Construction method and application for expressing CPD photolyase in bacillus subtilis
By constructing a co-expression system of CPD photorepair enzyme and 8-HDF synthase in Bacillus subtilis, the limited activity and safety issues of natural photorepair enzymes were solved, enabling the development of efficient and safe UV protection products and improving the repair efficiency of photorepair enzymes.
Patent Information
- Application Number
- CN202511114186.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-11-14
AI Technical Summary
The activity of natural photorepair enzymes in existing technologies is limited by coenzyme dependence and environmental conditions, which restricts their application potential in bioengineering. Furthermore, the purification of heterologous proteins introduces sensitization risks, and traditional E. coli systems do not meet the safety requirements for cosmetic raw material production.
A co-expression system of CPD photorepair enzyme and 8-HDF synthase was constructed in Bacillus subtilis. Through codon optimization and recombinant plasmid design, the efficient co-expression of photorepair enzyme and 8-HDF synthase was achieved. The safety and strong stress resistance of Bacillus subtilis were utilized to avoid the risks of heterologous protein purification.
This achievement enables efficient expression and enhanced activity of photorepair enzymes, providing highly safe UV protection products and filling the technological gap in photorepair enzyme expression in Bacillus subtilis. It has significant social value and market potential.
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Figure CN120944933A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to a method for constructing and applying CPD photorepair enzyme and 8-HDF synthase expressed in Bacillus subtilis. Background Technology
[0002] Ultraviolet (UV) radiation is one of the main environmental factors causing DNA damage to the skin. It disrupts the DNA double helix structure by inducing the formation of cyclobutanepyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) between adjacent pyrimidine bases in the DNA strand, leading to apoptosis, mutations, and even carcinogenesis. Photolyases are DNA repair proteins widely found in nature that specifically recognize and bind to pyrimidine dimers. Activated by visible light (300-500 nm), they efficiently cleave the dimers through electron transfer mechanisms, restoring the normal DNA structure. However, the activity of natural photolyases is limited by their coenzyme dependence and environmental conditions, restricting their potential applications in bioengineering.
[0003] Photorepair enzymes repair UV-induced DNA damage by absorbing visible light energy (300-500 nm), with their core mechanism relying on covalently bound flavin adenine dinucleotide (FAD) coenzymes. FAD acts as a photosensitive electron donor, transferring electrons to the pyrimidine dimer under light irradiation, achieving efficient damage repair. Studies have shown that some photorepair enzymes can also bind to a second coenzyme (such as 8-hydroxy-5-dezariboflavin, 8-HDF, or 6,7-dimethyl-8-trinitrouracil, DMRL), significantly enhancing their activity and repair efficiency by absorbing a broader spectrum of light energy and transferring energy to the reduced FAD.
[0004] Bacillus subtilis is an internationally recognized food-grade (GRAS) microorganism with advantages such as strong secretion capacity, no endotoxins, and a clear genetic background. Compared to traditional E. coli systems, it better meets safety requirements in cosmetic raw material production. This patent uses Bacillus subtilis as a host to achieve efficient co-expression of photorepair enzymes and 8-HDF synthase. This engineered strain can be directly used to produce cosmetic raw materials containing active photorepair enzymes, avoiding the sensitization risks introduced by heterologous protein purification. Simultaneously, the strong stress resistance of Bacillus subtilis ensures the stability of industrial fermentation, providing an innovative solution for developing UV protection products that combine high-efficiency repair and safety. Summary of the Invention
[0005] The purpose of this invention is to overcome the deficiencies in the prior art and provide a method for constructing CPD photorepair enzyme and 8-HDF synthase to express in Bacillus subtilis.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0007] A method for constructing CPD photorepair enzyme and 8-HDF synthase expressed in Bacillus subtilis, wherein the amino acid sequence of the photorepair enzyme is shown in SEQ ID No:2 and the amino acid sequence of the 8-HDF synthase is shown in SEQ ID No:4.
[0008] The first technical problem to be solved by this invention is to construct an expression vector containing the genes for photorepair enzyme and 8-HDF synthase.
[0009] The second technical problem to be solved by this invention is to construct a host cell containing the genes for photorepair enzyme and 8-HDF synthase.
[0010] The third technical problem to be solved by the present invention is the method for expressing the effect of the photorepair enzyme on the repair of damaged DNA.
[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention includes:
[0012] A photorepair enzyme gene having the nucleotide sequence of SEQ ID NO:1; and an 8-HDF synthase gene having the nucleotide sequence of SEQ ID NO:3, capable of synthesizing 8-HDF.
[0013] A protein encoded by a photorepair enzyme gene, the encoded protein having the amino acid sequence of SEQ ID NO:2.
[0014] A protein encoding an 8-HDF synthase having the amino acid sequence of SEQ ID NO:4.
[0015] A vector, wherein the vector is a recombinant plasmid containing the aforementioned photorepair enzyme gene and 8-HDF synthase.
[0016] A host, wherein the host is a recombinant engineered bacterium expressing photorepair enzyme, the engineered bacterium being Bacillus subtilis WB600 and containing the recombinant plasmid expression vector and nucleotide sequence.
[0017] The conditions for expressing photorepair enzyme protein by this engineered strain were: TB medium (24 g / L yeast extract, 12 g / L peptone, 12.5 g / L K2HPO4, 2.3 g / L KH2PO4), culture temperature of 35℃, and shake flask culture for 48 h.
[0018] The construction method of this invention involves searching the NCBI database to obtain the photorepair enzyme gene sequence (accession number: NC_007604.1, location: 111686-113140) and the 8-HDF synthase gene sequence (accession number: NZ_BMPR01000011.1), optimizing the codons, and then constructing them into the vector pP43NMK to obtain the plasmid NMK-P. 43 -PhoA and NMK-P 43 -ScFbiC. Primers were designed to tandemly express the photorepair enzyme synthesis gene PhoA and the 8-HDF synthase gene ScFbiC in the same vector. The promoter of PhoA is P. 43 The promoter for ScFbiC is P. yvyd Promoter. The recombinant expression plasmid NMK-P was constructed. 43 -PhoA-P yvyd The recombinant plasmid ScFbiC was transformed into Bacillus subtilis WB600 competent cells to construct the genetically engineered bacterium BS-NMK-P for photorepair enzyme expression. 43 -PhoA-P yvyd -ScFbiC.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0020] By constructing a genetically engineered bacterium BS-NMK-P for photorepair enzyme expression 43 -PhoA-P yvyd -ScFbiC is used to express photorepair enzymes, resulting in a faster photoreduction rate compared to photorepair enzymes without 8-HDF. Bacillus subtilis is an internationally recognized food-grade (GRAS) microorganism, possessing advantages such as being endotoxin-free and having a clear genetic background. Compared to traditional E. coli systems, it better meets safety requirements in cosmetic raw material production. This patent uses Bacillus subtilis as a host to achieve highly efficient co-expression of photorepair enzymes and 8-HDF synthase. This fills the technological gap in photorepair expression in Bacillus subtilis and opens up a new pathway for the prevention and treatment of UV-related diseases, possessing significant social value and market potential. Attached Figure Description
[0021] Figure 1 Electrophoresis image of the tandem expression plasmids of photorepair enzyme PhoA and 8-HDF synthase;
[0022] Figure 2 Plasmid map of tandem expression of the photorepair enzyme PhoA and 8-HDF synthase genes;
[0023] Figure 3Electrophoretic image of the proteins tandemly expressed in Bacillus subtilis, including photorepair enzymes PhoA and 8-HDF synthase.
[0024] Figure 4 The curve showing the changes in CPD photoproducts after expression and purification of the photorepair enzyme PhoA in Bacillus subtilis;
[0025] Figure 5 The curves showing the changes in CPD photoproducts after tandem expression and purification of photorepair enzymes PhoA and 8-HDF synthase in Bacillus subtilis. Detailed Implementation
[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0027] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0028] Example 1: Co-expression plasmid NMK-P 43 -PhoA-P yvyd - ScFbiC Construction
[0029] (1) Obtain the photorepair enzyme gene PhoA
[0030] The photorepair enzyme gene sequence (accession number: NC_007604.1, location: 111686-113140) and the 8-HDF synthase gene sequence (accession number: NZ_BMPR01000011.1) were obtained from the NCBI database. After codon optimization, they were constructed into the vector pP43NMK, respectively. The gene synthesis and plasmid construction steps were completed by Nanjing GenScript Biotech Co., Ltd. The obtained plasmids were named NMK-P, respectively. 43 -PhoA and NMK-P 43 -ScFbiC.
[0031] (2) Construction of co-expression plasmid NMK-P 43 -PhoA-P yvyd -ScFbiC
[0032] Preserved in the laboratory containing P yvydUsing the promoter plasmid as a template, primers F1 and RI were used to obtain the P-type promoter by PCR. yvyd The gene fragment of the promoter, P yvyd The promoter sequence has the nucleotide sequence of SEQ ID NO:5; with NMK-P 43 Using ScFbiC as a template, the gene fragment containing ScFbiC was obtained by PCR using primers F2 and R2; NMK-P 43 -PhoA template, using primers F3 and R3, PCR was performed to obtain NMK-P... 43 - A vector fragment linearized with PhoA and containing complementary sequences to the ScFbiC gene fragment at its 5' and 3' ends. The desired gene fragments were obtained by gel extraction according to the instructions of the Tiangen Biotech DNA Product Gel Extraction Kit, and three-fragment assembly was performed according to the instructions of the Gibson Assembly Recombinant Cloning Kit. Figure 1 and Figure 2 ).
[0033] The primers used for PCR are shown in Table 1 below:
[0034] Table 1
[0035]
[0036] The composition of the PCR reaction system is shown in Table 2;
[0037] Table 2
[0038]
[0039] The amplification conditions were as follows: pre-denaturation at 94℃ for 3 min; followed by denaturation at 94℃ for 0.5 min, annealing at 56℃ for 0.5 min, extension at 72℃ for 2.5 min, for 28 cycles; and finally extension at 72℃ for 5 min, followed by holding at 4℃.
[0040] (3) Validation of recombinant plasmids
[0041] 10 μL of recombinant plasmid ( Figure 3 The recombinant bacteria were added to competent cells of *E. coli* DH5α, incubated on ice for 20 min, and then heat-shocked at 42°C for 90 s. Subsequently, 1 mL of de LB liquid medium was added, and the culture was incubated at 37°C and 200 rpm for 1 h. The recombinant bacteria were then plated onto LB agar plates containing 50 μg / mL ampicillin (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder) and incubated overnight at 37°C for 12 h. Plasmids were extracted and sequenced by Suzhou Genewiz Biotechnology Co., Ltd.
[0042] Example 2: Expression and purification of photorepair
[0043] (1) Plasmid transformation: 10 μL of correctly sequenced recombinant plasmid was added to competent cells of Bacillus subtilis WB600 and cultured for 2 h at 37 ℃ and 200 rpm. The recombinant bacteria were then plated on Kana plates and incubated overnight at 37 ℃ for 12 h.
[0044] (2) Seed culture: Pick a single colony into 4 mL of LB medium containing 50 μg / mL kanamycin and incubate overnight at 37℃ and 200 rpm for 12-14 h.
[0045] Shake flask culture: Inoculate 1% of the culture into a 500mL Erlenmeyer flask containing 100mL of TB medium (TB medium contains 50μg / mL kanamycin), and culture at 33℃ and 200rpm for 48h.
[0046] (3) Cell disruption
[0047] The collected bacterial culture was centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cell pellet was collected. 15 mL of disruption buffer (50 mmol / L phosphate buffer, 200 mmol / L NaCl, pH adjusted to 7.2) was added to the bacterial pellet, and after thorough resuspending, the centrifuge tubes were placed in an ice bath and disrupted using an ultrasonic cell disruptor. The ultrasonic disruption conditions were: 1 s working time, 2 s stopping time, total time 20 min, and ultrasonic power 400 W. The disrupted bacterial culture was centrifuged at 4℃ and 8000 rpm for 15 min, the crude enzyme solution was collected, and filtered through a 0.45 μm microporous membrane for later use.
[0048] (4) Photorepair enzyme purification
[0049] First, the nickel ion affinity chromatography column was pretreated by thoroughly rinsing it with deionized water. Then, the column was equilibrated with a buffer containing 500 mmol / L NaCl and 50 mmol / L phosphate (pH 7.0) to ensure a suitable chromatographic environment. Next, the pre-prepared crude enzyme solution was filtered and loaded into the column. To effectively remove contaminating proteins, the column was first washed with a low-concentration imidazole buffer containing 500 mmol / L NaCl, 50 mmol / L imidazole, and 50 mM PBS (pH 7.0) until baseline equilibration was achieved. Subsequently, elution was performed with a high-concentration imidazole buffer containing 500 mmol / L NaCl, 500 mmol / L imidazole, and 50 mM PBS (pH 7.0) to specifically separate the target protein. Throughout the purification process, the operation was performed at a low temperature of 4°C to maintain protein stability and activity. The eluent corresponding to the elution peak was collected and dialyzed in dialysis buffer (50 mM PBS, pH 7.0) to remove high concentrations of imidazole. Finally, the obtained target protein was further identified by SDS-PAGE, and the results are as follows: Figure 3 As shown, the photorepair enzyme is expressed in soluble form in Bacillus subtilis.
[0050] Example 3: Enzyme activity detection of photorepair enzymes
[0051] (1) Photodamage to DNA
[0052] At room temperature (25℃), a 254nm ultraviolet lamp (12W power) was used to irradiate oligonucleotides containing 16-meric thymidine (Oligo(dT)) at a vertical distance of 10cm. 16 A solution with an initial concentration of 10 μmol / L was continuously irradiated for 30-40 minutes to induce the formation of UV damage products (labeled as UV-dT) mainly composed of cyclobutane pyrimidine dimers (CPD). 16 CPD exhibits a characteristic absorption peak at 265 nm due to its cyclized pyrimidine base structure. Through photoreactivation mediated by photorepair enzymes, the cyclobutane ring of CPD is specifically cleaved under activation by visible light of a specific wavelength (e.g., 370-450 nm), resulting in a significant increase in absorbance at 265 nm (A0). 265 The value increases as the repair process progresses. Based on this, A can be dynamically monitored. 265 The rate of increase (i.e., the decrease in CPD concentration per unit time, Δ[CPD] / Δt) is used to quantitatively characterize the catalytic activity of photorepair enzymes.
[0053] (2) Reaction system
[0054] The reaction system includes 5 μmol / L UV-dT 160.1 μmol / L photorepair enzyme, 1 mmol / L dithiothreitol (DTT), Protein Buffer to a final volume of 1 mL.
[0055] (3) In vitro photorepair of CPD
[0056] The above enzyme activity system was added to a quartz cuvette and incubated in the dark for 10 min. The system was then irradiated with a 440 nm UV lamp at a distance of 5 cm, and the absorbance at a visible light wavelength of approximately 265 nm was measured over a certain period to detect the in vitro CPD photorepair enzyme activity. The photorepair enzyme produced by *Bacillus subtilis* expressing only PhoA activated A within 25 min. 265 The value increased from 0.453 to 0.562 within 25 minutes. 265 The rate of increase was 0.00436 / min. The photorepair enzyme produced by Bacillus subtilis expressing both PhoA and ScFbiC increased A levels within 25 min. 265 The value increased from 0.453 to 0.832 within 25 minutes. 265 The rate of increase was 0.01516436 / min, which is 3.5 times that of PhoA expression alone.
[0057] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
Claims
1. A method for constructing CPD photorepair enzyme expressed in Bacillus subtilis, characterized in that, The nucleotide sequence of the photorepair enzyme is shown in SEQ ID No:
1.
2. The CPD photorepair enzyme expressed in Bacillus subtilis according to claim 1, characterized in that, The protein encoded by this photorepair enzyme has the amino acid sequence of SEQ ID NO:
3.
3. A nucleotide sequence of an 8-HDF synthase, characterized in that, The protein encoded by this 8-HDF synthase has the amino acid sequence of SEQ ID NO:
2.
4. The 8-HDF synthase according to claim 3, characterized in that, The protein encoding 8-HDF coenzyme has the amino acid sequence of SEQ ID NO:
4.
5. A carrier, characterized in that, The vector is a recombinant plasmid containing the pP43NMK recombinant plasmid containing the photorepair enzyme gene and the 8-HDF synthase gene.
6. A host, characterized in that, The recombinant engineered host photorepair enzyme is Bacillus subtilis WB600.
7. The Bacillus subtilis host according to claim 6, characterized in that, The host contains the recombinant plasmid as described in claim 5.
8. The method for constructing a photorepair enzyme expressed in Bacillus subtilis according to claim 1, characterized in that, Includes the following steps: (1) The photorepair enzyme gene sequence (accession number: NC_007604.1, location: 111686-113140) and the 8-HDF synthase gene sequence (accession number: NZ_BMPR01000011.1) were obtained from the NCBI database. After codon optimization, they were constructed into the vector pP43NMK to obtain the plasmid NMK-P. 43 -PhoA and NMK-P 43 -ScFbiC, which encodes the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively; (2) Design primers to express the photorepair enzyme synthesis gene PhoA and the 8-HDF synthase gene ScFbiC in tandem on the same vector. The promoter of PhoA is P 43 The promoter for ScFbiC is P. yvyd Promoter. The recombinant expression plasmid NMK-P was constructed. 43 -PhoA-P yvyd The recombinant plasmid ScFbiC was transformed into Bacillus subtilis WB600 competent cells to construct the genetically engineered bacterium BS-NMK-P for photorepair enzyme expression. 43 -PhoA-P yvyd -ScFbiC.
9. The application of the repair agent and safety protectant prepared by expressing CPD photorepair enzyme in Bacillus subtilis according to any one of claims 1-8.