A method for constructing a halophilic genetically engineered bacterium with improved prodigiosin production and application thereof
By constructing halophilic genetically engineered bacteria, cloning and expressing the lycopene synthase gene of halophilic archaea, the problem of low phytorubin yield was solved, and a significant increase in phytorubin production was achieved, laying the foundation for its industrial application.
Patent Information
- Application Number
- CN202311365088.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-20
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2043-10-20
AI Technical Summary
The low yield of halophilic archaea and halophilic bacteria synthesizing erythrin has become a bottleneck restricting their industrial application.
Halophilic genetically engineered bacteria were constructed using genetic engineering technology. The lycopene synthase gene HVO_2524 encoded by the genome of the halophilic archaea H. volcanii DS2 was cloned into the expression vector pTA-1228, and the recombinant plasmid was transformed into halophilic archaea H. volcanii H1424 cells. The expression was induced by the inducer tryptophan to increase the production of lycopene.
It significantly increased the production of phytorubrin in halophilic genetically engineered bacteria, making its synthesis capacity 2.6 times that of the initial strain, and further increased it to 1.3 times after the addition of inducers, providing the possibility for large-scale production of phytorubrin.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, specifically a method for constructing and applying a halophilic genetically engineered bacterium that can increase the production of erythromycin. Background Technology
[0002] Lycopene is a C-type lycopene synthesized from lycopene. 50 Carotenoids, bright red in color, have a structure containing 13 pairs of conjugated double bonds and 4 -OH groups. Currently, phytoerythrins are mainly produced by halophilic archaea and some halophilic bacteria, such as... Rubrobacter radiotolerance It is produced. Physiological activity tests revealed that phytoerythrin differs from C derived from plants and algae. 40 Carotenoids possess antioxidant, anticancer, and antibacterial activities, as well as other important physiological functions. However, the low yield of halophilic archaea and halophilic bacteria synthesizing erythroxins has become a bottleneck limiting their industrial application.
[0003] Phytorubin possesses strong antioxidant activity. Compared to β-carotene, which contains nine covalent double bonds, phytorubin contains more covalent double bonds, suggesting that its antioxidant capacity is higher than that of β-carotene. Studies have found that halophilic archaea... Halogeometricumrufum , Halogeometricumlimits , Haladaptatus litoreus , Haloplanus vescus , Halopelagiusinordinate , Halogranum rubrum and Haloferaxvolcani The antioxidant capacity of the synthesized mycoerythrin is significantly higher than that of β-carotene, thus confirming that mycoerythrin is currently the carotenoid with the strongest antioxidant properties.
[0004] Bacterial erythrin possesses anticancer activity. Studies have found that it originates from halophilic bacteria. Halobacterium halobium The erythromycin in *Hydroxypyrrolizin* has anti-proliferative activity against human cancer HepG2 cells, while halophilic archaea... Native The erythromycin produced by sp. M6 exhibits both antiviral and anticancer activities in vitro.
[0005] Bacterioerythrin possesses antibacterial activity. Studies have found that halophilic archaea... Haloferax sp. ME16, Halogeometricum sp. ME3 and Haloarcula The phytoerythrin synthesized by sp. BT9 can inhibit the growth and reproduction of human and fish pathogens. Therefore, phytoerythrin has antibacterial function.
[0006] Phytorubicin possesses other physiological functions. In addition to the functions mentioned above, phytorubicin also exhibits other physiological activities. Studies have found that phytorubicin can protect cells from gamma-ray radiation, UV radiation, and oxidative stress, and participate in DNA repair. Furthermore, phytorubicin can enhance cell membrane function, protecting microorganisms from DNA-damaging agents. Recent research shows that adding phytorubicin to freeze-thawed sperm cells significantly improves sperm cell integrity and motility by integrating into the sperm cell membrane. In industry, phytorubicin is used as a skin-protecting ingredient in personal care products. In the food industry, phytorubicin can be used as a coloring agent; a few micrograms of phytorubicin can completely color 100 kg of product. These physiological functions make phytorubicin a potentially valuable commercial application.
[0007] In summary, phytorubin is a carotenoid with important physiological functions, possessing antioxidant, anticancer, and antibacterial activities, and has significant potential applications in the pharmaceutical, cosmetic, and food industries. Currently, the low synthesis rate of phytorubin is a bottleneck limiting its industrial application. Summary of the Invention
[0008] This invention addresses the technical problem at hand and overcomes the shortcomings of existing technologies by providing a method for constructing and applying a halophilic genetically engineered bacterium that increases the production of erythromycin.
[0009] The purpose of this invention is to construct a halophilic genetically engineered bacterium that can increase the production of phytohexene using genetic engineering technology, and to further enhance the production of phytohexene by adding an inducer.
[0010] This invention provides a protein with lycopene synthase activity, the amino acid sequence of which is shown in SEQ ID NO.1.
[0011] The present invention further provides a gene encoding the protein having the above-mentioned lycopene synthase activity, the nucleotide sequence of which is shown in SEQ ID NO.2.
[0012] The present invention also provides the application of the constructed halophilic genetically engineered bacteria containing the gene encoding the protein with the above-mentioned lycopene synthase activity in the synthesis of lycopene.
[0013] In the above applications, the halophilic genetically engineered bacteria are halophilic archaea. Haloferaxvolcani DS2 is the chassis cell.
[0014] This invention provides a method for constructing the above-mentioned halophilic genetically engineered bacteria, comprising the following steps:
[0015] 1) PCR amplification of the gene as described in claim 2;
[0016] 2) Enzyme digestion of the PCR product and expression vector prepared in step 1);
[0017] 3) Ligate the enzyme-digested PCR product and the expression vector;
[0018] 4) The ligation product was transformed into competent E. coli cells to obtain recombinant plasmids;
[0019] 5) Transform the recombinant plasmid into halophilic archaea competent cells, verify and select successfully transformed recombinant halophilic bacteria for induction and culture to synthesize phytohexin.
[0020] This invention utilizes genetic engineering technology to clone halophilic archaea. H.volcani The lycopene synthase gene HVO_2524, encoded by the DS2 genome, was expressed into the expression vector pTA-1228, and then the recombinant plasmid was transformed into [the target vector]. H. volcanoes A halophilic genetically engineered bacterium was constructed using H1424 cells. By extracting and quantifying phytorubrin from the constructed bacterium, it was found that its ability to synthesize phytorubrin was significantly enhanced. In particular, the ability of the halophilic genetically engineered bacterium to synthesize phytorubrin was further enhanced after the addition of an inducer. Therefore, the halophilic genetically engineered bacterium constructed in this invention can significantly increase the yield of phytorubrin, thus providing an important method for the large-scale production and application development of phytorubrin.
[0021] In step 1) above, halophilic archaea are used. H.volcani Using DS2 genomic DNA as a template, PCR amplification was performed using forward and reverse primers, and the PCR amplification results were detected by agarose gel electrophoresis. The sequence of the forward primer was 5'-CCGGAATTCATGCTCAACGAATCACAGGTC-3', and the sequence of the reverse primer was 5'-CGCGGATCCTCAGTCCGAGCCCCAGCGGCT-3'.
[0022] In step 2) above, the expression vector is pTA-1228. The PCR amplification product obtained in step 1) and the pTA-1228 vector are subjected to double enzyme digestion (…). Eco RI / Bam HI) reaction. In step 3), the enzyme-digested PCR product and pTA-1228 vector fragment are recovered by agarose gel electrophoresis and ligation reaction is performed.
[0023] In step 4) above, the ligation product is transformed into competent cells. E. coli In DH5α, spread on LB agar plates containing ampicillin, 37 o After overnight incubation in C, clones were picked, and plasmids were extracted for sequencing verification, yielding positive clones.
[0024] In step 5) above, the cloned plasmid with the correct gene sequence is transformed into... H. volcanii In H1424 competent cells, the HVO_2524 gene was induced to express as described in claim 2 by adding tryptophan; phytorubin was extracted using acetone reagent; and the content of phytorubin was tested by spectrophotometry.
[0025] This invention constructs a recombinant plasmid containing lycopene synthase, which is transformed into halophilic archaea cells. Compared with the initial strain, the genetically engineered bacterium produces 2.6 times more lycopene. Furthermore, after induction with tryptophan, the lycopene production is 1.3 times that of the uninduced strain. Therefore, the halophilic genetically engineered bacterium constructed in this invention can significantly increase lycopene production, has market potential, and can generate significant economic benefits.
[0026] This invention provides the application of the halophilic genetically engineered bacteria constructed by the above method in increasing the production of phytohexene.
[0027] This invention utilizes genetic engineering technology to construct a halophilic genetically engineered bacterium expressing lycopene synthase. Compared to the initial strain, the constructed halophilic genetically engineered bacterium can increase the production of lycopene, and the production of lycopene synthesized by the constructed halophilic genetically engineered bacterium is further increased upon induction with tryptophan. Therefore, the halophilic genetically engineered bacterium constructed in this invention can significantly increase the synthesis of lycopene, thus providing an important strain for promoting the industrial application of lycopene. This invention is based on C... 50 It has significant value in the industrial application of carotenoids. Attached Figure Description
[0028] Figure 1 This is a schematic diagram showing the results of PCR amplification of the HVO_2524 gene.
[0029] Figure 2 This is a schematic diagram of the results of phenol-chloroform verification of positive clones.
[0030] Figure 3 It is the transformation of recombinant plasmid to H.volcani A schematic diagram illustrating the validation results of H1424 cells on a medium containing ampicillin.
[0031] Figure 4 This is a schematic diagram comparing the production of halophilic genetically engineered bacteria and the initial strain of synthetic erythrin.
[0032] Figure 5 This is a schematic diagram comparing the synthesis of halophilic genetically engineered bacteria with and without tryptophan induction to synthesize erythrin. Detailed Implementation
[0033] The technical solution of the present invention will be further described in detail below with reference to the embodiments: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.
[0034] The sources of the reagents and materials involved in the examples are as follows:
[0035] The OMEGA PCR kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the pTA-1228 vector was provided by Dr. Liu Xipeng of Shanghai Jiao Tong University; and the OMEGA gel extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. E. coli DH5α competent cells were obtained from TransGen Biotech Ltd.; the OMEGA plasmid extraction kit was obtained from Tiangen Biotech (Beijing) Co., Ltd. H. volcanii The H1424 bacterial cells were provided by Dr. Liu Xipeng of Shanghai Jiao Tong University.
[0036] Except for the biological materials and reagents specifically mentioned above, the other materials and reagents mentioned in this invention are commercially available and can be purchased by the public from domestic and international commercial channels, and will not be described in detail here. Example 1
[0037] (1) Design primers
[0038] Download the sequenced halophilic archaea from GenBank. H.volcani The DS2 genome encodes the HVO_2524 gene, which is an enzyme that synthases lycopene. H.volcani The amino acid sequence of DS2 lycopene synthase is shown in SEQ ID NO.1 (GenBank: ADE02816.1). H.volcani The nucleotide sequence of the HVO_2524 gene for DS2 lycopene synthase is shown in SEQ ID NO.2 (HVO_RS16875). A pair of primers containing two different restriction endonuclease sites was designed. The nucleotide sequence of the forward primer is shown in SEQ ID NO.3, specifically: 5'-CCG GAA TTC The nucleotide sequence of the reverse primer is ATGCTC AAC GAA TCA CAG GTC-3', as shown in SEQ ID NO.4, specifically: 5'-CGC GGA TCC TCA GTC CGA GCC CCA GCG GCT -3', where the underlined bases are respectively Eco RI and Bam HI restriction site.
[0039] (2) PCR amplification of the enzyme gene
[0040] a) Using the primer pairs described above, to download from GenBank... H.volcani PCR amplification was performed using the DS2 genome as a template.
[0041] The PCR reaction volume is 50 μL.
[0042] 10 μM forward primer 2 μL
[0043] 10 μM reverse primer 2 μL
[0044] H.volcani DS2 genomic DNA (50 ng / μL) 1 μL
[0045] ddH2O 20 μL
[0046] 2 × Phanta Max Master Mix 25 μL
[0047] PCR reaction cycling parameters: 95 o C, 3 min; 95 o C, 30 s; 55 o C, 30 s; 72 o C, 1 min; 34 cycles, 72 o C extends for 5 minutes.
[0048] b) Detection of the PCR amplification of the enzyme gene by agarose gel electrophoresis: After the reaction, 5 μL of the PCR product was subjected to 1.0% agarose gel electrophoresis. The results are shown in the figure below. Figure 1 .
[0049] c) Purification of PCR products: The PCR products were recovered and purified using an OMEGA PCR kit; specific steps are detailed in the kit's instructions. The concentration of the recovered PCR products was determined using a Nanodrop 2000 micro spectrophotometer.
[0050] (3) Digest the enzyme gene and expression plasmid vector.
[0051] The PCR product and pTA-1228 vector were double-digested separately. Bam HI / Eco RI) reaction.
[0052] The enzyme digestion reaction system is 20 μL:
[0053] PCR product or pTA-1228 vector 16 μL
[0054] 10 x Enzyme digestion buffer (Mg 2+ plus) 2 μL
[0055] Eco RI1 μL
[0056] Bam HI 1 μL
[0057] 37 o Incubate in a water bath at C for 2 hours. After enzyme digestion, the digestion products were analyzed by 1.0% agarose gel electrophoresis. The OMEGA gel recovery kit was used for gel extraction and purification; please refer to the instruction manual for specific steps. The concentration was determined using a Nanodrop 2000 micro spectrophotometer.
[0058] (4) Ligase gene and vector: The enzyme-digested PCR product and pTA-1228 vector were recovered by agarose gel electrophoresis and ligated.
[0059] The connection reaction system is 10 μL:
[0060] 10 x Ligation Buffer 1 μL
[0061] 2 μL of enzyme-digested pTA-1228 vector
[0062] 6 μL of PCR product after enzyme digestion
[0063] T4 DNA Ligase 1 μL
[0064] twenty two o C reaction 2 hr.
[0065] (5) Transformation of recombinant plasmids: The ligation product is transformed into... E. coli DH5α competent cells were plated on LB agar plates containing ampicillin and cultured at 37°C. o Incubate overnight at C. Select clones and extract plasmids for sequencing verification. The LB agar plate containing ampicillin was prepared by adding 10 g sodium chloride, 10 g peptone, 5 g yeast extract, and 15 g agar powder to 1 L of pure water, and incubating at 121°C. o The product is obtained by high-pressure sterilization and cooling.
[0066] Pipette 5 μL of ligation product into 50 μL E. coli In DH5α competent cells, mix well and incubate on ice for 30 min. 42 o Incubate in a still water bath at 37°C for 90 seconds, then immediately return to ice and continue the ice bath for 2 minutes. Add 200 μL of LB liquid medium and incubate at 37°C. o Incubate on a shaker at 150 rpm for 1 hour. Spread 100 μL of the culture onto an LB agar plate containing ampicillin at a final concentration of 50 μg / mL. Incubate at 37°C. oCultured in C for 12 to 16 hours, yielding 100 to 200 clones.
[0067] LB liquid medium: Add 10 g sodium chloride, 10 g peptone, and 5 g yeast extract to 1 L of pure water, and then reflux at 121°C. o C. Sterilize under high pressure and allow to cool before use.
[0068] Subsequently, positive clones were validated: four clones were selected and inoculated into 5 mL LB medium tubes containing 50 μg / mL ampicillin, and incubated at 37°C. o The culture was carried out overnight on a C-type shaker at 150 rpm. Plasmids were extracted using the OMEGA plasmid extraction kit and sequenced. The sequencing results were compared with the sequences annotated by NCBI to verify positive clones, yielding recombinant plasmids. Positive clones were verified using phenol-chloroform; the results are shown below. Figure 2 .
[0069] (6) Transformation of recombinant plasmids into H.volcani H1424 cells. Transformation of cloned plasmids with correct gene sequences into H1424 cells. H.volcani H1424 competent cells.
[0070] H.volcani The preparation method of H1424 competent cells is as follows: Halophilic archaea... H.volcani H1424 cells were streaked in Hv-Ca solid medium, and then single colonies were aseptically picked and transferred to 10 mL of Hv-YPC and incubated at 45°C. o C. Incubate overnight with shaking at 180 rpm. Then, inoculate the bacterial culture at a 10% inoculum into a 150 mL Erlenmeyer flask and incubate at 45°C. o C. Incubate with shaking at 175 rpm until OD 600 The pH value will be approximately 0.8 to 1.0, at which point the bacterial cells will appear pink. Collect the cells by centrifugation at 6000 x g for 15 min at room temperature, and discard the supernatant under aseptic conditions. Resuspend the bacterial cells in 10 mL of buffered protoplast solution, centrifuge at 6000 x g for 10 min, and discard the supernatant under aseptic conditions. Resuspend the bacterial cells in a buffered protoplast solution containing 15% glycerol, mixing thoroughly, ensuring gentle and slow operation. Finally, aliquot under aseptic conditions and store at -80°C. o Pending for use in C.
[0071] Preparation of Hv-YPC: Add 600 mL of 30% SW, 5 g of yeast extract, 1 g of peptone, and 1 g of acid-hydrolyzed casein to 400 mL of pure water. For solid culture medium, add 15 g of agar powder. oC. It can be used after high-pressure sterilization and cooling; the preparation method of 30% SW is to add 1,200 g NaCl, 150 g MgCl2·6H2O, 175 g MgSO4·7H2O, 35 g KCl, and 100 mL 1 M Tris-HCl pH 7.5 to 4 L of pure water, and then make up to 5 L.
[0072] Conversion method: The prepared H.volcani H1424 competent cells from -80 o Remove from the freezer (C), thaw at room temperature, and then place on ice until needed. The entire process is performed in a sterile environment. Add 100 μL... H.volcani Add 10 μL of 0.5 M EDTA (pH 8.0) to H1424 competent cells, gently tap to mix, and incubate at room temperature for 10 min to allow protoplast formation. Add 4 μL of 2 ng / μL target DNA, gently tap to mix, and incubate at room temperature for 2-5 min to incubate competent cells and target DNA. Add 100 μL of 60% PEG600, gently pipette to mix, and incubate at room temperature for 20 min. Add 1 mL of growth medium for recovery, and incubate at 25°C. o Centrifuge at 6000xg for 8 min at 37°C, and remove the supernatant under aseptic conditions. Resuspend the bacterial cells in 1 mL of freshly prepared growth medium and incubate at 37°C. o Incubate on a shaker at 45°C for 2-4 hours. After incubation, centrifuge at 6000 xg for 2 minutes, then transfer 100 μL to Hv-Ca medium and plate it at 45°C. o Cultured under C conditions for about one week.
[0073] Hv-Ca medium: Add 600 mL of 30% SW and 5 g of acid-hydrolyzed casein to 400 mL of pure water. For solid medium, add 15 g of agar. o It can be used after high-pressure sterilization and cooling.
[0074] The growth medium (i.e., the resuscitation solution) is prepared in a volume of 250 mL: 150 mL 30% SW, 25 mL 10×YPC, 1.5 mL 0.5 M CaCl2, and 37.5 g sucrose. After filtration and sterilization, it can be stored at room temperature.
[0075] The above transformation H. volcanii strain H1424 and H. volcanii Strain H1424 was inoculated onto Hv-Ca solid medium containing ampicillin for 45 minutes. o C was cultured, and after one week of culture, the transformation was verified to be successful. The results are shown below. Figure 3 .
[0076] (7) Induction and expression of HVO_2524 gene. HVO_2524 gene expression was induced by using tryptophan as an inducer.
[0077] The recombinant plasmid pTA-1228-HVO_2524 was transformed into... H.volcani In strain H1424, a single colony of the expression strain was inoculated into a test tube containing 5 mL of Hv-Ca liquid medium with 50 μg / ml ampicillin, and incubated at 45°C. o Cultured at C for 48 hours, then transferred at a 5% inoculum rate to 50 mL of Hv-Ca liquid medium containing 50 μg / mL ampicillin, and incubated at 45°C. o C-oscillation cultivation to OD 600 After approximately 0.5%, transfer to 50 mL of Hv-YPC liquid medium containing 50 μg / mL ampicillin at a 5% inoculation rate, and incubate at 45°C. o C-oscillation cultivation to OD 600 Add 3 mmol / L tryptophan when the concentration is approximately 0.5, at 45°C. o C was cultured for a total of 48 hours to express the recombinant protein.
[0078] (8) Extraction and determination of phytoerythrin
[0079] Studies have shown that phytorubin synthesized by halophilic archaea has an absorption wavelength of 494 nm. Therefore, a spectrophotometric method was used to determine the content of phytorubin. The method is as follows: Take 3 mL of the bacterial culture induced above into a centrifuge tube, centrifuge at 12000 rpm for 2 min, and remove the supernatant. Resuspend the bacterial culture in 3 mL of methanol and acetone at a 1:1 volume ratio, shake for 10 min, centrifuge at 12000 rpm for 3 min after shaking, and transfer the supernatant to a cuvette to measure the OD. 494 Record the reading using the formula: mg·L⁻¹ -1 = (OD 494 / 2540) x 10 4 The measured OD 494 The value was converted to μg. The yield of halophilic archaea synthesizing halophilic rubrin was defined as the ratio (μg / mg) of the extracted halophilic rubrin yield to the dry cell weight (DCW). The results are shown in […]. Figure 4 and Figure 5 .
[0080] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. The application of a halophilic genetically engineered bacterium containing a gene encoding a protein with lycopene synthase activity in the synthesis of lycopene, characterized in that, The lycopene synthase gene HVO_2524 was cloned into the expression vector pTA-1228, and the recombinant plasmid was transformed into pTA-1228 using a transformation method. H. volcanii H1424 cells were used to construct a halophilic genetically engineered bacterium; the nucleotide sequence of the lycopene synthase gene HVO_2524 is shown in SEQ ID NO.
2.
2. A method for constructing a halophilic genetically engineered bacterium as described in claim 1, characterized in that, Includes the following steps: 1) PCR amplification of the lycopene synthase gene HVO_2524; 2) Enzyme digestion of the PCR product and expression vector prepared in step 1); 3) Ligate the enzyme-digested PCR product and the expression vector; 4) The ligation product was transformed into competent E. coli cells to obtain recombinant plasmids; 5) Transform the recombinant plasmid into halophilic archaea. H. volcanii In H1424 competent cells, the successfully transformed recombinant halophilic bacteria were verified and selected for induction and culture to synthesize phytohexin.
3. The construction method according to claim 2, characterized in that, In step 1), halophilic archaea are used. H. volcanii Using DS2 genomic DNA as a template, PCR amplification was performed using forward and reverse primers, and the PCR amplification results were detected by agarose gel electrophoresis. The sequence of the forward primer was 5'-CCG GAA TTC ATG CTC AAC GAA TCACAG GTC -3', and the sequence of the reverse primer was 5'-CGC GGA TCC TCA GTC CGA GCC CCA GCG GCT -3'.
4. The construction method according to claim 2, characterized in that, In step 4), the ligation product is converted into competent cells. E. coli In DH5α, spread on LB agar plates containing ampicillin, 37 o After overnight incubation in C, clones were picked, and plasmids were extracted for sequencing verification, yielding positive clones.
5. The construction method according to claim 2, characterized in that, In step 5), the cloned plasmid with the correct gene sequence is transformed into... H. volcanii In H1424 competent cells, the expression of the lycopene synthase gene HVO_2524 was induced by adding tryptophan; lycopene was extracted using acetone reagent; and the content of lycopene was determined by spectrophotometry.
6. The application of the halophilic genetically engineered bacteria constructed by the method described in claim 2 in increasing the production of phytohexene.