineered strains

(1) Knocking out target gene

1) Amplification of homologous sequences of the Gene of interest

Designing according to genome data of bacillus amyloliquefaciens CGMCC No.11218, and amplifying by PCR to obtain homologous arm sequences up end and down end at two ends of a target gene sequence, respectively carrying out PCR amplification by taking the target gene-up-F, the target gene-up-R, the target gene-down-F and the target gene-down-R as two groups of primers (see a primer table 3), and taking the genome of CGMCC No.11218 as a template; the amplification reaction system is as follows:

primer F	2μL
		Primer R	2μL
DNA template	2μL
		PrimerStar enzyme	25μL
ddH ₂ O	19μL

The amplification procedure was set up as follows: pre-denaturation: 95 ℃ for 5min; denaturation: 95 ℃ for 30s; annealing: 45s at 56 ℃; extension: 72 ℃ for 5s; reacting for 30 cycles; extension: 72℃for 10min.

And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the sizes of electrophoresis bands at the up end and the down end are between 1000bp and 1500bp, and recovering the PCR product by a small amount of DNA recovery kit, namely the upstream and downstream homologous arm fragments of the target gene.

2) Linearization of expression vectors

The pQ-T2 plasmid was extracted, and the extraction procedure was described in the manual of the kit. And (3) performing agarose gel electrophoresis after double enzyme digestion of XbaI and SmaI, and recovering the product by a DNA gel recovery kit to obtain the linearization carrier sequence.

The double enzyme digestion system is as follows:

ddH ₂ O	25μL
		plasmid template	15μL
Q.cut Buffer	5μL
		Restriction endonuclease XbaI	2.5μL
Restriction endonuclease SmaI	2.5μL

After being evenly mixed, the mixture is digested in a water bath at 37 ℃ for 2 hours, after the reaction is finished, the digested product is subjected to agarose gel electrophoresis, 4260bp, and then the digested product is recovered by a small amount of DNA recovery kit: linear pQ-T2 plasmid.

3) Construction of knockout vectors

The linear vector fragment obtained by the cleavage and the upstream and downstream homology arms of the target gene are connected by seamless cloning to form a recombinant plasmid, and the knockout plasmid pQ-T2-delta sacB (shown in figure 1) is obtained by taking knockout sacB as an example.

The seamless cloned enzyme reaction system is as follows:

seamless cloning of enzymes	5μL
		Linear carrier	1μL
Insertion fragment	4μL

After being mixed uniformly, the mixture is reacted in a water bath at 50 ℃ for 15min.

4) Methylation modification of knockout vectors and electrotransformation into Bacillus amyloliquefaciens competent cells

The constructed knockout vector was transformed into EC135.P.Bam. Competent cells by transformation, and when the OD600 value of the culture solution was 0.2, 80. Mu.L of an aqueous solution of 50mg/mL arabinose was added for methylation induction, and cultured overnight at 30℃in a shaking table.

The methylation-modified knockout vector is electrotransferred into competent cells of bacillus amyloliquefaciens CGMCC No.11218 by using an electrotransfer instrument, and colony PCR is performed for verification by using a knockout vector verification primer XbaI-F, smaI-R.

5) Dual exchange authentication

After the successfully electrotransferred monoclonal is selected and cultured for 3-4 generations at 45 ℃, single colony is selected and cultured for 6-9 generations at 37 ℃ through dilution and coating, colony PCR verification is carried out on the single colony, and the used primers are target gene-up-F and target gene-down-R.

Each gene knockout method is the same as that described above, and the corresponding primers are shown in Table 3. The bacillus amyloliquefaciens CGMCC No.11218 knocks out epr, vpr, aprE, bpr, mpr, nprE, eps and pgs genes to obtain a strain BA delta 6 delta eps delta pgs; the bacillus amyloliquefaciens CGMCC No.11218 knocks out epr, vpr, aprE, bpr, mpr, nprE, eps, pgs and sacB to obtain the strain BA delta 6 delta eps delta pgs delta SacB.

(2) Introduction of the keratinase Gene kerk

The keratinase gene kerk is respectively transferred into strains CGMCC No.11218, BA delta 6 deltaeps delta pgs and BA delta 6 deltaeps delta pgs delta SacB.

1) The nucleotide sequence of the pLY-2 promoter and the Bacillus subtilis amyE signal peptide were synthesized by Suzhou Jin Weizhi Biotechnology Inc. with cleavage sites EcoRI and SmaI (SEQ ID NO: 12);

2) The gene kerk (sequence as shown in SEQ ID NO: 9) is synthesized with enzyme cutting sites SmaI and BamHI, and is subjected to gel cutting recovery under the conditions of 37 ℃ and 2 hours, wherein the system is as follows:

plasmid/fragment	20μL
		10×buffer	10μL
Restriction enzyme	5 mu L each
		ddH ₂ O	60μL

3) The recovered pLY-2 promoter is connected with the bacillus subtilis amyE signal peptide fragment, the keratinase gene kerk fragment and the pWB980 fragment under the connection condition of 16 ℃ for 6 hours or overnight, and the connection system is as follows:

keratin gene fragment	2.5μL
		pLY-2-SPamyE fragment	0.5μL
Linear pWB980 fragment	2μL
		Solution I	5.0μL

The connection products are respectively transferred into strains CGMCC No.11218, BA delta 6 deltaeps delta pgs and BA delta 6 deltaeps delta pgs delta SacB, and the method is as follows;

(1) selecting newly activated strains CGMCC No.11218, BA delta 6 eps delta pgs and BA delta 6 eps delta pgs delta SacB, and culturing overnight at 37 ℃ in 5mL LB liquid medium;

(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.

5) And extracting plasmids of the correct transformants, obtaining genetically engineered strains expressing keratinase, respectively naming WT-K, BA-K, BA delta 6 deltaeps delta pgs delta SacB-K, and verifying positive transformants.

Recombinant strains of aminopeptidase genes ywaD and alkaline protease genes AprE are constructed in the same way, and genetic engineering strains for expressing aminopeptidase and alkaline protease are respectively obtained, and are respectively named as WT-Y，BA-Y、BAΔ6ΔepsΔpgsΔsacB-Y、WT-A，BA-A、BAΔ6ΔepsΔpgsΔsacB-A.

Example 2: use of genetically engineered strains for producing keratinase

Shaking and fermenting: carrying out three-region lineation on genetic engineering strains WT-K, BA-K and BA delta 6 eps delta pgs delta SacB-K on an LB plate, inversely culturing overnight at 37 ℃, picking an activated single colony, carrying out shake culture for 12 hours at 37 ℃ and 220r/min in 5mL LB liquid culture medium, transferring the strain into 50mL LB liquid culture medium to OD600 reaching 0.8-1.0 at an inoculum size of 2%, transferring the strain into a baffle bottle of 100mL fermentation culture medium at an inoculum size of 2%, and carrying out shake culture for 48-60 hours at 37 ℃ and 220 r/min. Sampling at fixed point according to experimental requirement, centrifuging at 4deg.C at 12000r/min for 2min, collecting supernatant, and properly diluting to determine keratinase activity.

The enzyme activity of the genetically engineered strain BA delta 6 eps delta pgs delta SacB-K expressed keratinase is 3333.7 +/-53.8U/mL which is about 1.25 times that of the BA-K strain as shown in figure 3.

Engineering strains WT-Y、BA-Y、BAΔ6ΔepsΔpgsΔsacB-Y，WT-A、BA-A、BAΔ6ΔepsΔpgsΔsacB-A are subjected to fermentation culture for 60 hours by adopting the same method, and fermentation liquid supernatants are respectively taken, and the activity of aminopeptidase and alkaline protease is measured after proper dilution.

The genetic engineering strain BA delta 6 eps delta pgs delta SacB-Y is used for producing aminopeptidase by fermentation, and the enzyme activity of the aminopeptidase in fermentation liquor of the genetic engineering strain is 9357U/mL at most and is about 1.19 times of that of the BA-Y strain, as shown in figure 4.

The genetically engineered strain BAΔ6ΔepsΔpgsΔSacB-A is used for fermenting and producing keratinase, and the enzyme activity of the keratinase in fermentation liquor of the genetically engineered strain reaches 20046.2U/mL at most and is about 1.18 times that of the strain BA-A, as shown in figure 5.

Enzyme activity data of each strain (U/mL)

Therefore, the invention simplifies related genes on the genome of the bacillus amyloliquefaciens, finally obtains a host strain which is not influenced in growth and can efficiently heterologously express keratinase, aminopeptidase and alkaline protease, and provides an innovative thought for constructing high-yield chassis strains.

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.

Claims

1. A genetically engineered strain of bacillus amyloliquefaciens, wherein the genetically engineered strain does not express a strain on a bacillus amyloliquefaciens host genome: six extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene cluster eps, polyglutamic acid gene cluster pgs, and sacB gene.

2. The genetically engineered strain of bacillus amyloliquefaciens of claim 1, wherein the bacillus amyloliquefaciens host is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CGMCC No.11218.

3. The genetically engineered strain of bacillus amyloliquefaciens of claim 2, wherein the sacB gene has the amino acid sequence of SEQ ID NO:1, and a nucleotide sequence shown in the specification;

the extracellular protease gene epr has the sequence shown in SEQ ID NO: 7;

the polyglutamic acid gene cluster pgs has a sequence shown in SEQ ID NO:8, and a nucleotide sequence shown in SEQ ID NO.

4. The genetically engineered strain of bacillus amyloliquefaciens of claim 1, wherein the extracellular protease genes aprE, bpr, vpr, mpr, nprE, epr, extracellular polysaccharide gene clusters eps, polyglutamic acid gene clusters pgs, and sacB genes on the host genome are knocked out.

5. The use of the genetically engineered strain of bacillus amyloliquefaciens of any one of claims 1-4.

6. A genetically engineered bacterium for producing keratinase, which is characterized in that the genetically engineered bacterium is obtained by heterologously expressing keratinase on the basis of the genetically engineered bacillus amyloliquefaciens strain of claim 1;

the keratinase encoding gene kerk has the sequence shown in SEQ ID NO: 9.

7. A genetically engineered bacterium for producing aminopeptidase, which is characterized in that the genetically engineered bacterium is obtained by heterologously expressing aminopeptidase genes on the basis of the genetically engineered bacillus amyloliquefaciens strain of claim 1;

the coding gene ywaD of the aminopeptidase has the sequence shown in SEQ ID NO:10, and a nucleotide sequence shown in seq id no.

8. A genetically engineered bacterium for producing alkaline protease, which is characterized in that the genetically engineered bacterium is obtained by heterologously expressing alkaline protease on the basis of the genetically engineered bacillus amyloliquefaciens strain of claim 1;

the coding gene AprE of the alkaline protease has the sequence shown in SEQ ID NO:11, and a nucleotide sequence shown in seq id no.

9. The genetically engineered bacterium for producing keratinase according to claim 6 or the genetically engineered bacterium for producing aminopeptidase according to claim 7 or the genetically engineered bacterium for producing alkaline protease according to claim 8.