1. A promoter is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

2. A gene expression cassette comprising a promoter as claimed in claim 1, and a keratinase gene expressed from the promoter.

3. The gene expression cassette according to claim 2, wherein the nucleotide sequence of the keratinase gene is represented by SEQ ID No.2, or a nucleotide sequence having 95% or more homology with SEQ ID No. 2.

4. A vector plasmid containing the gene expression cassette of claim 2 or 3.

5. The vector plasmid of claim 4, wherein the vector is a vector suitable for Bacillus expression.

6. A recombinant bacterium which expresses keratinase and is characterized in that the recombinant bacterium promotes and expresses a keratinase gene by the promoter of claim 1.

7. The recombinant bacterium of claim 1, wherein the host of the recombinant bacterium is bacillus subtilis, bacillus cereus, bacillus licheniformis, bacillus megaterium, bacillus fragilis, bacillus clausii, bacillus alcalophilus, or bacillus thuringiensis.

8. A method for constructing a recombinant bacterium according to claim 6 or 7, comprising the steps of:

(1) amplifying a promoter sequence by taking a bacillus subtilis genome as a template, connecting two copy number promoter sequences in series, mutating ATGATA of a-35 region into TTGACA and TAAAAT of a-10 region into TATAAT to obtain a promoter with a nucleotide sequence shown as SEQ ID NO. 1;

(2) recovering the promoter fragment obtained in the step (1), and constructing the promoter fragment into a plasmid carrying the keratinase gene shown as SEQ ID NO.2 to obtain a recombinant plasmid;

(3) and (3) transforming the recombinant plasmid obtained in the step (2) into host bacteria to obtain the recombinant bacteria.

9. The method for producing keratinase by using the recombinant strain as claimed in claim 7, wherein the keratinase is obtained by inoculating the recombinant strain into a fermentation tank at an inoculation amount of 3-10%, and fermenting and producing under conditions of a fermentation temperature of 30-40 ℃, a rotation speed of 100-500 rpm, an air flow of 1-3 vvm and a pH value of 6.5-7.5.

10. Use of the promoter of claim 1 for enhancing the efficient expression and production of host-toxic proteases.

Background

The promoter is one of the most important elements for controlling the expression of foreign proteins, and can regulate the expression time and level of a target gene. As far as initial transcription conditions are concerned, existing natural promoters can be generally classified into three categories: constitutive promoters, inducible promoters and growth stage specific promoters. Strong constitutive promoters are mostly used for realizing high-level expression, and inducible promoters and growth period specific promoters realize the control of expression time and expression level, so that the promoter is a better choice for producing proteins with host toxicity. Phase-specific promoters are dependent on cell density to initiate gene expression compared to inducible promoters that are inducer dependent. Promoter PaprE specificity is expressed at late log phase; PsrfA specificity is expressed at the end of log and stationary phase. The two promoters hardly start or extremely weak expression of the promoter gene before the thalli reach the maximum biomass, thereby being beneficial to growth and accumulation of the thalli; reaching the late logarithmic phase or stable phase, being capable of rapidly inducing gene expression and being beneficial to the production of target products.

The keratinase has the function of specifically degrading natural keratin and is a protease with strong biological activity. The keratinase-producing microorganisms reported in the literature are mainly derived from bacteria, fungi or actinomycetes, and are mostly isolated from the accumulation of feathers, bird feathers or hair waste. Keratinase can treat keratin-containing materials more mildly and efficiently than other proteases, and at the same time, can improve the nutritional value of the treated keratin. At present, keratinase is widely applied to industries such as medicine, chemical industry, feed, textile, tanning and the like, and has important application values in the aspects of preparation of biological agents, utilization of waste biological resources and the like, so research on keratinase is concerned. With the intensive research on keratinase, researchers find that the expression of keratinase has certain host toxicity, and the over-expression and accumulation of keratinase can degrade various functional proteins in a host to destroy the normal growth and metabolic processes of the keratinase, influence the quantity of enzyme-producing bacteria and further reduce the yield of target products. However, no effective solution has been reported in domestic and foreign literature for this problem. Therefore, the efficient expression of keratinase becomes a bottleneck problem in the industrial mass production thereof.

Disclosure of Invention

In order to solve the technical problems, the invention provides a gene expression cassette for improving the growth of thalli and the potential of producing biological enzymes. According to the invention, the growth and enzyme production process of host bacteria is regulated and controlled based on a promoter engineering strategy, a period specific promoter, copy number optimization and core region sequence modification are respectively selected to construct a gene expression cassette for improving the thallus growth and the enzyme production potential of keratinase, and the enzyme production bacteria amount and the yield and activity of keratinase are effectively improved.

A nucleotide sequence of the promoter is shown as SEQ ID NO.1, and the promoter is a thallus density dependent self-induced promoter.

A gene expression cassette comprising said promoter, and a keratinase gene whose expression is promoted by said promoter.

In one embodiment of the invention, the nucleotide sequence of the keratinase gene is shown in SEQ ID NO.2, or a nucleotide sequence with more than 95% homology with SEQ ID NO. 2.

A vector plasmid of a gene expression cassette.

In one embodiment of the invention, the vector is a vector suitable for expression by bacillus.

The recombinant bacterium for expressing the keratinase uses the promoter to start and express a keratinase gene.

In one embodiment of the invention, the host of the recombinant bacterium is bacillus subtilis, bacillus cereus, bacillus licheniformis, bacillus megaterium, bacillus fragilis, bacillus clausii, bacillus alcalophilus, or bacillus thuringiensis.

A construction method of a recombinant bacterium comprises the following steps:

(1) amplifying a promoter sequence by taking a bacillus subtilis genome as a template, connecting two copy number promoter sequences in series, mutating ATGATA of a-35 region into TTGACA and TAAAAT of a-10 region into TATAAT to obtain a promoter with a nucleotide sequence shown as SEQ ID NO. 1;

(2) recovering the promoter fragment obtained in the step (1), and constructing the promoter fragment into a plasmid carrying the keratinase gene shown as SEQ ID NO.2 to obtain a recombinant plasmid;

(3) and (3) transforming the recombinant plasmid obtained in the step (2) into host bacteria to obtain the recombinant bacteria.

A method for producing keratinase by using recombinant bacteria comprises the steps of inoculating the recombinant bacteria into a fermentation tank in an inoculation amount of 3-10%, and carrying out fermentation production under the conditions of a fermentation temperature of 30-40 ℃, a rotation speed of 100-500 rpm, an air flow of 1-3 vvm and a pH value of 6.5-7.5 to obtain the keratinase.

An application of a promoter in improving the efficient expression and production of a protease with host toxicity.

In one embodiment of the invention, the host-toxic protease is keratinase.

The keratinase or the recombinant bacterium is applied to the animal husbandry, the leather making industry or the textile industry.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the invention provides a handleThe gene expression cassette for increasing the growth of the thallus and producing the biological enzyme potential effectively regulates and controls the growth and the enzyme production of host cells, improves the thallus density by 50 percent, and has the highest enzyme activity of 3500 U.mL^-1It is 2.9 times of the control bacterium. The components of the culture medium are optimized to improve the growth of host bacteria and increase the enzyme yield, so that the activity of the keratinase is further improved to 6878 U.mL^-1. The invention provides an effective strategy for the research of the high-efficiency expression and the enzyme production of the keratinase. The high-efficiency expression of the keratinase has certain toxicity to a host, and influences the growth and the enzyme production capacity of the host. The invention effectively controls the growth and the enzyme production process of the enzyme production engineering bacteria by using the thallus density dependent promoter, reduces the influence of enzyme production on a host, and thus improves the bacteria quantity and the enzyme production quantity. The invention provides an effective solution for industrially producing proteins and enzymes with certain host toxicity, such as keratinase and the like.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 shows the results of replacement of a bacterial density-dependent promoter according to the present invention;

FIG. 2 shows the research results of the copy number optimization of promoter according to the present invention;

FIG. 3 shows the results of sequence modification of the promoter core region in example 3;

FIG. 4 shows the effect of the carbon source species on the growth of the cells and the production of enzymes in example 4 of the present invention;

FIG. 5 shows the effect of glucose concentration on bacterial growth and enzyme production in example 4 of the present invention;

FIG. 6 shows the effect of addition of inorganic salts on cell growth and enzyme production in example 5 of the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Keratinase activity assay method:

the activity of the keratinase is determined by taking 1% (w/v) soluble keratin as a substrate, and the specific method is as follows:

adding 0.5mL of substrate to 0.5mL of enzyme solution diluted with Tris-HCl at pH 9.0, incubating in a water bath at 50 ℃ for 20min, adding 1mL of 4% TCA solution to stop the reaction, centrifuging the sample at 12000rpm for 5min, collecting 1mL of centrifuged supernatant, and adding 5mL of 0.4M Na₂CO₃And 1mL of a forlin phenol reagent, heating in a water bath at 40 ℃ for 20min for color development after uniformly mixing, cooling to room temperature, and then measuring the absorbance at 660 nm. In the control group, the enzyme solution and the TCA solution are mixed, incubated in a water bath at 50 ℃ for 20min, and then the substrate is added, and the rest operations are consistent with those of the experimental group. All experiments were performed on 3 replicates and the final results were expressed as mean ± standard deviation, calculated using the STDEV formula in Excel.

Definition of enzyme activity: in the reaction system, enzyme liquid hydrolyzes the substrate to ensure that each increase of 0.01 of absorbance value at 660nm is one enzyme activity unit, U.mL^-1。

Example 1: replacement of constitutive promoter by thallus density dependent promoter

Promoter replacement was performed by homologous recombination and primer design is shown in table 1. Using host cell B.subtilis WB600 genome as template, respectively amplifying promoter sequences containing homologous arms; using pMA5 plasmid as template, amplifying and removing original promoter P_{Hpa II}The whole plasmid sequence of (a); the promoter fragment is fused to the original promoter position in the plasmid using a homologous recombinase. Transforming the recombinant plasmid into a host cell B.subtilis WB600 to construct a recombinant strain which expresses keratinase under the control of two different thallus density-dependent promoters.

The growth curves of the wild host bacteria and the enzyme-producing engineering bacteria modified by the promoter are respectively detected, and the result shows that (figure 1) in the logarithmic phase of growth, the enzyme-producing strain containing the thallus density dependent promoter obviously grows more rapidly than the enzyme-producing strain of the constitutive promoter, and OD₆₀₀The value rises from 8 to over 10. The enzyme activity levels of different promoters are greatly different, and the bacterial enzyme is constitutively expressed in the first 36hThe activity is rapidly accumulated, and the maximum value is about 1200 U.mL after 48h^-1(ii) a And in promoter P_srfAAnd P_aprEThe expression level is lower within 24h under the guidance, and then the enzyme is rapidly produced to 60h to reach the highest enzyme activity, wherein P is_aprEThe effect of the modified bacteria is superior to that of P_srfAThe highest enzyme activity can reach about 2300 U.mL^-1。

TABLE 1 bacterial density-dependent promoter plasmid construction primers

Example 2: increasing keratinase production using tandem aprE promoter

To further increase the transcription strength, P is added_aprEThe copy number of the promoter is optimized. Respectively constructing a polypeptide containing one to four P by adopting a homologous recombination mode_aprERecombinant bacteria of the promoter. And screening the copy number of the promoter by taking the activity of the keratin enzyme in unit volume as a measurement standard. The results are shown in FIG. 2, compared to a single P_aprEAfter the series connection, the activity of the keratinase mediated by the multiple copies of the promoters is improved. Wherein the activity of the three-tandem body and the four-tandem body is higher than that of the two-tandem body in the first 36h of fermentation, the enzyme activity of the supernatant of the fermentation liquid of each recombinant bacterium reaches the peak value after 60h of fermentation culture, and the activity of the keratinase of the recombinant bacterium containing the two-tandem body promoter is the highest and can reach 3080 U.mL^-1Compared with single copy promoter, the promoter has 34% improvement.

Example 3: promoter core region sequence modification to improve promoter activity

aprE two-tandem promoter P is selected for use in the study_aprE-P_aprEFor the subjects, ATGATA in the-35 region and TAAAAT in the-10 region of the two aprE promoters were mutated to TTGACA and TATAAT, respectively. The results are shown in FIG. 3, and the growth of the mutant strain before and after the transformation is similar, but the mutant strain has a faster reduction in the concentration after the maximum concentration is reached. From the view of enzyme activity level, only a small amount of leakage expression exists in the first 24h of fermentation, and the promoter rapidly starts the transcription of keratinase in the late logarithmic phase (24-36h)And expression, the optimized enzyme activity is higher than that of an unoptimized control strain, and the highest enzyme activity can reach 3500 U.mL^-1. SDS-PAGE analysis results show that the expression quantity of the optimized target protein is increased.

Example 4: carbon source supplement for improving early accumulation of thalli

Taking TB culture medium as basic culture medium, respectively supplementing 1% of sucrose, soluble starch, glycerol, glucose, fructose, lactose and mannitol as additional carbon source. As shown in FIG. 4, among the carbon sources selected, glucose had the best effect of promoting the growth of the cells, the cell concentration after 12 hours of culture was close to 8, the maximum cell concentration after 60 hours of culture reached 13.6, and the enzyme activity reached the peak value of 3880 U.mL after 72 hours of culture^-1. After determining that glucose is the optimal carbon source, the influence of glucose with different concentrations on the growth of the thalli and the enzyme production is studied, and the result is shown in figure 5, 2% of glucose is added to ensure that the enzyme-producing strain reaches the maximum bacteria concentration of 15.6 after being cultured for 72h, and the enzyme activity can reach 4200 U.mL^-1Although the strain still shows an ascending trend after being cultured for 96 hours by adding 3% of glucose, the strain concentration is about 12, the acceleration is gentle, the enzyme production of the strain is seriously inhibited by adding 3% of glucose, and the enzyme activity is less than 1000 U.mL^-1。

Example 5: supplement of inorganic salt to promote growth of thallus and produce enzyme

0.1 g.L was added to each of the culture media^-1The results of the calcium chloride, the potassium sulfate, the zinc sulfate, the magnesium sulfate, the aluminum sulfate and the manganese sulfate are shown in figure 6, the enzyme activity is improved most after a small amount of potassium sulfate is added, and the highest enzyme activity can reach 6659 U.mL^-1. It can be presumed that a small amount of K was added to the medium⁺Can improve the enzyme activity. Further concentration optimization is carried out on the culture medium, and the result shows that the concentration of potassium sulfate in the culture medium is 0.5 g.L^-1When the enzyme activity reaches the maximum value of 6878 U.mL^-1。

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> a gene expression cassette for improving the growth of thallus and the potential of producing biological enzyme

<130> 2

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 668

<212> DNA

<213> (Artificial Synthesis)

<400> 1

cgataatatc cattgttctc acggaagcac acgcaggtca tttgaacgaa ttttttcgac 60

aggaatttgc cgggactcag gagcatttaa cctaaaaaag catgacattt cagcataatg 120

aacatttact catgtctatt ttcgttcttt tctgtatgaa aatagttatt tcgagtctct 180

acggaaatag cgagagttga catacctaaa tagagatata atcatctcaa aaaaatgggt 240

ctactaaaat attattccat ctattacaat aaattcacag aatagtcttt taagtaagtc 300

tactctgaat ttttttaaaa ggagagggta aagacgataa tatccattgt tctcacggaa 360

gcacacgcag gtcatttgaa cgaatttttt cgacaggaat ttgccgggac tcaggagcat 420

ttaacctaaa aaagcatgac atttcagcat aatgaacatt tactcatgtc tattttcgtt 480

cttttctgta tgaaaatagt tatttcgagt ctctacggaa atagcgagag ttgacatacc 540

taaatagaga tataatcatc tcaaaaaaat gggtctacta aaatattatt ccatctatta 600

caataaattc acagaatagt cttttaagta agtctactct gaattttttt aaaaggagag 660

ggtaaaga 668

<210> 2

<211> 1152

<212> DNA

<213> (Artificial Synthesis)

<400> 2

atgtgcgtga aaaagaaaaa tgtgatgaca agtgttttat tggctgtccc tcttctgttt 60

tcagcagggt ttggaggctc catggcaaat gccgagacgg tctccaaaac agatagtgaa 120

aaaagctata ttgttggttt taaagcctct gccaccacaa acagctctaa aaaacaagct 180

gtcattcaaa atggtggaaa actagaaaaa caataccgcc tcattaatgc tgcacaagtg 240

aaaatgtccg aacaagccgc caaaaaactt gaacatgacc ctagcattgc ttacgtagaa 300

gaagaccata aagcagaagc atatgcacaa accgtccctt atggaatccc tcaaatcaaa 360

gctccagctg tacacgctca aggttataaa ggtgctaatg tcaaagtagc tgtccttgat 420

actggaatcc acgctgcaca ccctgattta aatgttgcag gcggtgcgag cttcgtccct 480

tcagagccaa atgccaccca agactttcaa tcacatggaa ctcacgtagc tggaaccatt 540

gctgcccttg ataacacaat tggtgtactc ggggtcgctc caagcgcttc cctatatgct 600

gtaaaagtat tagaccgcta tggcgacgga caatacagct ggattattag cggtattgaa 660

tgggctgtag ccaataatat ggatgtcatc aatatgagct taggcggacc aagcggttca 720

actgcgctta aaaatgccgt cgatacagcg aataaccgtg gagtcgttgt tgtggcagcc 780

gcaggtaatt ctggctctag cggctctagc agtacagttg gctatccagc aaaatacgat 840

tctacaattg ctgttgccaa tgtaaacagt aacaatgtca gaaactcatc ttctagcgca 900

ggtcctgaat tagatgtttc tgcacctggt acttctattt taagtacagt gccaagcagt 960

ggatacactt cttatactgg aacatctatg gcgtctcctc atgtagcagg agcagcagcg 1020

cttattcttt ctaaaaaccc gaacctaaca aattcacagg ttcgccagcg cttagaaaat 1080

acagcgacac cgcttggtga ctcattctat tatggaaaag ggttaatcaa cgttcaagca 1140

gcttctaact aa 1152