CN-121975647-A - Genetically engineered bacterium for producing polydeoxyribonucleotide, construction method and application thereof

Abstract

The invention provides a genetically engineered bacterium for producing polydeoxyribonucleotide, a construction method and application thereof. The genetically engineered bacteria include kluyveromyces marxianus expressing salmon DNA polymerase I gene. The invention realizes the efficient, safe and controllable production of the PDRN by genetically engineering the edible probiotics Kluyveromyces marxianus, has the biological activity equivalent to that of the PDRN from a natural source, and is beneficial to the large-scale production and application of the PDRN.

Inventors

• CAI WEIFAN
• NI XIANPU
• HUANG XIUYAN
• CAI JIANPING
• WU WEI
• WEI QI

Assignees

• 上海加新生物科技有限公司

Dates

Publication Date

20260505

Application Date

20260206

Claims (10)

1. 1. A genetically engineered bacterium for producing polydeoxyribonucleotide is characterized in that, the genetically engineered bacteria include kluyveromyces marxianus expressing salmon DNA polymerase I gene.
2. 2. The genetically engineered strain for producing polydeoxyribonucleotides according to claim 1, wherein the nucleic acid sequence of the salmon DNA polymerase I gene comprises the sequence shown in SEQ ID No. 1.
3. 3. The method for constructing a polydeoxyribonucleotide-producing genetically engineered bacterium according to claim 1 or 2, characterized in that the method for constructing comprises: And inserting the salmon DNA polymerase I gene into an expression vector to obtain a recombinant vector, and transferring the recombinant vector into Kluyveromyces marxianus to obtain the genetically engineered bacterium for producing polydeoxyribonucleotide.
4. 4. Use of the genetically engineered bacterium for producing polydeoxyribonucleotide according to claim 1 or 2 for the preparation of polydeoxyribonucleotide.
5. 5. A method for preparing polydeoxyribonucleotide, characterized in that the method comprises: Culturing the genetically engineered bacterium for producing polydeoxyribonucleotide according to claim 1 or 2, collecting bacterial lysate, and purifying the product to obtain polydeoxyribonucleotide.
6. 6. The method for producing polydeoxyribonucleotide according to claim 5, characterized in that said culture medium contains glucose, yeast extract, peptone and trace elements.
7. 7. The method for preparing polydeoxyribonucleotide according to claim 6, characterized in that said medium contains 5% -10% glucose, 2% -4% yeast extract, 1% -2% peptone and 0.1% -0.5 mM trace element; Preferably, the trace elements include iron ions, zinc ions, and magnesium ions.
8. 8. The method for preparing polydeoxyribonucleotide according to any of the claims 5-7, characterized in that said cultivation conditions comprise a temperature of 28-32 ℃ and a pH of 6.5-7.0, and a dissolved oxygen of 30% or more.
9. 9. The method for preparing polydeoxyribonucleotide according to any of the claims 5-8, characterized in that said purification step comprises collecting the bacterial cells by centrifugation of the culture broth, crushing, collecting the supernatant, and subjecting the supernatant to ultrafiltration concentration, anion exchange chromatography and gel filtration chromatography in this order, and collecting polydeoxyribonucleotide.
10. 10. A polydeoxyribonucleotide, characterized in that it is prepared by the method for preparing a polydeoxyribonucleotide according to any of claims 5-9.

Description

Genetically engineered bacterium for producing polydeoxyribonucleotide, construction method and application thereof Technical Field The invention belongs to the technical field of bioengineering, relates to a genetically engineered bacterium for producing polydeoxyribonucleotide, and a construction method and application thereof, and is particularly suitable for large-scale preparation of Polydeoxyribonucleotide (PDRN) in the fields of medical cosmetology, regenerative medicine and health care products. Background Polydeoxyribonucleotide (PDRN) is a high purity, high molecular weight dna fragment extracted from a specific biological source. Nucleic acid substances with biological activities of repairing tissues, promoting cell proliferation, resisting inflammation and the like are widely applied to the fields of medical cosmetology, regenerative medicine and health care products. At present, polydeoxyribonucleotide is mainly extracted from fish germ cells, especially sperm cells of trout and salmon, and is a main source of PDRN extraction due to the abundant nucleic acid content. However, the salmon sperm extraction method has remarkable limitations that the extraction of PDRN is limited by raw materials (fish spermary) and has high price, the target fragments are required to be obtained through a complex degradation process after the extraction, the molecular weight distribution is difficult to precisely control (the distribution range is generally 100-5000 bp), and the risk of pathogen pollution of animal sources is easy to accompany. Therefore, attention is paid to development of alternative methods, which are currently used in common use, including chemical synthesis methods and biological synthesis methods, such as a phosphodiester method and a phosphoester method, by specific chemical reaction synthesis, however, the synthesis process is complex, the requirements on conditions are severe, the cost is high, and the synthesis efficiency of long-chain PDRN (> 500 bp) is low, the biological synthesis methods include a DNA amplification technology and a microbial fermentation method, wherein the DNA amplification technology uses genomic DNA as a template to amplify and then carries out ultrasonic and enzyme digestion treatment, the microbial fermentation method is used for preparing PDRN by constructing recombinant strains through microbial fermentation, such as CN119824022A, the preparation method comprises S1 for preparing a PDRN solution, S2 for complementing double chains of the PDRN to be flat ends, S3 for constructing recombinant plasmids, S4 for carrying out mixed culture of products of the step S3 and E.coli DH5 alpha, S6 for PCR amplification, recording strains with a length greater than 200bp, S7 for preparing recombinant strains, S8 for preparing recombinant strains, and S9 for preparing recombinant strains containing the PDRN sequences, and obtaining recombinant plasmids in the recombinant strains by extracting the recombinant strains, and obtaining recombinant plasmids in the recombinant strains, and obtaining recombinant plasmids by isolating the recombinant strains from the recombinant strains. However, the escherichia coli is a non-food-grade strain, the endotoxin content is high (generally more than 50 EU/mg), a detoxification process is required to be additionally added, the purity of PDRN is insufficient, and the quality requirements in the fields of medical cosmetology and regenerative medicine are difficult to meet. Kluyveromyces marxianus as one food-grade yeast has the advantage of fast growth rate, which is more than 2 times of that of Saccharomyces cerevisiae, is one of the fastest growing eukaryotes at present, has no endotoxin synthesis capability, and has obviously better safety than escherichia coli. However, no report on the preparation of PDRN by using Kluyveromyces marxianus expressed salmon DNA gene exists at present, and development of a PDRN efficient preparation technology based on the yeast has important significance in solving the bottleneck problem of the existing method. In view of the above, how to develop a safe, efficient and low-cost PDRN preparation method is a problem in the art. Disclosure of Invention Aiming at the defects and actual demands of the prior art, the invention provides a genetically engineered bacterium for producing polydeoxyribonucleotide, and a construction method and application thereof, so as to realize the PDRN production with controllable quality, low cost, safety and environmental protection. In order to achieve the above purpose, the invention adopts the following technical scheme: In a first aspect, the invention provides a genetically engineered bacterium for producing polydeoxyribonucleotide, the genetically engineered bacterium comprising Kluyveromyces marxianus expressing salmon DNA polymerase I gene )。 In the invention, the genetic engineering bacteria for producing polydeoxyribonucleotide are designed, the edible probiotics Kluyve