An improved engineered Saccharomyces cerevisiae strain, its construction method, and its application in PDRN preparation.

Abstract

This invention relates to an improved engineered Saccharomyces cerevisiae strain, its construction method, and its application in the preparation of PDRN, belonging to the field of microbial technology. The improved engineered Saccharomyces cerevisiae strain uses diploid Saccharomyces cerevisiae INVSC1 as the starting strain, knocking out the RNH1 and RNH201 genes, while simultaneously overexpressing the ORC1 and Cdc6 genes; wherein the nucleotide sequences of the RNH1, RNH201, ORC1, and Cdc6 genes are shown in SEQ ID NO. 1-4. This invention uses diploid Saccharomyces cerevisiae as the starting strain and constructs an improved engineered Saccharomyces cerevisiae strain through a targeted gene editing strategy of overexpressing the ORC1 and Cdc6 genes and knocking out the RNH1 and H2 genes. This strain retains the advantages of diploid yeast, such as genetic stability, high biomass, and short fermentation cycle, while also achieving increased intracellular DNA content, providing sufficient raw materials for high PDRN production, and maintaining a cell viability rate of over 80%, ensuring the stability of the fermentation process.

Description

Technical Field

[0001] This invention relates to an improved engineered strain of Saccharomyces cerevisiae, its construction method, and its application in the preparation of PDRN, belonging to the field of microbial technology. Background Technology

[0002] Polydeoxyribonucleic acid (PDRN) is a class of biological macromolecules derived from salmon testicular cells, composed of multiple base pairs linked by phosphodiester bonds. Essentially, it is a mixture of salmon genetic material fragments of varying sizes. As early as 1952, the Italian company Mastelli extracted PDRN fragments of different sizes from salmon testicular cells based on the observation that fishermen used salmon testes to treat wounds, and studied their pharmacological activities and related properties. Experimental results showed that nucleic acid fragments (PDRN) from male salmon testicular cells could significantly promote human cell regeneration, rapidly accelerate wound healing, and reduce scar formation. Subsequent research has further revealed that PDRN has anti-inflammatory, tissue repair, angiogenesis-promoting, anti-ischemic, and diabetic foot-improving effects, leading to its widespread application in medical aesthetics and pharmaceutical fields.

[0003] Currently, PDRN is mainly produced from two sources: extraction from salmon testes and preparation through microbial fermentation. However, with the continuous depletion of marine fishery resources, extracting PDRN from salmon testicular cells is no longer sufficient to meet the ever-increasing demand for PDRN raw materials in the new era. Furthermore, fuel spills, nuclear wastewater discharges, and coastal waste dumping caused by large ships constantly passing through ocean-going routes have all had a detrimental impact on the marine environment. This has led to problems such as heavy metal accumulation, chromosomal aberrations, and gene mutations in marine organisms, which have also significantly affected the safety of salmon sperm PDRN raw materials.

[0004] However, the technology for preparing PDRN using yeast as a raw material also has many shortcomings: On the one hand, commonly used haploid yeasts suffer from poor genetic stability, high mutation sensitivity, low biomass yield, and long fermentation cycles, which limits the production efficiency of PDRN. On the other hand, although diploid yeasts have certain advantages in genetic stability, the intracellular DNA content of natural diploid yeasts is limited, and their cell walls are thick and tightly cross-linked with dextran and mannan, making cell wall disruption difficult and increasing the extraction cost of PDRN. In addition, existing preparation processes mostly focus on the single purification of PDRN, neglecting the synergistic effect of natural active ingredients in yeast cells, resulting in the final product's functional activity not being fully realized.

[0005] Therefore, developing a highly active yeast PDRN preparation method based on diploid yeast, increasing intracellular DNA content, optimizing cell disruption and purification processes, and achieving synergistic effects between PDRN and yeast's natural active ingredients has significant practical implications and application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an improved engineered strain of Saccharomyces cerevisiae, its construction method, and its application in the preparation of PDRN.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides an improved Saccharomyces cerevisiae engineered strain, which is based on diploid Saccharomyces cerevisiae as the starting strain, knocking out the RNH1 gene and the RNH201 gene, while overexpressing the ORC1 gene and the Cdc6 gene. The nucleotide sequence of the RNH1 gene is shown in SEQ ID NO.1, the nucleotide sequence of the RNH201 gene is shown in SEQ ID NO.2, the nucleotide sequence of the ORC1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the Cdc6 gene is shown in SEQ ID NO.4.

[0008] According to a preferred embodiment of the present invention, the RNH1 and RNH201 genes encode endogenous nucleases, and their knockout can inhibit DNA degradation.

[0009] According to a preferred embodiment of the present invention, the ORC1 gene and Cdc6 gene are derived from Saccharomyces cerevisiae S288C, and respectively encode the replication origin recognition complex subunit 1 and cell cycle protein 6. ORC1 is a key subunit constituting the ORC complex, which binds to the DNA replication origin, initiates the assembly of pre-RC (pre-replication complex), and promotes the initiation of DNA replication. Cell cycle protein 6 (non-enzymatic) binds to the ORC complex and binds to the Mcm2-7 complex, enhancing the stability of pre-RC and regulating the timing of DNA replication initiation.

[0010] According to a preferred embodiment of the present invention, the diploid brewer's yeast is brewer's yeast INVSC1.

[0011] Secondly, the present invention provides a method for constructing the above-mentioned improved engineered brewer's yeast, comprising the following steps: Diploid Saccharomyces cerevisiae INVSC1 was selected as the starting strain. Overexpression vectors of ORC1 and Cdc6 genes were transferred into the strain INVSC1. At the same time, homologous recombination-mediated gene editing technology was used to knock out the RNH1 and RNH201 genes, thus constructing an improved Saccharomyces cerevisiae engineered strain.

[0012] Thirdly, the present invention provides the application of the above-mentioned improved brewer's yeast engineered strain in the production of PDRN.

[0013] Fourthly, the present invention provides a method for preparing PDRN, comprising the following steps: S1: Inoculate the above-mentioned improved brewing yeast engineered strain into the fermentation medium and culture it for 16-24 hours at 28-32℃, pH=5.0-6.0, aeration rate of 1.0-2.0 vvm, and oxygen content of 20-40%. S2: Collect the yeast cell pellet, wash it, and add PBS buffer or sterile water to obtain a cell suspension; add lyase mixture to the cell suspension, mix well, and lyse thoroughly to obtain a preliminary lysate; then use a high-pressure homogenizer to perform a second lysate on the preliminary lysate to obtain a cell lysate. S3: Centrifuge the cell lysis buffer to remove the precipitate, add ethanol to the supernatant, let stand, centrifuge a second time to collect the precipitate, wash the precipitate and dissolve it in TE buffer to obtain the crude nucleic acid extract; S4: First, the crude nucleic acid extract is ultrafiltered and desalted through an ultrafiltration membrane and dialysis bag. Then, the desalted retentate is freeze-dried to obtain highly active PDRN from diploid yeast.

[0014] According to a preferred embodiment of the present invention, in step S1, the fermentation medium is formulated as follows: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.5 g / L, pH=5.5; the fermentation conditions are: fermentation culture at 30℃, pH=5.5, and aeration rate of 1.5 vvm for 20 h, and the dissolved oxygen content is controlled at 30% during the fermentation process.

[0015] According to a preferred embodiment of the present invention, in step S2, the lysin mixture contains glucanase and mannanase in a mass ratio of 2:1; the total concentration of the lysin is 1.5~2.5% (w / v); the lysis conditions are: enzymatic hydrolysis at 35~40℃ and 150~200 r / min for 4~6 h; the secondary lysis conditions are: homogenization 2~3 times under a pressure of 80~100 MPa.

[0016] According to a preferred embodiment of the present invention, in step S3, the conditions for the second centrifugation are: centrifugation at 4°C and 10,000~12,000 r / min for 20~30 min; and the ethanol concentration is 60~70%.

[0017] According to a preferred embodiment of the present invention, in step S4, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa; the ultrafiltration conditions are: pressure 0.1~0.3 MPa, temperature 4~10℃; the dialysis bag has a molecular weight cutoff of 10 kDa, the dialysis time is 8~12 h, and the dialysis fluid is replaced 2~3 times during dialysis; the freeze-drying conditions are: freeze-drying at 0.01~0.05 MPa and -50℃~-40℃ for 24~36 h.

[0018] Fifthly, the present invention provides a PDRN prepared according to the above method.

[0019] Sixthly, the present invention provides the application of the above-mentioned PDRN in the preparation of anti-aging, sun protection, whitening, anti-inflammatory, cell regeneration promoting, tissue repair, wound healing promoting products and DNA origami products.

[0020] Beneficial effects: 1. This invention uses diploid Saccharomyces cerevisiae as the starting strain and employs a targeted gene editing strategy of overexpressing the ORC1 and Cdc6 genes and knocking out the RNH1 and RNH201 genes to construct an improved engineered Saccharomyces cerevisiae strain. This strain retains the advantages of diploid yeast, such as genetic stability, high biomass, and short fermentation cycle, while also increasing intracellular DNA content, providing sufficient raw materials for high PDRN production. Furthermore, the cell viability is maintained above 80%, ensuring the stability of the fermentation process.

[0021] 2. To address the technical challenges of thick and tightly cross-linked cell walls in diploid yeast, this invention employs a combined cell disruption process of "enzymatic hydrolysis + high-pressure homogenization." By optimizing the type, concentration, and hydrolysis conditions of the lysin, combined with secondary cell disruption via high-pressure homogenization, the efficiency of cell disruption is effectively improved, the cost of cell disruption is reduced, and the industry pain point of the difficulty in disrupting diploid yeast cell walls is solved.

[0022] 3. This invention innovatively uses an ultrafiltration membrane with a molecular weight cutoff of 10kDa for purification, breaking through the traditional approach of purifying PDRN alone. It simultaneously retains small molecule active peptides and β-glucan in yeast cells, and utilizes the synergistic effect of the three to significantly improve the functional activity of the product. The bioactivity is increased by more than 40% compared with single PDRN products, thus broadening the application scenarios of the product.

[0023] 4. The improved brewing yeast strain provided by this invention can produce 1.5 to 2 times the yield of haploid yeast per unit time. Moreover, the entire preparation process is simple, the conditions are mild, it is easy to scale up, the production efficiency is high, the cost is low, and it has good prospects for industrial application. Attached Figure Description

[0024] Figure 1 The graph shows the purity determination of the PDRN prepared in Example 2.

[0025] Figure 2 The image shows the agarose gel electrophoresis results of the PDRN prepared in Example 2.

[0026] Figure 3 The image shows the ELISA results of how PDRN prepared in Example 2 affects the secretion of IL-10 by macrophages.

[0027] Figure 4The image shows the qRT-PCR results of the effect of PDRN prepared in Example 2 on Arg-1 gene expression in macrophages.

[0028] Figure 5 The image shows the qRT-PCR results of the effect of PDRN prepared in Example 2 on the expression of VEGF gene in macrophages. Detailed Implementation

[0029] The invention will be further illustrated below with examples, but the scope of protection of the invention is not limited thereto.

[0030] Unless otherwise specified, the experimental procedures involved in the examples are standard practices in the art. Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0031] In this embodiment, the diploid Saccharomyces cerevisiae INVSC1 is available from Shanghai Preservation Microbial Co., Ltd., with product number SMHCC D25039.

[0032] The brewing yeast Saccharomyces cerevisiae S288C is available from Shanghai Preservation Microbial Co., Ltd., with product number SMHCC D10186.

[0033] The fermentation medium used in the examples was formulated as follows: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 1 g / L, MgSO4・7H2O 0.5 g / L, pH=5.5.

[0034] The formula for YPD liquid culture medium is: yeast extract 10g / L, peptone 20g / L, glucose 20g / L.

[0035] Example 1: Construction of an improved engineered brewer's yeast A method for constructing an improved engineered Saccharomyces cerevisiae involves selecting diploid Saccharomyces cerevisiae INVSC1 as the starting strain, transferring the overexpression vectors of the ORC1 and Cdc6 genes into the strain INVSC1, and simultaneously using CRISPR-Cas9 gene editing technology to knock out the RNH1 and RNH201 genes to construct the improved engineered Saccharomyces cerevisiae. The specific steps include the following.

[0036] 1. Gene Sequence Acquisition Genomic DNA was extracted from diploid Saccharomyces cerevisiae INVSc1. Specific primers ORC1-F / R and Cdc6-F / R were designed based on the homologous gene sequence of Saccharomyces cerevisiae S288C. The genomic DNA was then used as a template for PCR amplification to obtain the ORC1 and Cdc6 gene sequences, as shown in SEQ ID NO.3~4, respectively.

[0037] The specific sequences of primers ORC1-F / R and Cdc6-F / R are as follows: ORC1-F: 5'-CGGGATCCATGTCGAAGAAGTTTGTGGTG-3' (containing a BamHI restriction site, as shown in SEQ ID NO. 5) ORC1-R: 5'-CGGGTACCTTAGTTGGTGATGGTGATGGTG-3' (containing a KpnI restriction site, as shown in SEQ ID NO. 6) Cdc6-F: 5'-CGGGATCCATGTCGTTTGTTGAAGAAGCC-3' (containing a BamHI restriction site, as shown in SEQ ID NO. 7) Cdc6-R: 5'-CGGGTACCTTACTCGTCGGTGATGGTGAT-3' (containing a KpnI restriction site, as shown in SEQ ID NO. 8) PCR amplification program: pre-denaturation, 95℃ for 5 min; denaturation, 95℃ for 30 sec; annealing, 60℃ for 30 sec; extension, 72℃ for 55 sec (30 cycles); termination extension, 72℃ for 10 min; final incubation at 4℃.

[0038] PCR amplification system: Total volume 20 μL, upstream primer 1 μL, downstream primer 1 μL, template 1 μL, 2×phanta enzyme 10 μL, ddH2O 7 μL.

[0039] Finally, sequencing was used to verify the correctness of the ORC1 and Cdc6 gene sequences.

[0040] 2. Construction of overexpression vectors The expression vector pYES2 / CT, adapted to Saccharomyces cerevisiae S288C, was selected. BamHI and KpnI restriction sites were introduced at both ends of the coding regions of the ORC1 and Cdc6 genes, respectively. The ORC1 and Cdc6 gene sequences and the pYES2 / CT vector were then digested with BamHI / KpnI and ligated using T4 DNA ligase to construct the recombinant overexpression vectors pYES2-INV-ORC1 and pYES2-INV-Cdc6.

[0041] The recombinant overexpression vectors pYES2-INV-ORC1 and pYES2-INV-Cdc6 were then transformed into E. coli DH5α competent cells. Positive clones were screened on LB solid medium containing ampicillin. The gene insertion direction and sequence integrity were confirmed by enzyme digestion and sequencing to obtain the recombinant plasmids pYES2-INV-ORC1 and pYES2-INV-Cdc6.

[0042] 3. Target fragment acquisition Using the RNH1 and RNH201 genes from diploid Saccharomyces cerevisiae INVSC1 (as shown in SEQ ID NO. 1~2) as targets, knockout fragments containing homologous arms and selection markers were designed, as follows: (1) Upstream homologous arm: 500~600bp in length, homologous to the upstream non-coding regions of the RNH1 and RNH201 genes; the upstream homologous arm of the RNH1 gene is shown in SEQ ID NO.9, and the upstream homologous arm of the RNH201 gene is shown in SEQ ID NO.10. (2) Selection marker: The KanMX gene (G418 resistance) was used to meet the dual selection requirements of diploid strains; (3) Downstream homologous arm: 500-600bp in length, homologous to the downstream non-coding regions of RNH1 and RNH201 genes; the downstream homologous arm of RNH1 gene is shown in SEQ ID NO.11, and the downstream homologous arm of RNH201 gene is shown in SEQ ID NO.12. (4) The above three sequences (RNH1 gene and its upstream and downstream homologous arms, RNH201 gene and its upstream and downstream homologous arms) were amplified by overlap extension PCR to obtain complete RNH1 knockout cassette (INV-RNH1-KO) and RNH201 knockout cassette (INV-RNH201-KO), and the accuracy of the homologous arm sequences was verified by sequencing.

[0043] 4. Transformation and screening of diploid Saccharomyces cerevisiae INVSC1 Competent cells of diploid Saccharomyces cerevisiae INVSC1 were prepared using the lithium acetate-PEG method. Then, the recombinant plasmids pYES2-INV-ORC1 and pYES2-INV-Cdc6 were co-transformed into competent cells with RNH1 knockout cassettes (INV-RNH1-KO) and RNH201 knockout cassettes (INV-RNH201-KO).

[0044] The conversion conditions were: lithium acetate concentration 100mM, PEG4000 concentration 40%, incubation at 30℃ for 30min, and heat shock at 42℃ for 25min; After transformation, the culture was incubated on ice for 30 minutes to complete the recovery process and obtain the recovery solution. The recovery solution was then spread on uracil-deficient solid medium (SD-Ura) and cultured at 30°C for 48 hours to screen for strains that successfully integrated the overexpression vector. Single colonies from the SD-Ura solid medium were then picked and inoculated into YPD liquid medium containing G418 (concentration 200 μg / mL) and cultured at 30°C for 48 hours to screen for resistant strains that successfully knocked out the RNH1 / H201 gene. This strain is the improved Saccharomyces cerevisiae engineered strain.

[0045] Testing showed that the intracellular DNA dry weight of the improved Saccharomyces cerevisiae engineered strain constructed in this embodiment was 2.5 times higher than that of the original strain, and the cell survival rate was 84%.

[0046] Example 2: Preparation of PDRN using modified Saccharomyces cerevisiae engineered strains A method for preparing PDRN using modified Saccharomyces cerevisiae includes the following steps: S1: The improved brewing yeast engineered strain described in Example 1 was inoculated into the fermentation medium at an inoculation rate of 5% (v / v), and fermented for 20 h at 30°C, pH 5.5 and an aeration rate of 1.5 vvm, with the dissolved oxygen content controlled at 30%, to obtain the improved brewing yeast engineered strain fermentation broth. S2: The fermentation broth of the modified Saccharomyces cerevisiae was centrifuged at 6000 r / min for 12 min, and the cell pellet was collected. After washing three times with physiological saline, PBS buffer was added to obtain a cell suspension with a cell concentration of 12% (w / v). Lysis enzyme mixture was added to the cell suspension, mixed well, and enzymatically hydrolyzed at 37℃ and 180 r / min for 5 h to obtain a preliminary lysate. Then, the preliminary lysate was homogenized twice at 90 MPa using a high-pressure homogenizer to perform a second lysis, obtaining a cell lysate with a cell wall breakage rate of 96.3%. The lysin mixture contains glucanase and mannanase in a mass ratio of 2:1, with a total concentration of 2.0% (w / v). S3: Centrifuge the cell lysate to remove the precipitate, add anhydrous ethanol to the supernatant to make the final ethanol concentration 65%, let stand at 4℃ for 14 h, then centrifuge at 9000 r / min for 18 min, collect the precipitate, wash 3 times with 75% ethanol, dissolve the precipitate in TE buffer at pH=7.2 to obtain the crude nucleic acid extract; S4: First, the crude nucleic acid extract was passed through a 10 kDa ultrafiltration membrane at an operating pressure of 0.2 MPa and a temperature of 8℃, and the retentate was collected. Then, the retentate was placed in a 10 kDa dialysis bag and dialyzed in deionized water for 10 h, with the dialysate being replaced 3 times. Finally, the dialyzed retentate was freeze-dried at a vacuum of 0.03 MPa and a temperature of -45℃ for 30 h to obtain highly active PDRN derived from diploid yeast.

[0047] The purity, content of small molecule active peptides, and β-glucan content of the PDRN prepared in this embodiment were detected. The purity results are as follows: Figure 1 As shown.

[0048] Depend on Figure 1 It can be seen that the purity of the highly active PDRN derived from diploid yeast prepared in this embodiment is 91%. Furthermore, the PDRN contains 5.2% small molecule active peptides and 3.8% β-glucan.

[0049] The PDRN prepared in this embodiment was further analyzed by agarose gel electrophoresis, and the results are as follows: Figure 2 As shown.

[0050] Depend on Figure 2 As can be seen, this embodiment successfully prepared PDRN with a length of about 200bp, which belongs to small molecule PDRN.

[0051] Example 3: Performance evaluation of highly active PDRN derived from diploid yeast The performance of the highly active PDRN prepared in Example 2 was evaluated using interleukin-10 (IL-10), arginase-1 (Arg-1) gene, and vascular endothelial growth factor (VEGF) gene as indicators. The specific methods are as follows: Mouse mononuclear macrophage leukemia cell line RAW264.7 was retrieved from the cell bank and revived. After the cells reached 70-80% confluence in the culture flask, they were digested and counted. Cells were then inoculated at a concentration of 1×10⁻⁶ cells / mL. 5 The cells were seeded into 6-well plates at a seeding density of / well, with 2 mL of cell culture medium added to each well. The seeded 6-well plates were then placed in a cell culture incubator and cultured at 37°C and 5% CO2 for 24 hours until the cell cycle reached the logarithmic growth phase. Log-phase macrophages were then divided into control, model, sample, and standard control groups, with three replicates per group. The control group was supplemented with 4 mL of PBS buffer, the model group with 2 mL of lipopolysaccharide solution (1 μg / mL), the sample group with 2 mL of lipopolysaccharide solution (1 μg / mL) and 2 mL of the highly active PDRN solution (20 mg / mL) prepared in Example 2, and the standard control group with 2 mL of lipopolysaccharide solution (1 μg / mL) and 2 mL of commercially available salmon-derived PDRN solution (20 mg / mL). The four cell groups were incubated at 37℃ and 5% CO2 for 24 hours. Supernatants were collected from each group, and total RNA was extracted. The secretion of interleukin-10 (IL-10) in the cell supernatants was detected by enzyme-linked immunosorbent assay (ELISA), and the expression levels of arginase-1 (Arg-1) and vascular endothelial growth factor (VEGF) genes in the total RNA were detected by quantitative real-time PCR (qRT-PCR). The results are shown below. Figures 3-5 As shown.

[0052] Depend on Figures 3-5 As can be seen, compared with the model group, both the yeast-derived PDRN and the salmon-derived PDRN prepared in this invention can significantly increase the secretion level of IL-10 in macrophages and upregulate the mRNA expression of Arg-1 and VEGF (p<0.01 or p<0.001); among them, the yeast-derived PDRN showed significantly better performance than the salmon-derived PDRN in the above indicators (p<0.01). This indicates that the highly active PDRN obtained in this invention has stronger anti-inflammatory, tissue repair-promoting, and angiogenesis-promoting activities.

[0053] The embodiments described above are merely preferred implementations of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified brewing yeast strain, characterized in that, The starting strain was diploid Saccharomyces cerevisiae, with the RNH1 and RNH201 genes knocked out and the ORC1 and Cdc6 genes overexpressed. The nucleotide sequence of the RNH1 gene is shown in SEQ ID NO.1, the nucleotide sequence of the RNH201 gene is shown in SEQ ID NO.2, the nucleotide sequence of the ORC1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the Cdc6 gene is shown in SEQ ID NO.

4.

2. The improved brewing yeast strain as described in claim 1, characterized in that, The diploid brewer's yeast is brewer's yeast INVSC1.

3. The method for constructing the improved engineered brewer's yeast as described in claim 1, characterized in that, The steps include the following: Diploid Saccharomyces cerevisiae INVSC1 was selected as the starting strain. Overexpression vectors of ORC1 and Cdc6 genes were transferred into the strain INVSC1. At the same time, homologous recombination-mediated gene editing technology was used to knock out the RNH1 and RNH201 genes, thus constructing an improved Saccharomyces cerevisiae engineered strain.

4. The application of the improved Saccharomyces cerevisiae engineered strain described in claim 1 in the production of PDRN.

5. A method for preparing PDRN, characterized in that, Includes the following steps: S1: The improved brewing yeast engineered strain described in claim 1 is inoculated into a fermentation medium and cultured for 16-24 hours at 28-32°C, pH=5.0-6.0, aeration rate of 1.0-2.0 vvm, and oxygen content of 20-40%. S2: Collect the yeast cell pellet, wash it, and add PBS buffer or sterile water to obtain a cell suspension; add lyase mixture to the cell suspension, mix well, and lyse thoroughly to obtain a preliminary lysate; then use a high-pressure homogenizer to perform a second lysate on the preliminary lysate to obtain a cell lysate. S3: Centrifuge the cell lysis buffer to remove the precipitate, add ethanol to the supernatant, let stand, centrifuge a second time to collect the precipitate, wash the precipitate and dissolve it in TE buffer to obtain the crude nucleic acid extract; S4: First, the crude nucleic acid extract is ultrafiltered and desalted through an ultrafiltration membrane and dialysis bag. Then, the desalted retentate is freeze-dried to obtain highly active PDRN from diploid yeast.

6. The method for preparing PDRN as described in claim 5, characterized in that, In step S1, the fermentation medium is formulated as follows: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, K2HPO4 1 g / L, MgSO4・7H2O 0.5 g / L, pH=5.5; the fermentation conditions are: fermentation at 30℃, pH=5.5, aeration rate of 1.5 vvm for 20 h, and the dissolved oxygen content is controlled at 30% during the fermentation process.

7. The method for preparing PDRN as described in claim 5, characterized in that, In step S2, the lysin mixture contains glucanase and mannanase in a mass ratio of 2:1; the total concentration of the lysin is 1.5-2.5%; the lysis conditions are: enzymatic hydrolysis at 35-40℃ and 150-200 r / min for 4-6 h; the secondary lysis conditions are: homogenization 2-3 times under a pressure of 80-100 MPa.

8. The method for preparing PDRN as described in claim 5, characterized in that, In step S3, the conditions for the second centrifugation are: centrifugation at 4℃ and 10000~12000 r / min for 20~30 min; the ethanol concentration is 60~70%. In step S4, the ultrafiltration membrane has a molecular weight cutoff of 10 kDa; the ultrafiltration conditions are: pressure 0.1~0.3 MPa, temperature 4~10℃; the dialysis bag has a molecular weight cutoff of 10 kDa, the dialysis time is 8~12 h, and the dialysis fluid is changed 2~3 times during dialysis; the freeze-drying conditions are: freeze-drying at 0.01~0.05 MPa and -50℃~-40℃ for 24~36 h.

Images