CN108385170B - Regulatory sequence library of Bacillus subtilis F4 promoter - Google Patents

Abstract

The disclosure relates to a regulatory sequence library of a bacillus subtilis F4 promoter, which consists of regulatory sequences shown in SEQ ID NO. 1-3. The disclosure also provides a bacillus subtilis F4 promoter library consisting of promoters containing the regulatory sequences in the regulatory sequence library, a recombinant expression vector, a method for expressing foreign proteins, and application of the regulatory sequence library and the promoter library in preparation of recombinant expression vectors and/or expression plasmids. By the technical scheme, different intensities for starting transcription of the foreign protein can be obtained, so that the starting intensity of expression of the foreign protein can be reasonably selected and controlled according to actual production needs.

Description

Regulatory sequence library of Bacillus subtilis F4 promoter

Technical Field

The invention relates to the technical field of biology, in particular to a regulatory sequence library of a bacillus subtilis F4 promoter, a promoter library, a recombinant expression vector, a method for expressing a foreign protein and application of the regulatory sequence library and the promoter library.

Background

The bacillus subtilis is a gram-positive bacterium, and is an ideal strain for expressing and secreting foreign proteins in a prokaryotic expression system at present due to the characteristics of non-pathogenicity, strong protein secretion capacity and good fermentation foundation and production technology.

In genetic engineering, a promoter is one of the factors for realizing high-efficiency expression of a foreign gene. Because the host bacteria are selected from prokaryotes with strong division capability and much secreted protein, the method for searching an ideal engineering promoter by utilizing a prokaryote promoter regulation mechanism is particularly important for the fermentation industry.

A promoter with proper strength is required for realizing stable and high-efficiency expression of the foreign protein. The F4 promoter was truncated from Bacillus subtilis ybp promoter P16, and Cybera yunna et al (Yu, X., Xu, J., Liu, X., Chu, X., Wang, P., Tian, J., et al (2015). Identification of a high efficiency functional phase promoter in Bacillus subtilis scientific Reports,5,18405.) A truncation experiment found a fragment of only 103bp in length to function, and designated F4 promoter. The promoter can efficiently start the expression of downstream genes in the stable period of the growth of thalli. Compared with the constitutive promoter P43 and the self-inducible Gsib promoter which are commonly used in industry, the promoter has higher promoter activity, has natural advantages for researching the corresponding relation between the promoter sequence and the regulatory activity, and is an rare good material.

However, the use of a promoter cannot seek only the expression intensity, and the level of promoter activity is often not reflected in the expression level of a foreign protein. This is due to the fact that the properties of the two are not compatible with each other. Therefore, it is necessary to develop a library of promoter strength gradient, so as to design different promoter usage strategies for different exogenous proteins, different host cells, and different industrial production needs.

Disclosure of Invention

The purpose of the disclosure is to develop a promoter regulatory sequence library, so as to utilize the regulation mechanism of the bacillus subtilis promoter and provide a richer material source for industrial application of the bacillus subtilis promoter.

In order to achieve the above object, the present disclosure provides a regulatory sequence library of a bacillus subtilis F4 promoter, which consists of regulatory sequences shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No. 3.

The present disclosure also provides a promoter library consisting of promoters having the regulatory sequences SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, according to the first aspect of the present disclosure.

The invention also provides a recombinant expression vector, which can allow insertion of a reading frame and allows the inserted reading frame to express a protein in bacteria, wherein a promoter in the recombinant expression vector is selected from promoters in the promoter library disclosed by the disclosure.

The present invention also provides a method for expressing a foreign protein, wherein the method comprises: inserting the reading frame for coding the foreign protein into the recombinant expression vector to obtain recombinant expression plasmid, introducing the recombinant expression plasmid into competent bacteria to obtain an expression strain, and culturing the expression strain.

The invention also provides application of the bacillus subtilis F4 promoter regulatory sequence library in preparation of recombinant expression vectors and/or expression plasmids.

The invention also provides the application of the promoter library in the preparation of recombinant expression vectors and/or expression plasmids.

By the technical scheme, the control sequences or promoters with different foreign protein expression strengths can be selected according to the production purpose under the condition of no induction, and the starting strength of foreign protein expression is reasonably selected and controlled.

The regulatory sequence library and the promoter library provided by the disclosure can form a gradient library of promoter strength and can be used for expressing undefined foreign proteins, so that promoters which aim at different foreign proteins and different expression strains and meet different production requirements are determined.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 shows the result of functional relationship detection of promoter sequence using mApple as reporter gene; f4(P) represents a wild-type promoter, F4(C1) represents a promoter represented by SEQ ID No. 5; f4(C2) represents the promoter shown in SEQ ID NO. 6; f4(C3) represents the promoter shown in SEQ ID NO. 7.

Wherein, FIG. 1A shows the fluorescence intensity of mApple expressed as a reporter protein at different times.

FIG. 1B is a growth curve of different strains.

FIG. 1C shows the ratio of each mutant to wild-type expressed protein, where wild-type is 1.

FIG. 2 shows the results of the functional relationship between the promoter sequences of OPHC2 as a reporter gene;

wherein Free is WB600 no-load; f4(P) is wild type promoter; f4(C1) is a promoter shown as SEQ ID NO. 5; f4(C2) is a promoter shown as SEQ ID NO. 6; f4(C3) is a promoter shown in SEQ ID NO. 7.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The present disclosure provides a regulatory sequence library of the Bacillus subtilis F4 promoter, which consists of regulatory sequences shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No. 3.

In the present disclosure, the term "promoter" is used, unless otherwise specified, to refer to a DNA sequence located upstream of the 5' end of the structural gene that is specifically recognized by RNA polymerase and activates RNA polymerase. In the present invention, the promoter refers to a promoter of a prokaryote.

The Bacillus subtilis F4 promoter described in this disclosure was obtained by truncation of Bacillus subtilis ybp promoter P16, and sequence characteristics of this promoter are described in the article by Yucca et al (Yu, X., Xu, J., Liu, X., Chu, X., Wang, P., Tian, J., et al (2015) Identification of a high efficiency functional promoter in Bacillus subtilis scientific Reports,5,18405). The 103bp sequence of the wild type core segment of the promoter is shown in SEQ ID NO. 4. Having this core segment is a necessary condition for the nucleic acid to be able to function as a promoter, which itself may also function as a promoter; appropriate extension up to a length of 104-1000bp at the 5' end of the core fragment is also possible, as long as the nucleic acid obtained after extension is also capable of initiating transcription in B.subtilis.

The regulatory sequence of the present disclosure is located upstream of the transcription initiation site of the 5' end, and can affect the activity of the promoter for initiating transcription initiation, thereby affecting the expression amount of the target protein.

The regulatory sequence of the present disclosure is composed of 4 bases at the upstream and 4 bases at the downstream of a Bacillus subtilis F4 promoter-35 region sequence (-35TTGGATT-29), and the length is 15 bases.

In the disclosure, SEQ ID No.1 and SEQ ID No.2 can improve the strength of the promoter for promoting the expression of the target gene, and SEQ ID No.3 can reduce the strength of the promoter for promoting the expression of the target gene.

It is understood that nucleotide sequences that are in base complementary relationship to the sequences provided by the present disclosure also fall within the scope of the present disclosure.

Each nucleotide sequence in the promoter regulatory sequence library provided by the disclosure can be independently cloned to enter the upstream of a transcription initiation site of the promoter, so that different regulatory effects on the promoter can be provided, and the promoter can obtain different transcription initiation activities.

It is noted that the regulatory sequences in the library of promoter regulatory sequences provided by the present disclosure are not only suitable for regulating the activity of the Bacillus subtilis F4 promoter to initiate transcription, but are also suitable for regulating other promoters, for example, in a specific embodiment of the present disclosure, the regulatory sequences can be used for regulating a promoter obtained by mutating, replacing, truncating or extending a sequence based on the wild type F4 promoter, which is capable of initiating transcription in Bacillus subtilis; alternatively, for example, in another specific embodiment of the present disclosure, the regulatory sequences may be used to regulate the activity of the P43 promoter to initiate transcription, and may down-regulate, to varying degrees, the activity of the P43 promoter to initiate transcription.

In a second aspect of the present disclosure there is provided a library of the Bacillus subtilis F4 promoter consisting of promoters having the regulatory sequences SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, of the first aspect of the present disclosure.

Wherein, the promoters with the regulatory sequences SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 have different strength for regulating the expression of target genes.

In a specific embodiment of the present disclosure, the promoters with the regulatory sequences SEQ ID No.1 and SEQ ID No.2 have an up-regulated intensity to promote gene expression compared to the wild-type promoter. The promoter with the regulatory sequence SEQ ID NO.3 has a down-regulated strength to promote gene expression compared to the wild-type promoter.

Wherein, the length of each promoter in the Bacillus subtilis F4 promoter library is 104-1000bp, and the regulatory sequence is positioned at the position from-25 to-39 upstream of the transcription initiation site of the promoter.

In the present disclosure, there is no particular limitation on the sequence characteristics of the nucleic acid fragment other than the regulatory sequences in the promoter, and may be a promoter capable of promoting transcription in Bacillus subtilis obtained by mutating, replacing, truncating or extending the sequence based on the wild type of the Bacillus subtilis F4 promoter. For example, it may be a sequence having about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65% or 60% homology with the nucleotide sequence of the wild-type Bacillus subtilis F4 promoter shown in SEQ ID NO. 4.

In a preferred embodiment of the present disclosure, the promoter library consists of the nucleotides shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO. 7.

A third aspect of the present disclosure provides a recombinant expression vector capable of allowing insertion of a reading frame and allowing the inserted reading frame to express a protein in bacteria. The promoter in the recombinant expression vector is selected from the promoters in the promoter library of bacillus subtilis F4 described in the present disclosure.

Wherein the sequence of the reading frame of the protein to be expressed can be determined by means of interrogation or sequencing, and the DNA of the reading frame of the protein to be expressed can be obtained by conventional molecular biological means, such as full sequence synthesis, PCR amplification and restriction enzyme digestion. Generally, the recombinant expression vector may have conventional elements such as an origin of replication, a selection gene, a multiple cloning site, a transcription termination signal, and the like.

The present disclosure also provides a method of expressing a foreign protein, wherein the method comprises: inserting the reading frame for coding the foreign protein into the recombinant expression vector to obtain recombinant expression plasmid, introducing the recombinant expression plasmid into competent cells to obtain an expression strain, and then culturing the expression strain.

Wherein, the competent bacteria can be various conventionally used bioengineering bacteria, such as at least one of bacillus subtilis, escherichia coli, bacillus amyloliquefaciens and bacillus thuringiensis, preferably, the competent bacteria is bacillus subtilis.

Among them, each promoter in the promoter library of the present disclosure is capable of having higher transcriptional activity in the late log phase and the plateau phase of bacterial culture production than in other phases, and therefore, it is particularly preferred that the above-mentioned method for expressing a foreign protein comprises: culturing the expression strain to late log or plateau phase.

The present disclosure also provides the use of the bacillus subtilis F4 promoter regulatory sequence library as described above in the preparation of recombinant expression vectors and/or expression plasmids. The regulatory sequences upstream of the transcription start site of one or more of the promoters in the existing recombinant expression vectors and/or expression plasmids may be replaced by the regulatory sequences of the library of regulatory sequences of the present invention as described above using conventional genetic engineering means (e.g., restriction enzyme cleavage and/or PCR).

The present disclosure also provides the use of a promoter library as described above in the preparation of recombinant expression vectors and/or expression plasmids. One or more of the promoters in the existing recombinant expression vectors and/or expression plasmids may be replaced by promoters from the promoter libraries of the present invention as described above using conventional genetic engineering means (e.g., restriction and/or PCR).

The present invention will be described in further detail below with reference to examples. In the following examples, the Bacillus subtilis WB600 strain used for the preparation of competent bacteria was purchased from the Industrial microbial resources and information center, national colleges and universities, south China, university, at the name CICIM No. B0033.

Example 1

The entrusted reagent company synthesized the sequences shown in Table 1, and composed a regulatory sequence library and a promoter library.

TABLE 1

Test example 1

This test example serves to illustrate the effect of promoter regulatory sequences of the present disclosure on promoter transcriptional activity.

(1) mApple verifies the transcriptional activity of each sequence in the regulatory sequence library.

The regulatory sequences shown in the table 1 are respectively cloned into wild type bacillus subtilis F4 promoters (sequences shown in SEQ ID NO. 4), namely the regulatory sequences are respectively used for replacing nucleotide sequences at 40-54bp positions in the sequences shown in the SEQ ID NO.4 to obtain a promoter library, a report vector p43-G-F4-R: pUBC19 is constructed, and a report gene mApple is constructed in the report vector. The nucleotide sequence of the vector pUBC19 is shown in SEQ ID NO. 8.

And transforming the obtained report vectors into bacillus subtilis WB600 respectively, and amplifying to obtain a thallus sample after sequencing verification is correct.

Each sample was taken in a 3 x 6 pattern, i.e. 3 replicates, each replicate 6 times. Meanwhile, in order to reduce errors among the parallels, each sample is firstly streaked and activated, then 3 monoclonals with the same size are selected to be cultured in a 3mL LB liquid culture medium in an expanding way, and then the monoclonals are respectively inoculated in a 50mL LB liquid culture medium by 1% of inoculation amount and are fully and uniformly mixed, so that the differences among the samples and the parallels are consistent. In parallel, 8 samples (2 repeated samples at the outermost side are discarded because of the plate 'edge effect') are taken from the uniformly mixed culture medium by using a discharging gun, 200 mu L of culture medium is placed in a 96-well plate and cultured in a horizontal shaking table (37 ℃, 750rpm/min), the red fluorescence intensity and the cell density of the samples are measured every hour, the samples are continuously tracked for 20h, finally, the maximum average fluorescence values (relative fluorescence intensity/OD 600) of each expression strain and a wild type are compared, the wild type is 1, and each expression strain is converted into the percentage of the wild type, so that the influence of different regulatory sequences on the transcription activity of the promoter is measured. The results are shown in FIG. 1 (C).

Test example 2

The expression strain was constructed according to the method of test example 1. The only difference is that the reporter gene is replaced by the catalytic organophosphate hydrolase gene ophc 2.

Streaking strains such as an expression strain, a wild type (positive control), WB600 (negative control) and the like, selecting three clones (3 parallels) with smaller body states and consistent individuals in each strain, putting the three clones in 3mL LB liquid culture medium (containing 10 mu g/mL Kana), culturing for 12h at 37 ℃ and 220rpm, inoculating 3 LB liquid culture medium containing the same concentration of antibiotics in each parallel in an inoculation amount of 1%, uniformly mixing, putting the mixture in a shaking table at 37 ℃ and 220rpm, culturing for 20h, wherein each sample is taken once (1mL of shaking fermentation liquor) for 12h and 20h, centrifuging and taking supernatant. The methyl parathion hydrolase activity is detected by using methyl parathion pesticide. The results are shown in FIG. 2.

The above results indicate that promoters with regulatory sequences in the regulatory sequence libraries provided by the present disclosure can form different expression intensity gradients. Wherein, compared with a wild promoter, the activity of the promoter with the SEQ ID NO.1 in the library is up-regulated by 56.4% when the reporter gene is mApple, the activity of the reporter gene is up-regulated by 36.9% at 12h and up-regulated by 24.3% at 20h when the reporter gene is ophc 2; compared with the wild promoter, the promoter with the SEQ ID NO.2 has 79.9 percent of activity up-regulated when the reporter gene is mApple, 48.9 percent of activity up-regulated when the reporter gene is ophc2, and 63.8 percent of activity up-regulated when the reporter gene is 20 h; compared with the wild promoter, the activity of the promoter with the SEQ ID NO.3 is reduced by 69% when the reporter gene is mApple, 94.4% when the reporter gene is ophc2, and 98.6% when the activity of the reporter gene is reduced by 12 h.

Test example 3

This test example was used to examine the effect of promoter regulatory sequences of the present disclosure on the transcriptional activity of the P43 promoter.

The P43-egfp fragment was amplified using a primer having a homologous end to the Free-pUBC19 fragment, using the plasmid P43-G-F4-G pUBC19 as a template. A reporter vector P43-egfp: pUBC19 containing the reporter gene egfp was constructed and transformed into Bacillus subtilis WB 600. The correct clones were selected for testing.

The effect of the promoter regulatory sequence on the transcriptional activity of the P43 promoter was examined using the mApple fluorescence intensity method as in test example 1.

Compared with the wild P43 promoter, the activity of the promoter containing the regulatory sequence SEQ ID NO.1 and the activity of the promoter containing the regulatory sequence SEQ ID NO.2 are reduced to different degrees, and the activity of the promoter containing the regulatory sequence SEQ ID NO.3 is reduced by about 30 percent.

The above results indicate that the P43 promoter having the promoter regulatory sequence in the promoter regulatory sequence library provided in the present disclosure can form different expression intensity gradients.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

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gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140

tttttgagca actggatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat 4200

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ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 4320

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4380

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taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4560

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4620

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4680

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4740

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gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4860

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4920

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4980

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5040

gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5100

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5160

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5220

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5280

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5340

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 5400

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 5460

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 5520

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 5580

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 5640

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 5700

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 5760

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 5820

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 5880

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 5940

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6000

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6060

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6120

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6180

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6240

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6300

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 6360

aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 6420

gtc 6637

Claims (9)

1. The regulatory sequence library of the bacillus subtilis F4 promoter is characterized by consisting of regulatory sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

2. The library of regulatory sequences of the Bacillus subtilis F4 promoter of claim 1, wherein SEQ ID No.1 and SEQ ID No.2 are capable of increasing the strength of the promoter to promote the expression of the target gene, and SEQ ID No.3 is capable of decreasing the strength of the promoter to promote the expression of the target gene.

3. Promoter library, characterized in that it consists of promoters with the regulatory sequences SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, according to claim 1, which are capable of initiating transcription in Bacillus subtilis.

4. The promoter library of claim 3, wherein the promoters having the regulatory sequences SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 have different strengths to regulate the expression of the gene of interest.

5. The promoter library of claim 3 or 4, wherein each promoter in the promoter library has a length of 104-1000bp, and the regulatory sequence is located at a position-25 to-39 upstream of the transcription initiation site of the promoter.

6. A recombinant expression vector capable of allowing insertion of a reading frame and allowing expression of a protein from the inserted reading frame in bacteria, wherein the promoter in the recombinant expression vector is selected from the group consisting of promoters from the promoter library of any one of claims 3 to 5.

7. A method of expressing a foreign protein, the method comprising: inserting the reading frame encoding the foreign protein into the recombinant expression vector of claim 6 to obtain a recombinant expression plasmid, introducing the recombinant expression plasmid into a competent bacterium to obtain an expression strain, and then culturing the expression strain.

8. The method of claim 7, wherein the competent bacteria is Bacillus subtilis.

9. Use of a library of regulatory sequences according to any of claims 1 to 2 or a library of promoters according to any of claims 3 to 5 for the preparation of recombinant expression vectors and/or expression plasmids.

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