High-induction-activity promoter derived from tagatose operon of bacillus licheniformis

Abstract

The invention discloses a high-induction-activity promoter derived from a tagatose operon of bacillus licheniformis, and belongs to the field of gene engineering. The promoter PfruK2 capable of being induced by a carbon source in a PTS (Pleurotropin Tyrosine Sequence) way is screened, the induced carbon substrate spectrum capable of being responded by the promoter is wide, the promoter is not affected by a glucose-mediated CCR effect, the induction intensity is high, and the induction peak value is improved by more than 50% compared with that of a quorum sensing strong promoter PLan.

Description

TECHNICAL FIELD

[0001] The present application relates to a high-inducible promoter derived from the tagatose operon of Bacillus licheniformis, belonging to the field of genetic engineering. BACKGROUND

[0002] Bacillus licheniformis is a gram-positive facultative anaerobic endospore-forming bacterium. The basis for producing target products using Bacillus licheniformis as a chassis cell is the promoter element. At present, the promoters widely used in Bacillus licheniformis include the constitutive strong promoter P 43 , the quorum-sensing strong promoter P Lan , the xylose-inducible promoter P xylA , and the mannitol-inducible promoter P mtlA . These elements are far from sufficient for the development of Bacillus licheniformis. Taking Bacillus subtilis as an example, there are more than 20 efficient constitutive promoters in Bacillus subtilis, and the inducible promoter elements almost cover all known sugar alcohol induction systems. However, the promoter elements in Bacillus subtilis are not suitable for Bacillus licheniformis, and therefore, new efficient promoter elements suitable for Bacillus licheniformis need to be developed.

[0003] Mining high-strength promoters from sugar metabolic pathways is a strategy for developing promoters. For example, the methanol-inducible promoter widely used in Pichia pastoris. It has been reported that wild-type Bacillus licheniformis can grow using tagatose as a carbon source, while wild-type Escherichia coli and Bacillus subtilis cannot use tagatose. Therefore, Bacillus licheniformis has a tagatose biological element that is not possessed by the model microorganisms Escherichia coli and Bacillus subtilis. This system is expected to be developed as an expression system suitable for Bacillus licheniformis. In 2013, Heiden et al. reported the tagatose operon system of Bacillus licheniformis, which consists of a transcriptional regulator, 1-phosphofructokinase FruK2, fructose phosphotransferase FruA2, and tagatose diphosphate aldolase GatY (see A pathway closely related to the D-tagatose pathway of Gram-Negative Enterobacreria identified in the Gram-Positive bacterium Bacillus licheniformis). However, there is still a lack of promoter elements in Bacillus licheniformis, and the problem of glucose-mediated carbon catabolite repression (CCR) of promoter elements derived from sugar operons (such as xylose-inducible promoters and mannitol-inducible promoters). SUMMARY

[0004] The present application provides a promoter PfruK2 derived from a tag sugar operon of Bacillus licheniformis and not subject to CCR effect mediated by glucose and its application. The present application constructs a carbon source induction expression system of Bacillus licheniformis PTS pathway by constructing the promoter PfruK2 to an expression vector, and adds a cheap PTS pathway carbon source (such as glucose, fructose, sucrose, mannose, mannitol, maltose, tag sugar, etc.) as an inducer in practical application, thereby saving cost.

[0005] The present application provides a promoter PfruK2, which has a nucleotide sequence shown in SEQ ID NO. 1.

[0006] The present application also provides an expression vector containing the promoter PfruK2.

[0007] In an embodiment, the expression vector comprises pHY-PLK300.

[0008] The present application also provides a genetically engineered bacterium containing the promoter PfruK2 or the expression vector.

[0009] In an embodiment, the genetically engineered bacterium takes Bacillus licheniformis CICIM B1522 as a host, and expresses a target protein by using a plasmid pHY-PLK300 containing the promoter PfruK2.

[0010] In an embodiment, the target protein includes but is not limited to an enzyme with biological catalytic activity.

[0011] In an embodiment, the target protein includes a fluorescent protein.

[0012] The present application also provides an application of the promoter PfruK2 in regulating protein expression.

[0013] In an embodiment, the application is to connect a gene encoding the protein to a downstream of the promoter PfruK2, and to initiate transcription by the promoter PfruK2.

[0014] In an embodiment, the application further includes culturing a genetically engineered bacterium expressing the protein in a culture medium to a logarithmic growth phase, and inducing protein expression by a PTS pathway carbon source.

[0015] In an embodiment, the application is to culture the genetically engineered bacterium for 5-8 hours, and to induce protein expression by a PTS pathway carbon source.

[0016] In an embodiment, the PTS pathway carbon source includes one or more of glucose, fructose, sucrose, mannose, mannitol, maltose, and tag sugar.

[0017] Advantages:

[0018] The present application first discovers the promoter PfruK2 which can be induced by the carbon source (glucose, fructose, sucrose, mannose, mannitol, maltose, tagatose) of PTS pathway, and the carbon substrate spectrum is wide. The promoter is not affected by the CCR effect mediated by glucose, and is affected by the induction effect mediated by glucose, and the induction intensity is high, and the induction peak is more than 50% higher than that of the group induction strong promoter P Lan . BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a schematic diagram of tagatose operon of Bacillus licheniformis.

[0020] Figure 2 It is a map of recombinant plasmid pHY-P T1 -mCherry, pHY-PfruK2-mCherry.

[0021] Figure 3 It is a graph of tagatose concentration and fluorescence value of recombinant strain carrying pHY-PfruK2-mCherry.

[0022] Figure 4 It is the fluorescence value of recombinant strain carrying pHY-PfruK2-mCherry in response to different carbon source inducers. DETAILED DESCRIPTION

[0023] Red fluorescent protein detection method: centrifugal collection of sample, 200 μL supernatant is added to the enzyme-labeled plate, and placed in the enzyme-labeled instrument for detection, the detection method is: selecting the detection area, shaking the enzyme-labeled plate for 10 s, the excitation wavelength is 532 nm, the absorption light is 588 nm, and the gain value is 100. Sample fluorescence value (AU / mL) = (experimental group fluorescence value-control (carrying pHY-PLK300 plasmid recombinant strain) fluorescence value) * dilution multiple.

[0024] Example 1: Construction of tagatose operon-based induced expression system

[0025] The tagatose operon in Bacillus licheniformis is shown in Figure 1 , and the tagatose operon comprises two separate promoters P T1 (nucleotide sequence as SEQ ID NO. 2) and PfruK2 (nucleotide sequence as SEQ ID NO. 1). In the tagatose operon, the promoter P T1 is responsible for the transcription of transcription factors, and PfruK2 is responsible for the transcription of structural genes. With Bacillus licheniformis CICIM B1391 genome as template, primer P T1-F / PT1-R ;P fruK2-F / P fruK2-R PCR amplification of P T1 and PfruK2. The P T1 and PfruK2 were respectively homologously recombined with the pHY300PLK plasmid linearized by HindIII / XhoI, to obtain plasmids pT and pF. The mCherry fragment (shown in SEQ ID NO. 3) was amplified by mCherry-F / mCherry-R, and homologously recombined with the plasmids pT and pF linearized by KpnI / SalI, to obtain plasmids pTM and pFM Figure 2 ) respectively. Based on the original strain Bacillus licheniformis CICIM B1522, the amyL gene was knocked out by referring to the method in the paper “Efficient genome editing in Bacillus licheniformis mediated by a conditional CRISPR / Cas9 system”, and the aprE gene was knocked out by referring to the method in “Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout”, to obtain Bacillus licheniformis CICIM B1522ΔamyL,ΔaprE.

[0026] The constructed recombinant plasmid pTM and the recombinant plasmid pF were respectively electroporated into Bacillus licheniformis CICIM B1522ΔamyL,ΔaprE, to obtain target strains PTM and PFM respectively.

[0027] P T1-F :tatggaaaaacgctttgcccAAGCTTacttgaaatatccggctctc;

[0028] P T1-R :GCAAACTTTTTGATGTTCATCTCGAgattaaaacccccttttatag;

[0029] P fruK2-F :tatggaaaaacgctttgcccAAGCTTaaacctttacacaacctgta;

[0030] P fruK2-R:GCAAACTTTTTGATGTTCATCTCGAggctgtatcccccctttatct;

[0031] mCherry-F:CGCATCATCATCATCATCATGGTACCatggtgagcaagggcgagga;

[0032] mCherry-R:AATCCGCCTGGCTGAATGTCGACttacttgtacagctcgtccatgc.

[0033] Example 2: Fermentation effects of recombinant strains regulated by different promoters

[0034] Prepare fermentation medium: 20 g / L peptone, 12 g / L yeast powder, 10 g / L K2HPO4, 1.36 g / L KH2PO4, 5 g / L (NH4)2SO4, 10 g / L urea, 2 g / L MgSO4.7H2O, 10 g / L sodium glutamate.

[0035] The recombinant strains PTM and PFM constructed in Example 1 were cultured under the following two conditions:

[0036] 1. Without tagatose: inoculate the strain into the fermentation medium (initial OD after inoculation = 0.1) and culture at 37°C and 220 rpm for 48 h;

[0037] 2. Induction with 0.75% tagatose: inoculate the strain into the fermentation medium (initial OD after inoculation = 0.1), culture at 37°C, 220 rpm for 6 h, add tagatose to a final concentration of 7.5 g / L, and continue to culture for a total of 48 h;

[0038] The fluorescence values ​​of the fermentation broth cultured for 48 hours under the above two conditions were detected respectively. Figure 3 As shown, the fluorescence value of the PTM strain after 48 hours of fermentation without tagatose was 2521 AU / mL; after 48 hours of fermentation with tagatose, the fluorescence value was 6493 AU / mL, a 157.5% increase compared to the sample without tagatose. The fluorescence value of the PFM strain was 22383 AU / mL without tagatose and 36934 AU / mL with tagatose, a 65.01% increase compared to the sample without tagatose.

[0039] The above results showed that the PFM strain showed a higher tagatose response value, indicating that the expression level of PfruK2 was higher than that of P T1 , so the PFM strain was selected for further study.

[0040] Example 3: Fermentation effect of recombinant strains under different induction conditions

[0041] Next, the addition concentration of tagatose was tested. The PFM strain was inoculated into the fermentation medium at an inoculation amount of 3%, and after 6h of culture at 37℃, 2.5, 5, 7.5, 10, 15, and 20g / L of tagatose was added respectively, with no addition of tagatose as a control. The results showed that when the concentration of tagatose was less than 10g / L, the red fluorescent protein fluorescence value was positively correlated with the addition concentration of tagatose (R 2 = 0.94); within the range of 10-20g / L, the red fluorescent protein fluorescence value was basically unchanged. The optimal addition condition was 1.0% tagatose, and the maximum response value was 48523AU / mL.

[0042] Example 4: Verification of the response ability of recombinant bacteria to different carbon sources

[0043] The strain PFM constructed in Example 1 was cultured in the fermentation medium at 37℃ for 6h, and 10g / L of different carbon sources was added respectively as an inducer, and cultured for 48h. The results showed that when the fermentation medium contained PTS pathway carbon sources, i.e. glucose, fructose, sucrose, tagatose, mannose, mannitol or maltose, the promoter could be induced, while the non-PTS pathway carbon sources (such as xylose, arabinose, etc.) could not induce the promoter. As shown in Table 1, the induction intensity from high to low was glucose, fructose, sucrose, tagatose, mannose, mannitol, and maltose. Figure 4 The promoter P fruK2 was not affected by the CCR effect mediated by glucose, and had a very high regulation intensity in the presence of glucose.

[0044] Comparative example

[0045] The quorum-sensing strong promoter P Lan (nucleotide sequence as shown in SEQ ID NO. 4) was constructed into a recombinant plasmid according to the method of Example 1, and transformed into Bacillus licheniformis CICIM B1522ΔamyL,ΔaprE. The strain was cultured in the fermentation medium at 37℃, 220rpm for 48h, and the results showed that the red fluorescent protein fluorescence value in the fermentation broth of the strain regulated by P Lan was 35292AU / mL, which was much lower than the response value (48523AU / mL) of the strain PFM.

[0046] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. Promoter PfruK2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

2. An expression vector containing the promoter PfruK2 according to claim 1.

3. The expression vector according to claim 2, characterized in that Includes pHY-PLK300 plasmid.

4. A genetically engineered bacterium containing the promoter PfruK2 according to claim 1 or the expression vector according to claim 2 or 3.

5. The genetically engineered bacterium according to claim 4, characterized in that Bacillus licheniformis CICIM B1522 is used as a host, and the expression vector according to claim 3 is used to express the target protein.

6. The genetically engineered bacterium according to claim 5, characterized in that The target protein includes but is not limited to an enzyme with biocatalytic activity.

7. Use of the promoter PfruK2 according to claim 1 in regulating protein expression.

8. The use according to claim 7, characterized in that The application is to connect the gene encoding the protein to the downstream of the promoter PfruK2, and initiate transcription from the promoter PfruK2.

9. The use according to claim 7, characterized in that The application further comprises culturing the genetically engineered bacteria expressing the protein in a culture medium to a logarithmic growth phase, and inducing protein expression using a PTS pathway carbon source.

10. The use according to claim 9, characterized in that The PTS pathway carbon source includes one or more of glucose, fructose, sucrose, mannose, mannitol, maltose, and tagatose.

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