Construction method for simultaneously expressing 6-4 photolyase and 8-HDF synthetase in bacillus subtilis

Abstract

The invention provides a construction method for simultaneously expressing 6-4 photolyase and 8-HDF synthetase in bacillus subtilis. According to the invention, bacillus subtilis is used as an expression host, and codons of a photolyase PhoB gene and an 8-HDP synthetase ScFbiC gene are optimized. The optimized gene sequences respectively correspond to SEQ ID NO: 1 and SEQ ID NO: 2. According to the invention, two gene segments are connected with a vector pP43NMK, plasmids NMK-P43-PhoB and NMK-P43-ScFbiC are constructed, tandem expression of PhoB and ScFbiC is realized, and the plasmids NMK-P43-PhoB-Pyvyd-ScFbiC are further constructed. The plasmid is transformed into a bacillus subtilis WB600 cell, and the genetically engineered bacterium NMK-P43-PhoB-Pyvyd-ScFbiC is successfully constructed, so that the genetically engineered bacterium NMK-P43-PhoB-Pyvyd-ScFbiC is obtained. The constructed bacillus subtilis 6-4 photolyase expression system has the application potential in the field of cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to a method for constructing and applying a method for simultaneously expressing 6-4 photorepair enzyme and 8-HDF synthase in Bacillus subtilis. Background Technology

[0002] Ultraviolet (UV) radiation induces photochemical reactions between adjacent pyrimidine bases (such as thymine) in the DNA chain, primarily forming two types of damage products: cyclobutanepyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). 6-4PP is formed by the cross-linking of the C5-C6 double bond of a pyrimidine base with the C4 position of an adjacent pyrimidine base via a ketone group, leading to severe local distortion of the DNA double helix structure. Compared to CPD, 6-4PP has a stronger steric hindrance effect, significantly increasing the difficulty of repair and making it more prone to base mismatches and genomic instability, which is closely related to the risk of skin cancer. 6-4 photolyases are photoactivators that specifically recognize 6-4PP. They are activated by absorbing visible light at wavelengths of 300-500 nm, catalyzing the breakage of pyrimidine-pyrimidinone cross-links and restoring the original DNA base sequence. However, natural 6-4 photolyases are limited by coenzyme binding efficiency and light energy utilization range, facing problems such as low repair efficiency and poor environmental adaptability in practical applications.

[0003] The function of 6-4 photorepair enzyme depends on its coenzyme system: flavin adenine dinucleotide (FAD) acts as the core coenzyme, directly participating in electron transfer reactions to cleave 6-4PP via the photoexcited state (FADH-); the second coenzyme, 8-hydroxy-5-dezazoflavin (8-HDF), extends the light absorption spectrum to the near-ultraviolet region (e.g., 350-400 nm) and transmits the captured light energy through... The resonant energy transfer (FRET) mechanism delivers energy to the photodiode (FAD), thereby enhancing the enzyme's light energy utilization efficiency. Experiments show that the binding of 8-HDF can increase the activity of 6-4 photorepair enzymes several times. However, the synthesis and assembly efficiency of 8-HDF in natural enzyme systems is low, and the addition of exogenous coenzymes presents problems such as high cost and poor stability (e.g., photolysis or oxidative degradation), which severely restricts its large-scale application.

[0004] Bacillus subtilis, a food-grade (GRAS) microorganism certified by the U.S. Food and Drug Administration (FDA), possesses characteristics such as being endotoxin-free, exhibiting highly efficient intracellular protein folding, and having a mature genetic manipulation system. This patent utilizes Bacillus subtilis as a host to achieve efficient co-expression of 6-4 photorepair enzyme and 8-HDF synthase. This engineered strain can be directly used to produce cosmetic raw materials containing active 6-4 photorepair enzyme, 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 a system that simultaneously expresses 6-4 photorepair enzyme and 8-HDF synthase 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 a 6-4 photorepair enzyme and an 8-HDF synthase that simultaneously express in Bacillus subtilis, wherein the amino acid sequence of the 6-4 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 second 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 third 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 fourth technical problem to be solved by the present invention is the method for expressing the effect of the 6-4 photorepair enzyme on DNA repair.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention includes:

[0012] A 6-4 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 6-4 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 6-4 photorepair enzyme gene and 8-HDF synthase.

[0016] A host, wherein the host is a recombinant engineered bacterium expressing 6-4 photorepair enzyme, the engineered bacterium being Bacillus subtilis WB600 and containing the recombinant plasmid expression vector and nucleotide sequence.

[0017] The conditions for expressing the 6-4 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 obtaining the photorepair enzyme gene sequence (accession number: NC_007604.1, location: 1204598-1206109) and the 8-HDF synthase gene sequence (accession number: NZ_BMPR01000011.1) from the NCBI database, optimizing their codons, and constructing these two genes into the vector pP43NMK to obtain plasmids NMK-P43-PhoB and NMK-P43-ScFbiC. Their corresponding amino acid sequences are SEQ ID NO:2 and SEQ ID NO:4, respectively.

[0019] Based on this, specific primers were designed to tandemly express the photorepair enzyme gene PhoB and the 8-HDF synthase gene ScFbiC. PhoB is expressed by P… 43 The startup sub-driver, ScFbiC, is driven by P. yvyd Promoter-driven. The recombinant plasmid obtained was NMK-P43-PhoB-Pyvyd-ScFbiC. Subsequently, this recombinant plasmid was transformed into Bacillus subtilis WB600 competent cells, successfully constructing the genetically engineered strain BS-NMK-P43-PhoB-Pyvyd-ScFbiC for photorepair enzyme expression.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] By constructing a genetically engineered bacterium BS-NMK-P for expressing 6-4 photorepair enzyme. 43 -PhoB-P yvyd-ScFbiC, experimental results show that compared with 6-4 photorepair enzyme without 8-HDF, this system can achieve a faster photoreduction reaction. Bacillus subtilis, as an internationally recognized food-grade (GRAS) microorganism, has advantages such as being endotoxin-free and having a well-defined genetic background, making it safer for use in cosmetic raw material production compared to traditional Escherichia coli expression systems. This study selected Bacillus subtilis as the host to achieve efficient co-expression of 6-4 photorepair enzyme and 8-HDF synthase. This technology not only fills the gap in the expression of 6-4 photorepair enzyme in Bacillus subtilis but also provides a new research direction for the prevention and treatment of ultraviolet-related diseases, possessing significant social value and broad market prospects. Attached Figure Description

[0022] Figure 1 Plasmid map of tandem expression of genes 6-4 photorepair enzyme and 8-HDF synthase;

[0023] Figure 2 Electrophoretic image of proteins tandemly expressed in Bacillus subtilis: 6-4 photorepair enzyme and 8-HDF synthase.

[0024] Figure 3 The curve showing the change in 6-4 photorepair enzyme after expression and purification in Bacillus subtilis and repair of 6-4 photoproducts;

[0025] Figure 4 The curves showing the changes in 6-4 photorepair enzyme and 8-HDF synthase after tandem expression and purification in Bacillus subtilis to repair 6-4 photoproducts. 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 -PhoB-P yvyd - ScFbiC Construction

[0029] (1) Obtain the photorepair enzyme gene PhoB

[0030] The 6-4 photorepair enzyme gene sequence (accession number: NC_007604.1, location: 1204598-1206109) 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. Gene synthesis and plasmid construction were performed by Nanjing GenScript Biotech Co., Ltd. The obtained plasmids were named NMK-P, respectively. 43 -PhoB and NMK-P 43 -ScFbiC.

[0031] (2) Construction of co-expression plasmid NMK-P 43 -PhoB-P yvyd -ScFbiC

[0032] Using laboratory-preserved P yvyd Using a plasmid containing the promoter (whose sequence corresponds to SEQ ID NO:5) as a template, polymerase chain reaction (PCR) technology was successfully employed, and specific primers F1 and R1 were used to amplify a gene containing the promoter.

[0033] P yvyd Gene fragments of the promoter. Subsequently, NMK-P 43 Using -ScFbiC as a template, and binding primers F2 and R2, through...

[0034] The gene sequence containing ScFbiC was obtained by PCR. Furthermore, NMK-P... 43 Using PhoA as a template and primers F3 and R3, linearized NMK-P was obtained by PCR. 43 The PhoA vector fragment has complementary sequences at its 5' and 3' ends to the ScFbiC gene fragment, respectively, to facilitate subsequent cloning operations. All obtained gene fragments were purified and recovered according to the operating instructions of Tiangen Biotech's DNA Product Purification Gel Recovery Kit.

[0035] Then, following the detailed steps of the Gibson assembly recombinant cloning kit, the assembly of the three fragments was completed (Figure 1).

[0036] 1).

[0037] The primers used for PCR are shown in Table 1 below:

[0038] Table 1

[0039]

[0040] The composition of the PCR reaction system is shown in Table 2;

[0041] Table 2

[0042]

[0043] 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℃.

[0044] (3) Validation of recombinant plasmids

[0045] 10 μL of recombinant plasmid ( Figure 3 The recombinant bacteria were transformed into competent *E. coli* DH5α cells, treated on ice for 20 min, and then heat-shocked at 42℃ for 90 s. 1 mL of LB broth was then added, and the cells were incubated at 37℃ and 200 rpm for 1 h. The transformed recombinant bacteria were inoculated onto LB agar plates containing 50 μg / mL ampicillin (broth composition: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder), and then inverted and incubated at 37℃ for 12 h. The plasmid was extracted and sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing analysis.

[0046] Example 2: Expression and purification of photorepair

[0047] (1) Plasmid transformation

[0048] 10 μL of the recombinant plasmid, confirmed by sequencing, was added to competent Bacillus subtilis WB600 cells and cultured at 37°C and 200 rpm for 2 h. The transformed recombinant bacteria were then plated on Kanamycin-containing plates and incubated upside down in a 37°C incubator overnight for 12 h.

[0049] (2) Recombinant bacterial culture

[0050] Single colonies were picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 200 rpm for 12–14 h. Subsequently, the seed culture was inoculated at a rate of 1% into 500 mL Erlenmeyer flasks containing 100 mL of TB medium containing 50 μg / mL kanamycin, and cultured on a shaker at 33°C and 200 rpm for 48 h.

[0051] (3) Cell disruption

[0052] 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 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.

[0053] (4) Purification of photorepair enzyme

[0054] 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 filtered crude enzyme solution was loaded onto the column. To remove contaminating proteins, elution was performed using a low-concentration imidazole buffer containing 500 mmol / L NaCl, 50 mmol / L imidazole, and 50 mmol / L PBS (pH 7.0) until baseline equilibration was achieved. Subsequently, elution was performed using a high-concentration imidazole buffer containing 500 mmol / L NaCl, 500 mmol / L imidazole, and 50 mmol / L PBS (pH 7.0) to specifically separate the target protein. The entire purification process was performed at a low temperature of 4°C to maintain protein stability and activity. The eluent was collected and dialyzed against 50 mmol / L PBS (pH 7.0) buffer to remove the high concentration of imidazole. Finally, the purified target protein was identified using SDS-PAGE, and the results are shown in the figure. Figure 2 It can be seen that when 6-4 photorepair enzyme is expressed alone, there is a target band. When 8-HDF synthase and 6-4 photorepair enzyme are expressed in tandem, the purified enzyme contains a small amount of 8-HDF synthase in addition to 6-4 photorepair enzyme.

[0055] Example 3: Enzyme activity detection of photorepair enzymes

[0056] (1) Photodamage to DNA

[0057] At room temperature (25℃), a solution containing 16-polythymidine oligonucleotide (Oligo(dT)16, initial concentration 10 μmol / L) was irradiated with a 254 nm UV lamp (12 W). The vertical distance between the UV lamp and the sample was 10 cm, and the irradiation was continuous for 30-40 minutes, inducing the formation of UV damage products (labeled UV-dT16) mainly composed of cyclobutane pyrimidine dimers (CPD). Among them, the 6-4 photorepair product (6-4PP) has a characteristic absorption peak at 325 nm due to its cyclized pyrimidine base structure. Through the photoreactivation reaction mediated by 6-4 photorepair enzyme, the pyrimidine-pyrimidinone cross-linking bond is specifically broken under activation by visible light of a specific wavelength (e.g., 370-450 nm), resulting in a linear decrease in the absorbance (A325) of the solution at 325 nm during the repair process. Based on this, the rate of decrease of A325 (i.e., 6- ) per unit time can be dynamically monitored.

[0058] The catalytic activity of 6-4 photorepair enzyme was quantitatively characterized by the change in 6-4PP concentration, Δ[6-4PP] / Δt).

[0059] (2) 6-4 photorepair reaction system

[0060] The reaction system consisted of 5 μmol / L UV-dT16, 0.1 μmol / L photorepair enzyme, and 1 mmol / L dithiothreitol (DTT), and was brought to a final volume of 1 mL with protein buffer.

[0061] (3) In vitro photorepair of 6-4 photorepair products

[0062] The above enzyme activity system was added to a quartz cuvette and incubated in the dark for 10 min. Then, the repair system was irradiated with a 440 nm UV lamp at a distance of 5 cm, and the absorbance of visible light near 325 nm was measured at regular time intervals to detect the activity of the 6-4 photorepair enzyme in vitro. The 6-4 photorepair enzyme produced by Bacillus subtilis expressing only PhoB reduced A within 40 min. 325 The value decreased from 0.0459 to 0.0423 within 40 minutes. 325 The rate of decrease was 0.00009 / min. The photorepair enzyme produced by Bacillus subtilis expressing both PhoA and ScFbiC reduced A levels within 40 min. 265 The value decreased from 0.0459 to 0.0313 within 40 minutes. 325 The rate of increase was 0.00365 / min, which is 4.1 times that of PhoB expression alone.

[0063] 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 a system that simultaneously expresses 6-4 photorepair enzyme and 8-HDF synthase in Bacillus subtilis, characterized in that, The nucleotide sequence of the 6-4 photorepair enzyme is shown in SEQ ID No:

1.

2. The 6-4 photorepair enzyme expressed in Bacillus subtilis according to claim 1, characterized in that, The protein encoded by this 6-4 photorepair enzyme has the amino acid sequence of SEQ ID NO:

3.

3. The 8-HDF synthase expressed in Bacillus subtilis according to claim 1, 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 encoded protein 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 of the 6-4 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 Bacillus subtilis to simultaneously express 6-4 photorepair enzyme and 8-HDF synthase according to claim 1, characterized in that, Includes the following steps: (1) The photorepair enzyme gene sequence (accession number: NC_007604.1, location: 1204598-1206109) and the 8-HDF synthase gene sequence (accession number: NZ_BMPR01000011.1) were obtained from the NCBI database and their codons were optimized. These two genes were then constructed into the vector pP43NMK to obtain plasmids NMK-P43-PhoB and NMK-P43-ScFbiC. Their corresponding amino acid sequences are SEQ ID NO:2 and SEQ ID NO:4, respectively. (2) Based on this, specific primers were designed to express the photorepair enzyme gene PhoB and the 8-HDF synthase gene ScFbiC in tandem. PhoB is expressed by P… 43 The startup sub-driver, ScFbiC, is driven by P. yvyd Promoter-driven. The recombinant plasmid obtained was NMK-P43-PhoB-Pyvyd-ScFbiC. Subsequently, this recombinant plasmid was transformed into Bacillus subtilis WB600 competent cells, successfully constructing the genetically engineered strain BS-NMK-P43-PhoB-Pyvyd-ScFbiC for photorepair enzyme expression.

9. The application of the repair agent and safety protectant prepared by simultaneously expressing 6-4 photorepair enzyme and 8-HDF synthase in Bacillus subtilis according to any one of claims 1-8.

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