Propionibacterium acnes VB208 gene editing method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a propionibacterium acnes VB208 gene editing method and application of the propionibacterium acnes VB208 gene editing method, the propionibacterium acnes are preserved in the China General Microbiological Culture Collection Center (CGMCC) on February 20, 2023, and the preservation number is CGMCC No.26576. The propionibacterium acnes VB208 gene editing method comprises the following steps of: selecting propionibacterium acnes VB208 gene editing method; the propionibacterium acnes obtained through screening has the characteristics of being capable of occupying the skin surface, high in transformation efficiency, high in gene modification potential and high in foreign protein expression efficiency, can be used as an engineering strain, can be used for establishing a genetic manipulation system suitable for the strain, and is wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a method for gene editing of Propionibacterium acnes VB208 and its application. Background Art

[0002] Propionibacterium acnes ( Propionibacterium acnes ) is one of the most common bacteria on the human skin and is usually present in the pilosebaceous unit. In traditional understanding, Propionibacterium acnes is considered a pathogenic bacterium due to its association with the onset of acne; however, with the progress of genomics and microbiology research, people have gradually realized that there are significant genetic background differences among different strains of Propionibacterium acnes, and these differences may lead to its different roles in skin health and diseases.

[0003] Although a large number of studies have described the pathogenic characteristics of Propionibacterium acnes, its beneficial aspects, especially the potential benefits of specific strains, have not been fully characterized. Relevant research shows that some strains of Propionibacterium acnes may be beneficial to skin health and can even antagonize the infection of other pathogenic bacteria (such as Staphylococcus aureus), which suggests that specific strains of Propionibacterium acnes can be used as beneficial bacteria to play a role in solving skin problems. However, at present, a genetic operation system applicable to these beneficial strains has not been established, which limits their wide application in the fields of biotechnology and medicine.

[0004] Therefore, finding a special strain of Propionibacterium acnes that can occupy positions on the skin surface, has high transformation efficiency, and can efficiently express foreign proteins, and establishing a genetic operation system applicable to this strain is of great significance for the application of Propionibacterium acnes beneficial bacteria in developing new skin health-related products and treatment means. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. To this end, the present invention provides a method for gene editing of Propionibacterium acnes VB208 and its application. The Propionibacterium acnes screened by the present invention has the characteristics of being able to occupy positions on the skin surface, high transformation efficiency, gene modification potential, and efficient expression of foreign proteins. At the same time, it can be used as an engineering strain, and a genetic operation system applicable to this strain is established, with broad application prospects.

[0006] In the first aspect of the present invention, the present invention provides a Propionibacterium acnes VB208. According to an embodiment of the present invention, the Propionibacterium acnes was deposited with the China General Microbiological Culture Collection Center on February 20, 2023, and the deposit number is CGMCC No. 26576.

[0007] Deposit Information: Strain Name: VB208 Classification and Naming: Propionibacterium acnes Date of Deposit: February 20, 2023 Depositary Institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms Deposit Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101 Deposit Number: CGMCC No. 26576 The newly isolated strain of Propionibacterium acnes ( Propionibacterium acnes ) VB208 obtained by screening in the present invention has the characteristics of occupying space on the skin surface, high transformation efficiency, potential for genetic modification, and high-efficiency expression of foreign proteins. At the same time, it can be used as an engineering strain, and a genetic operation system suitable for this strain can be established using it, with broad application prospects.

[0008] In the second aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, it includes the Propionibacterium acnes VB208 described in the first aspect.

[0009] Those skilled in the art can understand that the characteristics and advantages described above for Propionibacterium acnes VB208 also apply to this composition and will not be elaborated herein.

[0010] In the third aspect of the present invention, the present invention provides a microbial inoculant. According to an embodiment of the present invention, it includes the Propionibacterium acnes VB208 described in the first aspect or the composition described in the second aspect.

[0011] Those skilled in the art can understand that the characteristics and advantages described above for Propionibacterium acnes VB208 also apply to this microbial inoculant and will not be elaborated herein.

[0012] In the fourth aspect of the present invention, the present invention provides the use of the Propionibacterium acnes VB208 described in the first aspect, the composition described in the second aspect, or the microbial inoculant described in the third aspect in the preparation of daily chemical products or pharmaceuticals.

[0013] Those skilled in the art can understand that the characteristics and advantages described above for Propionibacterium acnes VB208 also apply to this use and will not be elaborated herein.

[0014] In the fifth aspect of the present invention, the present invention provides the application of the Propionibacterium acnes VB208 described in the first aspect in genetic engineering or metabolic engineering. According to an embodiment of the present invention, the newly isolated strain of Propionibacterium acnes VB208 obtained by screening in the present invention has high transformation efficiency and potential for genetic modification, indicating that it can obtain new functions through gene insertion, replacement, or knockout, etc., and provides a technical basis for the development of engineering strains with specific functions.

[0015] In the sixth aspect of the present invention, the present invention provides a genetically engineered strain for expressing a target gene. According to an embodiment of the present invention, the genetically engineered strain is obtained by the following method: introducing a vector containing a target gene expression cassette into Propionibacterium acnes VB208 described in the first aspect to obtain a genetically engineered strain capable of expressing the target gene, wherein the vector containing the target gene expression cassette has upstream and downstream homologous arms designed for the genome of Propionibacterium acnes VB208 described in the first aspect. The genetically engineered strain for expressing the target gene according to the embodiment of the present invention can stably and highly express foreign genes or proteins, and has broad application prospects.

[0016] In the seventh aspect of the present invention, the present invention provides a method for producing a foreign protein. According to an embodiment of the present invention, the method includes: using the genetically engineered strain for expressing a target gene described in the sixth aspect to express the foreign protein; wherein the target gene encodes the expression of the foreign protein. According to the method of the embodiment of the present invention, the genetically engineered strain constructed by the foregoing method is used to produce the foreign protein, providing an efficient and low-cost biosynthesis method, which can be applied to the production of proteins with specific functions, such as drugs, bioactive factors, etc., and has broad application prospects.

[0017] In the eighth aspect of the present invention, the present invention provides the application of the genetically engineered strain for expressing a target gene described in the sixth aspect in the expression of a foreign protein. According to an embodiment of the present invention, the target gene encodes the expression of the foreign protein.

[0018] Those skilled in the art can understand that the features and advantages described above for the genetically engineered strain for expressing a target gene also apply to this application, and will not be repeated here.

[0019] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of the embodiments in conjunction with the accompanying drawings, wherein: Figure 1 It is the electron microscopy result of Propionibacterium acnes VB208 in Example 1 of the present invention; Figure 2 It is the map of the recombinant plasmid p2107-mcherry in Example 3 of the present invention. Detailed Description of the Embodiments

[0021] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0022] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0023] The endpoints and any values disclosed in this text for a range are not limited to that precise range or value. These ranges or values should be understood to include values close to those ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this text.

[0024] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0025] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may but do not necessarily occur, and this description includes the cases where such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0026] In this text, the term "milk tube" refers to a litmus milk biochemical tube, the composition of which is skim milk powder and litmus, and is used to determine the fermentation ability of bacteria to milk.

[0027] In this text, the term "Propionibacterium acnes" is a Gram-positive bacillus, anaerobic or facultatively anaerobic, mainly inhabiting the skin hair follicles and sebaceous glands. Among them, the new strain of Propionibacterium acnes VB208 is isolated and screened from the facial skin surface of healthy people, which does not belong to pathogenic bacteria and has potential probiotic properties.

[0028] Propionibacterium acnes VB208 The present invention provides a Propionibacterium acnes VB208. According to an embodiment of the present invention, the Propionibacterium acnes was deposited with the China General Microbiological Culture Collection Center on February 20, 2023, and the deposit number is CGMCC No. 26576.

[0029] According to an embodiment of the present invention, the 16S rDNA sequencing result of the Propionibacterium acnes VB208 is as shown in SEQ ID NO: 1.

[0030] Propionibacterium acnes screened by the present invention ( Propionibacterium acnesThe new strain VB208 of Propionibacterium acnes has the characteristics of occupying space on the skin surface, high transformation efficiency, potential for genetic modification, and efficient expression of foreign proteins. At the same time, it can be used as an engineering strain, and a genetic operation system suitable for this strain can be established with broad application prospects.

[0031] In this article, the term "Propionibacterium acnes ( Propionibacterium acnes ) VB208" and "Propionibacterium acnes VB208" are synonymous.

[0032] Composition The present invention provides a composition. According to an embodiment of the present invention, it includes the aforementioned Propionibacterium acnes VB208.

[0033] Those skilled in the art can understand that the characteristics and advantages described above for Propionibacterium acnes VB208 also apply to this composition and will not be elaborated herein.

[0034] Microbial inoculant The present invention provides a microbial inoculant. According to an embodiment of the present invention, it includes the aforementioned Propionibacterium acnes VB208 or the aforementioned composition.

[0035] In some specific embodiments, the forms of the microbial inoculant include two types: microbial liquid inoculant and microbial solid inoculant. Exemplarily, when the form of the microbial inoculant is a microbial solid inoculant, it includes but is not limited to freeze-dried powder, etc.

[0036] It should be noted that in the microbial inoculant of the present invention, Propionibacterium acnes VB208 can exist in the form of live bacteria and / or non-live bacteria.

[0037] In this article, the "form of live bacteria" refers to the existence form of Propionibacterium acnes VB208 with the ability of metabolism, reproduction, or replication.

[0038] Exemplarily, the live bacteria can be immobilized bacteria. In this article, "immobilized bacteria" refers to live bacteria immobilized on a carrier, which can carry out life activities such as growth, development, reproduction, inheritance, and metabolism within a certain spatial range.

[0039] In this article, the "form of non-live bacteria" refers to the existence form of Propionibacterium acnes VB208 without the ability of metabolism, reproduction, and replication, including but not limited to dried bacterial cells. Exemplarily, the microbial inoculant is freeze-dried powder.

[0040] In some specific embodiments, Propionibacterium acnes VB208 exists in the form of live bacteria, dried bacterial cells, immobilized bacteria, or any other form.

[0041] In some specific embodiments, the dried bacterial cells are obtained by freeze-drying Propionibacterium acnes VB208.

[0042] Those skilled in the art can understand that the characteristics and advantages described above for Propionibacterium acnes VB208 also apply to this microbial agent and will not be elaborated here.

[0043] Use The present invention provides the use of the aforementioned Propionibacterium acnes VB208, the aforementioned composition or the aforementioned microbial agent in the preparation of daily chemical products or pharmaceuticals.

[0044] Those skilled in the art can understand that the characteristics and advantages described above for Propionibacterium acnes VB208 also apply to this use and will not be elaborated here.

[0045] Application in genetic engineering or metabolic engineering The present invention provides the application of the aforementioned Propionibacterium acnes VB208 in genetic engineering or metabolic engineering. According to the embodiments of the present invention, the newly obtained strain of Propionibacterium acnes VB208 has high transformation efficiency and potential for genetic modification, indicating that it can obtain new functions through gene insertion, replacement or knockout, etc., and provides a technical basis for the development of engineered strains with specific functions.

[0046] Engineered strain expressing a target gene The present invention provides an engineered strain expressing a target gene. According to the embodiments of the present invention, the engineered strain is obtained by the following method: introducing a vector containing a target gene expression cassette into the aforementioned Propionibacterium acnes VB208 to obtain an engineered strain capable of expressing the target gene, wherein the vector containing the target gene expression cassette has upstream and downstream homologous arms designed for the genome of the aforementioned Propionibacterium acnes VB208. The engineered strain expressing the target gene according to the embodiments of the present invention can stably and efficiently express foreign genes or proteins and has broad application prospects.

[0047] In some specific embodiments, the method of introducing a vector containing a target gene expression cassette into the aforementioned Propionibacterium acnes VB208 may include conjugation transfer, electroporation, chemical transformation, etc.

[0048] Exemplarily, when the target gene is a gene encoding the expression of a fluorescent protein, the method of conjugation transfer can be selected to construct an engineered strain expressing the gene encoding the fluorescent protein. At this time, Escherichia coli with a clear genetic background, simple technical operation and simple culture conditions can be preferentially selected as the donor bacterium, which can further improve the construction efficiency and success rate of the engineered strain expressing the target gene.

[0049] Method for producing exogenous protein The present invention provides a method for producing an exogenous protein. According to an embodiment of the present invention, the method includes: using the aforementioned engineered strain expressing a target gene to express the exogenous protein; wherein, the target gene encodes the expression of the exogenous protein. According to the method of the embodiment of the present invention, the exogenous protein is produced by the engineered strain constructed by the aforementioned method, providing an efficient and low-cost biosynthesis method, which can be applied to the production of proteins with specific functions, such as drugs, bioactive factors, etc., and has a wide application prospect.

[0050] Application of an engineered strain expressing a target gene in the expression of an exogenous protein The present invention provides an application of the aforementioned engineered strain expressing a target gene in the expression of an exogenous protein. According to an embodiment of the present invention, the target gene encodes the expression of the exogenous protein.

[0051] Those skilled in the art can understand that the features and advantages described above for the engineered strain expressing the target gene also apply to this application, and will not be elaborated herein.

[0052] Unless otherwise specified, the formula of the No. 22 agar medium (No. 22 solid medium) used in the embodiments of the present invention is (g / L): hemin 0.001 wt%, Tween 80 0.1 wt%, tryptone 0.82 wt%, bacteriological peptone 0.25 wt%, soy peptone 0.1 wt%, glucose 0.58 wt%, yeast extract FM860 0.5 wt%, sodium chloride 0.17 wt%, L-cystine 0.04 wt%, agar 1.5 wt%, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) 0.3 wt%. After preparation, the pH of the medium is adjusted to 7.5 and sterilized at 121°C for 30 min.

[0053] Unless otherwise specified, the formula of the No. 22 liquid medium used in the embodiments of the present invention is (g / L): tryptone 0.82 wt%, bacteriological peptone 0.25 wt%, soy peptone 0.1 wt%, glucose 0.58 wt%, yeast extract 0.5 wt%, sodium chloride 0.17 wt%, disodium hydrogen phosphate 0.08 wt%, L-cystine 0.04 wt%, hemin 0.001 wt%, Tween 80 0.1 wt%, Tris-HCl 0.3 wt%. After preparation, the pH of the medium is adjusted to 7.5 and sterilized at 121°C for 30 min.

[0054] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0055] Example 1: Obtaining of Propionibacterium acnes VB208 The Propionibacterium acnes VB208 of the present invention was isolated and screened from the facial skin surface of healthy people.

[0056] Process of strain collection and isolation: Immerse a sterile sampling cotton swab in 3 mL of sterile 40% glycerol for 30 s, then wipe the facial skin with the sterile sampling cotton swab for 30 s, and then put the cotton swab into the glycerol tube and stir for 30 s to obtain a sample. Take 1 mL of the sample and dilute it serially to 10 -2 、10 -4 、10 -6 . Respectively take 100 μL and spread it evenly on the No. 22 agar medium plate. Wait for single colonies to grow, and repeat the streak purification culture for multiple times until the colony morphology in the petri dish is consistent. After examination under the microscope and no contaminating bacteria are found, the isolated and purified strains are numbered pa01~pa10, and the Propionibacterium acnes strain numbered pa02 is named VB208. The morphological identification of the strain is carried out by electron microscopy observation, and in combination with the results of PCR amplification and sequencing analysis of the 16S rDNA gene of the strain, the strain classification is further determined.

[0057] The electron microscopy results of Propionibacterium acnes VB208 are shown in Figure 1 .

[0058] Send the strain VB208 to Hangzhou Qingke Biotechnology Co., Ltd. for 16S rDNA sequence detection.

[0059] The 16S rDNA sequencing result of Propionibacterium acnes VB208 is as shown in SEQ ID NO: 1.

[0060] The results showed that: The 16S rRNA sequence of strain VB208 was compared with strains in GenBank for homology. The highest homology of strain VB208 with Propionibacterium acnes IGM5-4 ( Propionibacterium acnes ) was 99%.

[0061] Final identification determined that: The isolated strain VB208 was a strain of Propionibacterium acnes, named Propionibacterium acnes VB208, and was deposited in the "China General Microbiological Culture Collection Center" on February 20, 2023. The deposit address was No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number was CGMCC No. 26576.

[0062] Example 2: Identification of the physiological and biochemical characteristics of Propionibacterium acnes VB208 Propionibacterium acnes VB208 was cultured using No. 22 agar medium, and then the physiological and biochemical characteristics of Propionibacterium acnes VB208 were identified with reference to "Bergey's Manual of Determinative Bacteriology". The specific detection contents were as follows: 1. Catalase test After normal 24-hour culture, a small loop of Propionibacterium acnes VB208 colonies was taken with a sterile inoculation loop and smeared on a glass slide with 3% hydrogen peroxide dropped. Observation was immediately made. If bubbles were generated, it was positive (+), and if no bubbles were present, it was negative (-).

[0063] 2. Glucose semi-solid test The glucose semi-solid medium was purchased from Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park, and the product number was GB025.

[0064] Preparation of the Propionibacterium acnes VB208 bacterial suspension: Take the glycerol tube stored in the -80°C refrigerator, after thawing, pipette 100 μl of the bacterial liquid into No. 22 medium, and culture at 37°C for 24 h. The bacterial liquid concentration was 10 8 cfu / ml.

[0065] Add 100 μL of the Propionibacterium acnes VB208 bacterial suspension (concentration 10 8 cfu / ml) to a biochemical tube containing sufficient glucose semi-solid medium, and culture at 37°C for 24 h. If it turned yellow, it was positive (+), and if it turned gray-purple, purple or magenta, it was negative (-).

[0066] 3. Glucose fermentation test The glucose fermentation medium was purchased from Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park, and the product number was GB117.

[0067] The preparation steps of the Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 bacterial suspension" section of the glucose semi-solid experiment.

[0068] Add 100 μL of the Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into a biochemical tube containing sufficient glucose fermentation medium, and incubate at 37 °C for 24 h. If it turns yellow, it is positive (+); if it turns blue or blue-green, it is negative (-).

[0069] 4. Milk decomposition experiment Take a test tube containing 2 mL of sterile water. Use an inoculation needle to pick 3 colonies of Propionibacterium acnes VB208 from the plate and transfer them into the test tube containing sterile water. Grind them carefully to make a bacterial suspension, and add 100 μL of the bacterial suspension into the milk tube; place the milk tube in an incubator at 37 °C for 24 h. After the incubation, observe. If the milk shows stratification and turns pink or solidifies, it is positive (+); if there is no color change, it is negative (-).

[0070] 5. Indole test Peptone water medium, purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number HB5218.

[0071] The preparation steps of the Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 bacterial suspension" section of the glucose semi-solid experiment.

[0072] Add 100 μL of the Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into the peptone water medium, incubate at 37 °C for 24 h, then add 0.2 mL of Kovacs indole reagent. If a red color appears in the upper layer, it is a positive indole reaction (+); otherwise, it is a negative reaction (-).

[0073] 6. Cellobiose / maltose / sucrose fermentation experiment Cellobiose biochemical tube, purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number SN067.

[0074] Maltose fermentation tube, purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number GB079.

[0075] Sucrose fermentation tube, purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number GB169.

[0076] The preparation steps of the Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 bacterial suspension" section of the glucose semi-solid experiment.

[0077] Add 100 μL of Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into the cellobiose biochemical tube, maltose fermentation tube, and sucrose fermentation tube respectively, and culture at 37°C for 24 h. If it turns yellow, it is positive (+); if it turns purple or purple-gray, it is negative (-).

[0078] 7. L-Arabinose fermentation experiment The L-arabinose biochemical tube is purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, with the product number GB188.

[0079] The preparation steps of Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0080] Add 100 μL of Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into the L-arabinose biochemical tube, and culture at 37°C for 24 h. If it turns yellow, it is positive (+); if it turns blue or blue-green, it is negative (-).

[0081] 8. Glycerol fermentation experiment The glycerol biochemical tube is purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, with the product number GB064.

[0082] The preparation steps of Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0083] Add 100 μL of Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into the glycerol biochemical tube, and culture at 37°C for 24 h. If it turns yellow, it is positive (+); if it turns blue or blue-green, it is negative (-).

[0084] 9. Gelatin liquefaction experiment The gelatin biochemical tube is purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, with the product number GB066.

[0085] The preparation steps of Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0086] Add 100 μL of Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into the gelatin biochemical tube, and culture at 37°C for 24 h. After taking it out, place it in a 4°C refrigerator for 30 min and then observe. If it is still liquid, the experimental result is positive (+); otherwise, it is negative (-).

[0087] 10. Esculin Hydrolysis Experiment The esculin biochemical tube was purchased from Haibo Biotechnology Co., Ltd. in Qingdao High-tech Industrial Park, with the product number GS021.

[0088] The preparation steps of the Propionibacterium acnes VB208 bacterial suspension are the same as those in the "Preparation of Propionibacterium acnes VB208 Bacterial Suspension" section of the glucose semi-solid experiment.

[0089] Add 100 μL of the Propionibacterium acnes VB208 bacterial suspension (concentration: 10 8 cfu / ml) into the esculin biochemical tube, incubate at 37°C for 24 h. After inoculation, add sterile liquid paraffin to cover the liquid surface of the medium. If it turns black, it is positive (+); if it turns purple or purple-gray, it is negative (-).

[0090] The results of the identification of the physiological and biochemical characteristics of Propionibacterium acnes VB208 are shown in Table 1.

[0091] Table 1 Results of the Identification of the Physiological and Biochemical Characteristics of Propionibacterium acnes VB208

[0092] The above results indicate that Propionibacterium acnes VB208 can produce catalase, can utilize glucose, D-cellobiose and glycerol as carbon sources, and can decompose esculin to produce glucose and esculetin.

[0093] Example 3: Investigation of the Conjugative Transfer of Different Strains of Propionibacterium acnes 1. Construction of Plasmid p2107-mcherry (1) Extraction of the genomic DNA of Propionibacterium acnes VB208 The extraction kit used was the Bacterial DNA Kit D3146-02 from Meiji Bio. Operate according to the extraction steps of the kit to obtain the whole genome of Propionibacterium acnes VB208, and use agarose gel electrophoresis to detect the integrity and purity of the DNA.

[0094] (2) PCR Amplification of Homologous Arms Amplify the upstream and downstream homologous arms homologous to the genome of Propionibacterium acnes VB208 (designed according to the common sequence of Propionibacterium acnes) by PCR for subsequent homologous recombination. After amplification, obtain the homologous arms and verify the size and purity of the homologous arms by agarose gel electrophoresis.

[0095] (3) Synthesis of the Erythromycin Resistance Gene and the mcherry Fluorescent Labeling Gene According to the codon preference of Propionibacterium acnes VB208, the sequences of the erythromycin resistance gene and the mcherry fluorescent marker gene were optimized and synthesized (completed by Nanjing Genscript Biotechnology Co., Ltd.), and the erythromycin resistance gene and the mcherry fluorescent marker gene were obtained.

[0096] (4)Construct the recombinant plasmid p2107-mcherry Plasmid p2107 contains the gene elements required for Gram-positive bacterial conjugation transfer. Therefore, plasmid p2107 was selected as the vector, and the homologous arms obtained in step (2), the erythromycin resistance gene and the mcherry fluorescent marker gene obtained in step (3) were ligated to plasmid p2107 using Gibson assembly technology to obtain a recombinant plasmid. The recombinant plasmid was named p2107-mcherry, and the structure and sequence correctness of the recombinant plasmid p2107-mcherry were verified.

[0097] The map of the recombinant plasmid p2107-mcherry is shown in Figure 2 。

[0098] 2. Conjugation transfer experiment (1)Selection and treatment of donor strains Use E.coli S17 and E.coli ET12567 as donor strains. The recombinant plasmid p2107-mcherry obtained in step 1 was transformed into the donor strains by electroporation to obtain donor strains E.coli S17’ and E.coli ET12567’. They were cultured using liquid LB medium. After 24 h of culture, single colonies of the donor bacteria E.coli S17’ and E.coli ET12567’ were inoculated into 50 mL of liquid LB medium (supplemented with 400 μg / ml erythromycin antibiotic) and cultured at 37°C for 18 h; then they were transferred to 50 mL of liquid LB medium (supplemented with 400 μg / ml erythromycin antibiotic) at 2% respectively. When the optical density (OD) of the donor bacteria E.coli S17’ and E.coli ET12567’ 600 reached 1.0 (about 5 h), 50 mL of the donor bacteria E.coli S17’ and E.coli ET12567’ were centrifuged at 5000 rpm for 10 min to collect the bacterial cells, washed twice with 20 mL of liquid LB medium, centrifuged at 5000 rpm for 10 min to obtain the donor bacterial cell pellet, and resuspended with LB liquid medium to obtain the resuspension of the donor bacteria E.coli S17’ and the resuspension of the donor bacteria E.coliET12567' resuspension.

[0099] Preparation of liquid LB medium (g / L): Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L. After preparation, adjust the pH of the medium to 7.5 and sterilize at 121 °C for 30 min.

[0100] (2) Selection and treatment of recipient strains The recipient strains are Propionibacterium acnes pa01 - pa10, among which pa02 is the preserved strain Propionibacterium acnes VB208, and Propionibacterium acnes pa01, pa03 - pa10 are other strains obtained by screening and numbered from the facial skin surface of healthy people in the same batch; inoculate the recipient strains pa01 - pa10 into No. 22 liquid medium respectively, culture at 37 °C for 24 h, collect by centrifugation and wash twice, then resuspend the recipient bacteria pa01 - pa10 with No. 22 liquid medium to obtain recipient bacteria resuspensions 1 - 10 respectively.

[0101] (3) Conjugative transfer Heat shock the recipient bacteria resuspensions 1 - 10 (incubate in a metal bath at 40 °C) for 1 min, and then mix them with the donor bacteria E.coli S17' resuspension or the donor bacteria E.coli ET12567' resuspension evenly according to the ratio of recipient bacteria to donor bacteria = 1:10. After mixing, the number of recipient bacteria is 10 8 CFU. Drop the mixed solution onto the well - dried No. 22 solid medium plate (extra adding 10 mM MgCl2) respectively, and place it in an anaerobic incubator at 37 °C for co - culture for 24 h, then transfer and coat them on the resistance screening plate respectively, and count the number of transformants. Among them, the conjugative transfer frequency = number of transformants / number of recipient bacteria.

[0102] The conjugative transfer results of different strains of Propionibacterium acnes are shown in Table 2.

[0103] Table 2 Conjugative transfer results of different strains of Propionibacterium acnes

[0104] The results show that the higher the conjugative transfer frequency, the higher the transformation efficiency. Only the strain Propionibacterium acnes pa02 (Propionibacterium acnes VB208) can perform genetic manipulation and has the highest conjugative transfer efficiency.

[0105] Example 4: Detection of the fluorescence protein expression content of the recombinant strain Propionibacterium acnes VB208' Select Propionibacterium acnes pa02 (Propionibacterium acnes VB208) as the recipient strain, E.coliUsing S17 as the donor strain, repeat the conjugation transfer experiment steps in Example 2. When co-cultured for 24 h, use 1 mL of sterile water to rinse the mixed bacteria of Propionibacterium acnes pa02- E.coli S17' from the plate, and spot the mixed bacteria on a solid medium plate No. 22 (extra adding 10 μg / mL erythromycin and 25 μg / mL nalidixic acid), and culture anaerobically at 37°C for 6 d to obtain the recombinant strain pa02' (Propionibacterium acnes VB208'), and observe it under the iBright™ FL1500 imaging system. The excitation light wavelength is set to 587 nm, and it can be observed that the recombinant strain pa02' emits red fluorescence, indicating that the recombinant plasmid p2107-mcherry has been successfully integrated into the target strain Propionibacterium acnes pa02. Then, detect the fluorescence protein expression content of the recombinant strain Propionibacterium acnes VB208' by the following specific method: Inoculate the recombinant strain Propionibacterium acnes VB208' (experimental group) and the strain Propionibacterium acnes VB208 (control group) into the liquid medium No. 22 respectively. After culturing for 6 d, take 1 mL of the bacterial liquid, centrifuge to obtain the bacterial cell precipitate, resuspend the bacterial cells with PBS buffer, centrifuge after cell disruption and take the supernatant, and detect the fluorescence protein content with the mCherry ELISA kit (product number: ab221829) after 100-fold dilution respectively.

[0106] The detection results of the fluorescence protein content are shown in Table 3.

[0107] Table 3 Detection results of fluorescence protein content

[0108] The above results show that the recombinant strain Propionibacterium acnes VB208' can highly express foreign proteins.

[0109] Example 5: Investigation of antibiotic sensitivity of different strains of Propionibacterium acnes Inoculate Propionibacterium acnes pa01~pa10 into the liquid medium No. 22 (extra adding 10 mg / mL of antibiotics) respectively. Among them, pa02 is the preserved strain Propionibacterium acnes VB208, and Propionibacterium acnes pa01, pa03~pa10 are other strains screened and numbered from the facial skin surface of healthy people in the same batch; after culturing at 37°C for 42 h, measure OD 600 , so as to verify the sensitivity of Propionibacterium acnes pa01~pa10 to three antibiotics, namely erythromycin, tetracycline and cephalosporin. Among them, when OD 600 is greater than or equal to the OD of the control group 600 , it means that the antibiotic is insensitive (+), that is, the strain can grow normally in the medium containing antibiotics and has drug resistance. When OD 600 is less than the OD of the control group600 In the case of being sensitive to antibiotics (-), that is, the strain cannot grow normally in the culture medium containing antibiotics and does not have drug resistance; the control group culture medium is No. 22 liquid medium without any antibiotics added.

[0110] The results of the investigation on the antibiotic sensitivity of different strains of Propionibacterium acnes are shown in Table 4.

[0111] Table 4 Results of the investigation on the antibiotic sensitivity of different strains of Propionibacterium acnes

[0112] In Table 4: + indicates insensitivity to antibiotics; - indicates sensitivity to antibiotics The above results show that among the Propionibacterium acnes pa01 - pa10 screened and numbered from the facial skin surface of healthy people, only the preserved strain Propionibacterium acnes VB208 (number: pa02) does not have drug resistance to conventional antibiotics. Due to its sensitivity to antibiotics, it is safer when used in skin drugs or care products.

[0113] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0114] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A strain of Propionibacterium acnes ( Propionibacterium acnes )VB208, characterized in that, The Propionibacterium acnes was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on February 20, 2023, with the deposit number CGMCC No.26576.

2. A composition, characterized in that It comprises the Propionibacterium acnes VB208 described in claim 1.

3. A microbial agent, characterized in that: The method comprises the Propionibacterium acnes VB208 according to claim 1 or the composition according to claim 2.

4. Use of the Propionibacterium acnes VB208 according to claim 1, the composition according to claim 2 or the microbial agent according to claim 3 in the preparation of daily chemicals or medicines.

5. Use of the Propionibacterium acnes VB208 according to claim 1 in genetic engineering or metabolic engineering.

6. An engineered strain expressing a target gene, characterized in that: The engineered strain is obtained by the following method: The vector containing the target gene expression cassette is introduced into the Propionibacterium acnes VB208 described in claim 1 to obtain an engineered strain capable of expressing the target gene, wherein the vector containing the target gene expression cassette has upstream and downstream homology arms designed for the genome of the Propionibacterium acnes VB208 described in claim 1.

7. A method for producing exogenous protein, characterized in that: The method comprises: Using the engineered strain for expressing the target gene according to claim 6 to express the exogenous protein; Wherein, the target gene encodes the expression of the exogenous protein.

8. Use of the engineered strain expressing the target gene according to claim 6 in the expression of foreign proteins, characterized in that: The target gene encodes the expression of the exogenous protein.

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