An artificially engineered xylose-inducible promoter and use thereof
By modifying the Bacillus subtilis Pxyl promoter, the inducible expression intensity of the xylose-inducible promoter Pxyl4 was enhanced and the background leakage was reduced, solving the problem of insufficient expression intensity of existing promoters and achieving efficient regulation of gene and protein synthesis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING AGRICULTURAL UNIVERSITY
- Filing Date
- 2022-08-23
- Publication Date
- 2026-06-19
AI Technical Summary
Bacillus subtilis lacks xylose-inducible promoters with excellent characteristics. Existing promoters have low expression intensity after induction and high background expression levels, which is difficult to meet the requirements of efficient gene expression and protein synthesis.
By modifying the Pxyl promoter of Bacillus subtilis strain 168, including modifying the -35 and -10 regions, adding mRNA stability sequences, and random mutations of operators, a novel xylose-inducible promoter, Pxyl4, was designed and obtained, which improved the induction expression intensity and reduced the background leakage.
It achieved a more than 30-fold increase in the expression intensity of the promoter Pxyl4 under induced conditions and a 700-fold reduction in background leakage under non-induced conditions, making it suitable for efficient regulation of gene expression and protein synthesis.
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Figure CN115948395B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biohigh technology and discloses an artificially modified xylose-inducible promoter and its applications. Background Technology
[0002] Bacillus subtilis is an important microorganism. On the one hand, these bacteria are used in theoretical research as representatives of Gram-positive bacteria; on the other hand, they are developed into cell factories for protein expression or compound synthesis. Promoters are commonly used genetic elements in both theoretical research and cell factory development. Promoters mediate gene transcription and generally include constitutive promoters and inducible promoters. The former works without induction, while the latter requires physical or chemical induction to function. Because inducible promoters can flexibly regulate the expression of target genes, they play an important role in gene function verification and cell factory construction. A high-quality inducible promoter should possess the following characteristics: low background expression level under non-induction conditions and high expression level under induction conditions. However, there is currently a lack of high-quality inducible promoters in Bacillus subtilis, and this problem needs to be solved through discovery or modification.
[0003] The xylose-inducible promoter P in Bacillus subtilis xyl This is a commonly used inducible promoter in this type of bacteria. The promoter includes three key sequences: the -35 region, the -10 region, and the operator. Figure 1 The -35 and -10 regions are used to bind RNA polymerase to initiate transcription. The operator can be bound by the repressor protein XylR, thereby preventing transcription. In the absence of xylose, XylR binds to the operator site, inhibiting transcription and allowing the promoter P to... xyl In the off state; upon addition of xylose, xylose can bind to XylR, causing the latter to detach from the operator site, thereby relieving inhibition and allowing P to... xyl It is in the enabled state (Journal of bacteriology. 1989. 171: 3840-3845). Although the promoter P xyl It is frequently used in Bacillus subtilis, but the promoter still suffers from low expression intensity after induction.
[0004] This invention uses Bacillus subtilis strain 168 P xyl Using the starting sequence, a new xylose-inducible promoter P was obtained through modifications to the -35 and -10 regions, the addition of mRNA stability sequences, and random mutations of the operator. xyl4 P xyl4It combines the advantages of high induced expression intensity and low background expression level, and has great application potential in protein recombinant expression and synthetic biology. Summary of the Invention
[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing an artificially modified xylose-inducible promoter.
[0006] Another object of the present invention is to provide applications of the promoter.
[0007] In view of this, the present invention uses a method of rational design plus random mutation to target P. xyl Through modification, a novel xylose-induced promoter with high induction level and low background leakage was obtained.
[0008] The objective of this invention can be achieved through the following technical solutions:
[0009] An artificially modified xylose-inducible promoter P xyl4 The sequence is shown in SEQ ID NO.10.
[0010] The artificially modified xylose-inducible promoter P described in this invention xyl4 Applications in the construction of recombinant expression vectors.
[0011] The artificially modified xylose-inducible promoter P described in this invention xyl4 Application in constructing recombinant expression vectors for expressing exogenous proteins.
[0012] The artificially modified xylose-inducible promoter P described in this invention xyl4 Application in the expression of recombinant proteins in Bacillus subtilis.
[0013] As a preferred embodiment of the present invention, the artificially modified xylose-inducible promoter P xyl4 Recombinant expression of mussel adhesion proteins Pvfp5 or Mfp-3.
[0014] A recombinant expression vector comprising the xylose-inducible promoter P described in this invention. xyl4 .
[0015] As a preferred embodiment of the present invention, the recombinant expression vector further comprises the encoding gene of mussel adhesion protein Pvfp5 or Mfp-3.
[0016] Beneficial effects:
[0017] The artificially modified promoter P obtained in this invention xyl4 It exhibits high induced expression levels and the lowest background leakage (a difference of over 700-fold between induced and non-induced conditions); relative to the starter promoter P... xyl P xyl4The strength increased more than 30 times under induced conditions. P xyl4 It can be used for gene expression regulation or efficient protein synthesis in Bacillus subtilis. Attached Figure Description
[0018] Figure 1 xylose promoter P in Bacillus subtilis xyl Genetic map and sequence
[0019] Figure 2 Random mutations in the spacer region of the operator
[0020] Figure 3 The expression of GFP protein mediated by four modified promoters
[0021] Figure 4 promoter P xyl4 Mediating the expression of mussel adhesion protein Pvfp5
[0022] M is the marker; lane 1 is an empty host that does not express the target protein; lane 2 is the promoter P. xyl4 Intracellular expression of Pvfp5 adhesion protein mediated by [the study / initiative].
[0023] Figure 5 promoter P xyl4 Mediating the expression of mussel adhesion protein Mfp-3
[0024] M is the marker; lane 1 is the promoter P. xyl4 Intracellular expression of Mfp-3 mediated by [the medium / substance]. Detailed Implementation
[0025] Example 1
[0026] This invention utilizes the xylose-induced promoter P from Bacillus subtilis type strain 168 (GenBank accession number: NC_000964.3). xyl (SEQ ID NO.1) is the starting sequence. For example... Figure 1 As shown, P xyl Including the -35 region "TTGAAA", the -10 region "TAAGAT", and the operator " TTAGTTTGTT TAAAC AACAAACTAA "These three key sites. The five bases TAAAC in the middle of the operator are called the spacer, and the spacer is flanked by inverted repeat sequences."
[0027] In order to improve P xyl The induction level, firstly, P xylThe -35 region “TTGAAA” and the -10 region “TAAGAT” were modified to “TTGACA” and “TATAAT” respectively, resulting in SEQ ID NO.2. Next, SEQ ID NO.2 was compared with P from Bacillus thuringiensis. cryIIIa A portion of the promoter (SEQ ID NO. 3) was fused to obtain SEQ ID NO. 4. The xylose-inducible promoter obtained after this modification was named P. xyl1 (SEQ ID NO.4). A biotechnology company was commissioned to synthesize SEQ ID NO.4, bleomycin resistance (SEQ ID NO.5), and replicons of the pBS72 plasmid (Plasmid. 2003. 49: 53-62.) (SEQ ID NO.6), as well as the green fluorescent protein gene gfp (SEQ ID NO.7). Then, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 were ligated using a homologous recombination kit (purchased from Novizan Biotechnology Co., Ltd.). The ligation products were transformed into competent cells of Bacillus subtilis strain 168 (Journal of bacteriology. 1961. 81: 741-746.), and plated on LB agar plates containing 20 mg / L bleomycin (LB medium formulation: 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride), yielding P... xyl1 Regulated green fluorescent protein expression plasmid pPX1-P xyl1 -gfp.
[0028] It has been reported that the TAAAC sequence in the interstitial spacer region affects the affinity between the operator and the repressor protein xylR (Molecular and general genetics. 1992. 232: 415-422.). Therefore, we then enhanced the affinity between the operator and xylR by mutating the interstitial spacer sequence, thereby reducing P. xyl1 Background leakage expression levels under non-induced conditions. To alter the spacer region "TAAAC" using a random mutation approach, pPX1-P... xyl1 Using -gfp as a template, fragment P was amplified with primers 5N-F / 5N-R. xylNThe spacer region “TAAAC” was transformed into “NNNNN” using primer 5N-F; the pPX1 vector fragment was amplified using primers P3 / P4; then the two fragments were fused using a multimerization method (Methods in molecularbiology. 2014. 111: 183-192.); the fusion product was transformed into Bacillus subtilis 168 competent cells (Journal of bacteriology. 1961. 81: 741-746.) and plated on LB agar plates containing 20 mg / L bleomycin to obtain a random mutant library of the central spacer sequence. Transformants were spotted onto LB agar plates and LB + 1% xylose agar plates, respectively. Figure 2 Transformants exhibiting weak fluorescence on LB plates (without inducer, -xylose) and strong fluorescence on LB plates with 1% xylose (with inducer, +xylose) were screened. Cell fluorescence intensity was further quantified by flow cytometry. Finally, mutant P, exhibiting high induced expression levels and low background leakage, was selected. xyl2 (SEQ ID NO.8), where P xyl2 The spacer region mutated from "TAAAC" to "GGTCC". It carries the xylose promoter P. xyl2 The plasmid was named pPX2-P xyl2 -gfp.
[0029] To further reduce background leakage under non-induced conditions, we added an operator to the xylose promoter. Using pPX1-P xyl1 Using -gfp as a template, fragment A and fragment B were amplified with primer pairs P3 / P4 and P5 / P6, respectively. The two fragments were then ligated using the multimer method described above (Methods in molecular biology. 2014.111:183-192.) to obtain plasmid pPX3-P. xyl3 -gfp, the promoter P carried by this plasmid xyl3 (SEQ ID NO.9) contains a dual operator ( TTAGTTTGTT TAAAC AACAAACTAA ). with pPX2-P xyl2 Using -gfp as a template, fragments C and D were amplified using primer pairs P3 / P4 and P7 / P8, respectively. These two fragments were then ligated using the multimer method described above (Methods in molecularbiology. 2014.111:183-192.) to obtain plasmid pPX4-P. xyl4- gfp (SEQ ID NO.13), the plasmid carries the promoter P xyl4 (SEQ ID NO.10) contains a double mutant operator ( TTAGTTTGTT GGTCC AACAAACTAA The pPX2 vector was amplified using primers, and the two sequences were amplified into multimers. The constructed double-mutant operator region ( TTAGTTTGTT GGTCC AACAAACTAA The resulting promoter is named P. xyl4 (SEQ ID NO.10).
[0030] Example 2: Regulation of green fluorescent protein expression in Bacillus subtilis using an artificially modified promoter
[0031] The four plasmids (pPX1-P) constructed above were used. xyl1 -gfp, pPX2-P xyl2 -gfp, pPX3-P xyl3 -gfp and pPX4-P xyl4 Transformants (-gfp) were transformed into Bacillus subtilis 168 competent cells. The four transformants were divided into induction and non-induction groups and cultured at 37℃ and 200 rpm until OD600. 600 =0.8, the induction group was induced with a final concentration of 1% xylose. After 10 h of induction, the green fluorescence distribution of each sample was measured using a BDBiosciences Accuri C6 flow cytometer. 10 measurements were taken for each sample. 5 Individual cells. For example... Figure 3 As shown, compared to the other three modified promoters, promoter P xyl4 The fluorescence intensity was highest after induction, and the background leakage was very low under non-induction conditions. This is relative to the starting promoter P. xyl P xyl4 The strength increased more than 30 times under induced conditions. P xyl4 The drop is approximately 700 times under induced and non-induced conditions.
[0032] Example 3: Using promoter P xyl4 Mediating the expression of mussel adhesion protein Pvfp5
[0033] Pvfp5 (approximately 10 kDa) is a mussel foot protein that exhibits high adhesion to solid surfaces in moist environments, making it a potential protein material (Nature Communications. 2015.6:8737.). We used Pvfp5 in Bacillus subtilis... xyl4 Mediates Pvfp5 recombination expression. Using pPX4-P xyl4Using the -gfp plasmid as a template, the pPX4 vector fragment F4 was amplified using primers ZT5-F / ZT5-R4; the Pvfp5 fragment (SEQ ID NO.11, synthesized according to Bacillus subtilis codon preference) was amplified using primers Pvfp5-F / Pvfp5-R; the Pvfp5 fragment and F4 were homologously ligated (kit purchased from Novizan Biotechnology Co., Ltd.), and the ligation product was then transformed into Bacillus subtilis 168 competent cells to obtain strain 168 (pPX4-P xyl4 -Pvfp5). The induction conditions for this strain were the same as in "Example 2". The induced cells were disrupted using an ultrasonic homogenizer. The sample was mixed with protein loading buffer and then subjected to high-temperature denaturation, followed by SDS-PAGE analysis. Figure 4 As shown, P xyl4 The mediated expression level of Pvfp5 is very high, accounting for more than 40% of the total intracellular protein.
[0034] Example 4: Using promoter P xyl4 Mediating the expression of mussel adhesion protein Mfp-3
[0035] Mfp-3 (approximately 6 kDa) is another mussel adhesion protein. Studies have shown that this protein participates in the surface binding between the matrix and the adhesion patch, thereby mediating mussel adhesion (Nature Chemical Biology. 2011.7:588-90.). Similarly, we used P in Bacillus subtilis. xyl4 Mediates the expression of this protein. Using pPX4-P xyl4 Using the -gfp (SEQ ID NO. 13) plasmid as a template, the pPX4 vector fragment F4-2 was amplified with primers ZT3-F / ZT3-R4; the mfp3 fragment (SEQ ID NO. 12, referred to Bacillus subtilis codon preference synthesis) was amplified with primers Mfp-3-F / Mfp-3-R; ligation, transformation, induction, and protein detection methods were the same as above. Figure 5 As shown, in P xyl4 Under its mediation, Mfp-3 accounts for more than 40% of the total intracellular protein.
[0036] Table 1 Primers involved in this invention
[0037]
Claims
1. An artificially modified xylose-inducible promoter P xyl4 Its characteristics The sequence is shown in SEQ ID NO.
10.
2. The artificially modified xylose-inducible promoter P as described in claim 1 xyl4 Applications in the construction of recombinant expression vectors.
3. The application according to claim 2, characterized in that, The artificially modified xylose-inducible promoter P as described in claim 1 xyl4 Application in constructing recombinant expression vectors for expressing exogenous proteins.
4. The artificially modified xylose-inducible promoter P as described in claim 1 xyl4 Application in the expression of recombinant proteins in Bacillus subtilis.
5. The application according to claim 4, characterized in that... The artificially modified xylose-inducible promoter P as described in claim 1 xyl4 Recombinant expression of mussel adhesion proteins Pvfp5 or Mfp-3.
6. A recombinant expression vector, characterized in that... Contains the xylose-inducible promoter P as described in claim 1 xyl4 .
7. The recombinant expression vector according to claim 6, characterized in that... It also contains genes encoding mussel adhesion proteins Pvfp5 or Mfp-3.