Propionibacterium acnes vb208 gene editing method and applications thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a gene editing method of propionibacterium acnes VB208 and application thereof. Propionibacterium acnes ) on February 20, 2023, and is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.26576. The propionibacterium acnes screened by the present application has the characteristics of skin surface occupation, high conversion efficiency, gene modification potential and efficient expression of exogenous proteins, and can be used as an engineering strain. Furthermore, a genetic operation system suitable for the strain can be established by using the propionibacterium acnes, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a method for editing the VB208 gene of Propionibacterium acnes and its application. Background Technology

[0002] Propionibacterium acnes ( Propionibacterium acnes Propionibacterium acnes is one of the most common bacteria on human skin, typically found in the pilosebaceous unit. Traditionally, it was considered a pathogen due to its association with acne development; however, with advancements in genomics and microbiology, it has become increasingly clear that significant genetic background differences exist among different strains of Propionibacterium acnes, which may lead to different roles in skin health and disease.

[0003] Although numerous studies have described the pathogenic characteristics of *Propionibacterium acnes*, its beneficial aspects, especially the potential benefits of specific strains, have not been fully characterized. Related research shows that certain *Propionibacterium acnes* strains may be beneficial to skin health and even antagonistic to infections caused by other pathogens (such as *Staphylococcus aureus*), suggesting that specific *Propionibacterium acnes* strains can be used as beneficial bacteria to address skin problems. However, the lack of a genetic manipulation system suitable for these beneficial strains limits their widespread application in the biotechnology and medical fields.

[0004] Therefore, finding a specific Propionibacterium acnes strain that can occupy the skin surface, has high conversion efficiency, and efficiently express exogenous proteins, and establishing a genetic manipulation system suitable for this strain, is of great significance for the application of beneficial Propionibacterium acnes bacteria in the development of novel skin health-related products and treatments. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides a method for gene editing of Propionibacterium acnes VB208 and its applications. The Propionibacterium acnes strain screened by this invention possesses characteristics such as skin surface occupancy, high transformation efficiency, genetic modification potential, and efficient expression of exogenous proteins. Furthermore, it can be used as an engineered strain, and a genetic manipulation system suitable for this strain can be established, showing broad application prospects.

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

[0007] Preservation information:

[0008] Strain name: VB208

[0009] Classification: Propionibacterium acnes

[0010] Deposit date: February 20, 2023

[0011] Deposit unit: China General Microbiological Culture Collection Center

[0012] Deposit address: No. 3, Beichen West Road, Chaoyang District, Beijing, China, 100101

[0013] Deposit number: CGMCC No. 26576

[0014] The new strain of Propionibacterium acnes (P. acnes) VB208 screened by the application has the characteristics of skin surface occupation, high conversion efficiency, genetic modification potential, and efficient expression of exogenous proteins, and can be used as an engineering strain, and a genetic operation system suitable for the strain can be established using the strain, and the application prospect is wide. Propionibacterium acnes

[0015] In a second aspect of the application, a composition is provided. According to embodiments of the application, the composition comprises the P. acnes VB208 of the first aspect.

[0016] It will be appreciated by persons skilled in the art that the features and advantages described above for the P. acnes VB208 also apply to the composition, and will not be repeated here.

[0017] In a third aspect of the application, a microbial inoculant is provided. According to embodiments of the application, the microbial inoculant comprises the P. acnes VB208 of the first aspect or the composition of the second aspect.

[0018] It will be appreciated by persons skilled in the art that the features and advantages described above for the P. acnes VB208 also apply to the microbial inoculant, and will not be repeated here.

[0019] In a fourth aspect of the application, the P. acnes VB208 of the first aspect, the composition of the second aspect, or the microbial inoculant of the third aspect is used in the preparation of a daily chemical product or a pharmaceutical product.

[0020] It will be appreciated by persons skilled in the art that the features and advantages described above for the P. acnes VB208 also apply to the use, and will not be repeated here.

[0021] ​In a fifth aspect, the application provides an application of the P. acnes VB208 in genetic engineering or metabolic engineering. According to an embodiment of the application, the P. acnes VB208 strain screened by the application has high transformation efficiency and genetic modification potential, which indicates that the strain can obtain new functions through gene insertion, replacement or knockout, and provides a technical basis for developing an engineered strain with specific functions.

[0022] In a sixth aspect, the application provides an engineered strain expressing a target gene. According to an embodiment of the application, the engineered strain is obtained by introducing a vector containing a target gene expression cassette into the P. acnes VB208 of the first aspect, to obtain an engineered strain capable of expressing a target gene, wherein the vector containing the target gene expression cassette has upstream and downstream homologous arms designed for the genome of the P. acnes VB208 of the first aspect. According to the engineered strain expressing a target gene of an embodiment of the application, an exogenous gene or protein can be stably and efficiently expressed, and the application prospect is wide.

[0023] In a seventh aspect, the application provides a method for producing an exogenous protein. According to an embodiment of the application, the method comprises: using the engineered strain expressing a target gene of the sixth aspect to express an exogenous protein, wherein the target gene encodes the expression of the exogenous protein. According to the method of an embodiment of the application, the engineered strain obtained by the foregoing method is used to produce an exogenous protein, which provides an efficient and low-cost biosynthesis method, and can be applied to produce proteins with specific functions, such as drugs, bioactive factors, etc., and the application prospect is wide.

[0024] In an eighth aspect, the application provides an application of the engineered strain expressing a target gene of the sixth aspect in expression of an exogenous protein. According to an embodiment of the application, the target gene encodes the expression of the exogenous protein.

[0025] It can be understood by those skilled in the art that the features and advantages described above for the engineered strain expressing a target gene also apply to the application, and will not be described here.

[0026] Additional aspects and advantages of the application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0027] The above and / or additional aspects and advantages of the application will become apparent and be readily understood by considering the following detailed description, including the accompanying drawings, in which:

[0028] Figure 1Electron microscope result of Propionibacterium acnes VB208 in Example 1 of the present application;

[0029] Figure 2 Map of recombinant plasmid p2107-mcherry in Example 3 of the present application. DETAILED DESCRIPTION

[0030] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0031] It should be noted that the terms "first", "second" are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0032] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. Individual endpoints of the ranges, the endpoints of the ranges and individual points, and the individual points can be combined with each other to form one or more new ranges or values, which should be considered as specifically disclosed herein.

[0033] In this document, the terms "comprising" or "including" are open-ended, that is, the inclusion of the indicated steps or components is not a limitation.

[0034] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0035] In this document, the term "milk tube" refers to a litmus milk biochemical tube, which is composed of skim milk powder and litmus, and is used to determine the fermentation ability of bacteria on milk.

[0036] In this document, the term "Propionibacterium acnes" is a gram-positive bacillus, anaerobic or facultative anaerobic, mainly inhabiting in skin hair follicles and sebaceous glands. The new strain of Propionibacterium acnes VB208 is isolated and screened from the surface of the face skin of healthy people, which is not a pathogenic bacterium and has potential probiotic properties.

[0037] Propionibacterium acnes VB208

[0038] This invention proposes a Propionibacterium acnes VB208. According to an embodiment of the invention, the Propionibacterium acnes was deposited on February 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26576.

[0039] According to an embodiment of the present invention, the 16S rDNA sequencing results of Propionibacterium acnes VB208 are shown in SEQ ID NO: 1.

[0040] The Propionibacterium acnes obtained by screening in this invention ( Propionibacterium acnes The new VB208 strain has the characteristics of occupying sites on the skin surface, high transformation efficiency, genetic modification potential and efficient expression of exogenous proteins. It can also be used as an engineered strain and a genetic manipulation system suitable for this strain can be established, with broad application prospects.

[0041] In this article, the term "Propionibacterium acnes" is used. Propionibacterium acnes "VB208" and "Propionibacterium acnes VB208" are synonyms.

[0042] Composition

[0043] This invention proposes a composition. According to embodiments of the invention, it comprises the aforementioned Propionibacterium acnes VB208.

[0044] Those skilled in the art will understand that the features and advantages described above for Propionibacterium acnes VB208 also apply to this composition, and will not be repeated here.

[0045] Microbial agents

[0046] This invention provides a microbial inoculant. According to embodiments of the invention, it includes the aforementioned Propionibacterium acnes VB208 or the aforementioned composition.

[0047] In some specific embodiments, the microbial agent may be in the form of either liquid or solid microbial agents. For example, when the microbial agent is in the form of solid microbial agents, it may include, but is not limited to, lyophilized powder.

[0048] It should be noted that in the microbial agent of the present invention, Propionibacterium acnes VB208 can exist in the form of live bacteria and / or non-live bacteria.

[0049] In this article, "live bacterial form" refers to the form in which Propionibacterium acnes VB208 exists and has the ability to metabolize, reproduce, or replicate.

[0050] Exemplarily, the live bacteria can be immobilized bacteria. In the present disclosure, the "immobilized bacteria" refers to the live bacteria immobilized on a carrier, which can grow, develop, reproduce, inherit and metabolize in a certain spatial range.

[0051] In the present disclosure, the "non-live bacteria form" refers to the form of P. acnes VB208 that does not have the ability of metabolism, reproduction and replication, including but not limited to dry bacteria, and exemplarily, the microbial agent is a freeze-dried powder.

[0052] In some specific embodiments, the P. acnes VB208 is in the form of live bacteria, dry bacteria, immobilized bacteria or any other form.

[0053] In some specific embodiments, the dry bacteria are obtained by freeze-drying treatment of the P. acnes VB208.

[0054] It is understood by those skilled in the art that the features and advantages described above for the P. acnes VB208 also apply to the microbial agent, which will not be repeated herein.

[0055] Use

[0056] The present disclosure provides the use of the aforementioned P. acnes VB208, the aforementioned composition or the aforementioned microbial agent in the preparation of daily-use or pharmaceutical products.

[0057] It is understood by those skilled in the art that the features and advantages described above for the P. acnes VB208 also apply to the use, which will not be repeated herein.

[0058] Application in genetic engineering or metabolic engineering

[0059] The present disclosure provides the application of the aforementioned P. acnes VB208 in genetic engineering or metabolic engineering. According to the embodiments of the present disclosure, the newly screened P. acnes VB208 strain has high transformation efficiency and gene modification potential, which indicates that it can obtain new functions through gene insertion, replacement or knockout, and provides a technical basis for the development of engineering strains with specific functions.

[0060] Engineering strains expressing target genes

[0061] The application provides an engineered strain for expressing a target gene. According to an embodiment of the application, the engineered strain is obtained by introducing a vector containing a target gene expression cassette into the aforementioned P. acnes VB208, wherein the vector containing the target gene expression cassette has upstream and downstream homologous arms designed according to the genome of the aforementioned P. acnes VB208, so as to obtain the engineered strain capable of expressing the target gene. The engineered strain for expressing the target gene according to the embodiment of the application can stably and efficiently express an exogenous gene or protein, and has a wide application prospect.

[0062] In some specific embodiments, the method for introducing the vector containing the target gene expression cassette into the aforementioned P. acnes VB208 can include conjugative transfer, electroporation and chemical transformation.

[0063] For example, when the target gene is a gene for expressing a fluorescent protein, the method of conjugative transfer can be selected to construct the engineered strain for expressing the gene for expressing the fluorescent protein, and at this time, the Escherichia coli with a clear genetic background, simple technical operation and simple culture condition can be preferentially selected as the donor bacteria, so as to further improve the construction efficiency and success rate of the engineered strain for expressing the target gene.

[0064] Method for producing an exogenous protein

[0065] The application provides a method for producing an exogenous protein. According to an embodiment of the application, the method comprises: using the aforementioned engineered strain for 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 application, the engineered strain obtained by the aforementioned method is used to produce the exogenous protein, so as to provide an efficient and low-cost biosynthesis method, which can be applied to produce proteins with specific functions, such as drugs and bioactive factors, and has a wide application prospect.

[0066] Application of the engineered strain for expressing a target gene in expression of an exogenous protein

[0067] The application provides the application of the aforementioned engineered strain for expressing a target gene in expression of an exogenous protein. According to an embodiment of the application, the target gene encodes the expression of the exogenous protein.

[0068] It can be understood by those skilled in the art that the features and advantages described above for the engineered strain for expressing a target gene are also applicable to the application, and will not be repeated here.

[0069] Unless otherwise specified, the 22 agar medium (22 solid medium) used in the examples of the present application has the following formulation (g / L): hematin chloride 0.001 wt%, Tween 80 0.1 wt%, tryptone 0.82 wt%, bacterial peptone 0.25 wt%, soybean peptone 0.1 wt%, glucose 0.58 wt%, yeast extract powder FM860 0.5 wt%, sodium chloride 0.17 wt%, L-cystine 0.04 wt%, agar 1.5 wt%, Tris-HCl 0.3 wt%, and the pH of the prepared medium is adjusted to 7.5, and the medium is sterilized at 121°C for 30 min.

[0070] Unless otherwise specified, the 22 liquid medium used in the examples of the present application has the following formulation (g / L): tryptone 0.82 wt%, bacterial peptone 0.25 wt%, soybean peptone 0.1 wt%, glucose 0.58 wt%, yeast extract powder 0.5 wt%, sodium chloride 0.17 wt%, disodium hydrogen phosphate 0.08 wt%, L-cystine 0.04 wt%, hematin chloride 0.001 wt%, Tween 80 0.1 wt%, Tris-HCl 0.3 wt%, and the pH of the prepared medium is adjusted to 7.5, and the medium is sterilized at 121°C for 30 min.

[0071] The solutions of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer is specified for the reagent or instrument used, it is a conventional product that can be commercially available.

[0072] Example 1: Obtaining of Propionibacterium acnes VB208

[0073] The Propionibacterium acnes VB208 of the present application is obtained by isolation and screening from the surface of the facial skin of healthy people.

[0074] Collection and isolation process of the strain: a sterile sampling cotton swab was immersed in 3 mL of sterile 40% glycerol for 30 s, and then the facial skin was wiped with the sterile sampling cotton swab for 30 s, and then the cotton swab was placed in the glycerol tube and stirred for 30 s to obtain the sample. 1 mL of the sample was gradiently diluted to 10 -2 、10 -4 、10 -6, respectively, 100 μL of each was uniformly coated on a 22 agar medium plate, and after single colonies grew, multiple streaking and purification culture was repeated until the colony morphology in the plate was consistent, and after no mixed bacteria were observed under a microscope, the isolated and purified strains were numbered pa01~pa10, and the strain numbered pa02 was named VB208. Morphological identification of the strain was performed by electron microscopy observation, and the classification of the strain was further determined by combining the PCR amplification and sequencing analysis results of the 16S rDNA gene of the strain.

[0075] The electron microscopy results of the P. acnes VB208 are shown in Figure 1 .

[0076] The strain VB208 was sent to Hangzhou Qikexi Biotechnology Co., Ltd. for 16S rDNA sequence detection.

[0077] The 16S rDNA sequencing results of the P. acnes VB208 are shown in SEQ ID NO: 1.

[0078]

[0079] The results show that: comparing the 16S rRNA sequence of strain VB208 with the strains in GenBank, the homology of strain VB208 with Propionibacterium acnes IGM5-4 is the highest, which is 99%. Propionibacterium acnes

[0080] Finally, it is identified that the isolated strain VB208 is a strain of Propionibacterium acnes, named Propionibacterium acnes VB208, and preserved in the "China General Microbiological Culture Collection Center" on February 20, 2023, the address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 26576.

[0081] Example 2: Physiological and biochemical characteristics identification of Propionibacterium acnes VB208

[0082] The 22 agar medium was used to culture Propionibacterium acnes VB208, and then the physiological and biochemical characteristics of Propionibacterium acnes VB208 were identified according to the "Berger's Bacterium Identification Manual", and the detection contents were as follows:

[0083] 1. Contact enzyme experiment

[0084] After normal 24 h culture, a small ring of Propionibacterium acnes VB208 colony was taken with a sterile inoculation ring and smeared on a glass slide with 3% hydrogen peroxide, and observed immediately. If bubbles are produced, it is positive (+), and if there are no bubbles, it is negative (-).

[0085] 2. Glucose semi-solid experiment

[0086] Glucose semi-solid medium, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., product number GB025.

[0087] Preparation of Propionibacterium acnes VB208 bacterial suspension: take the glycerol tube stored in the-80℃ refrigerator, after thawing, take 100 μl of bacterial solution in the 22 medium, culture at 37℃ for 24 h, the concentration of bacterial solution is 10 8 cfu / ml.

[0088] Add 100 μL of Propionibacterium acnes VB208 bacterial suspension (concentration of 10 8 cfu / ml) to a biochemical tube containing sufficient glucose semi-solid medium, and culture at 37℃ for 24 h. If it is yellow, it is positive (+), and if it is gray purple, purple or purple red, it is negative (-).

[0089] 3. Glucose fermentation experiment

[0090] ​Glucose fermentation medium, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., item number GB117.

[0091] The preparation of P. acnes VB208 bacterial suspension was the same as the "Preparation of P. acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0092] Add 100 μL of P. acnes VB208 bacterial suspension (concentration of 10 8 cfu / ml) to the biochemical tube containing sufficient glucose fermentation medium, and incubate at 37°C for 24 h. If yellow color appears, it is positive (+), and if blue or blue-green color appears, it is negative (-).

[0093] 4. Milk decomposition experiment

[0094] Take a test tube containing 2 mL of sterile water, use the inoculation needle to pick 3 P. acnes VB208 colonies from the plate into the test tube containing sterile water, and carefully grind to prepare a bacterial suspension. Add 100 μL of the bacterial suspension to the milk tube. Place the milk tube at 37°C for 24 h. After incubation, observe the changes. If the milk layering changes to pink and solidifies, it is positive (+), and if there is no color change, it is negative (-).

[0095] 5. Indole experiment

[0096] Proteose peptone water medium, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., item number HB5218.

[0097] The preparation of P. acnes VB208 bacterial suspension was the same as the "Preparation of P. acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0098] Add 100 μL of P. acnes VB208 bacterial suspension (concentration of 10 8 cfu / ml) to the proteose peptone water medium, and incubate at 37°C for 24 h. Then add 0.2 mL of Kovacs indigo substrate reagent. If red color appears in the upper layer, it is an indigo substrate positive reaction (+), otherwise it is a negative reaction (-).

[0099] 6. Cellobiose / maltose / sucrose fermentation experiment

[0100] Cellobiose biochemical tube, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., item number SN067.

[0101] Maltose fermentation tube, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., item number GB079.

[0102] Sucrose fermentation tube, purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., item number GB169.

[0103] The preparation of P. acnes VB208 bacterial suspension was the same as the "Preparation of P. acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0104] 100 μL of P. acnes VB208 bacterial suspension (10 8 cfu / ml) was added to the cellobiose biochemical tube, the maltose fermentation tube, and the sucrose fermentation tube, respectively, and incubated at 37°C for 24 h. If it was yellow, it was positive (+), and if it was purple or purple-gray, it was negative (-).

[0105] 7. L-arabinose fermentation experiment

[0106] L-arabinose biochemical tubes were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., with the item number GB188.

[0107] The preparation of P. acnes VB208 bacterial suspension was the same as the "Preparation of P. acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0108] 100 μL of P. acnes VB208 bacterial suspension (10 8 cfu / ml) was added to the L-arabinose biochemical tube, and incubated at 37°C for 24 h. If it was yellow, it was positive (+), and if it was blue or blue-green, it was negative (-).

[0109] 8. Glycerol fermentation experiment

[0110] Glycerol biochemical tubes were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., with the item number GB064.

[0111] The preparation of P. acnes VB208 bacterial suspension was the same as the "Preparation of P. acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0112] 100 μL of P. acnes VB208 bacterial suspension (10 8 cfu / ml) was added to the glycerol biochemical tube, and incubated at 37°C for 24 h. If it was yellow, it was positive (+), and if it was blue or blue-green, it was negative (-).

[0113] 9. Gelatin liquefaction experiment

[0114] Gelatin biochemical tubes were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., with the item number GB066.

[0115] The preparation of P. acnes VB208 bacterial suspension was the same as the "Preparation of P. acnes VB208 bacterial suspension" section in the glucose semi-solid experiment.

[0116] Add 100 μL of P. acnes VB208 bacterial suspension (concentration of 10 8 8 If it is still liquid, the experimental result is positive (+), otherwise it is negative (-).

[0117] 10. Escin hydrolysis experiment

[0118] Escin biochemical tubes were purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., and the item number was GS021.

[0119] The preparation steps of P. acnes VB208 bacterial suspension were the same as the "preparation of P. acnes VB208 bacterial suspension" in the glucose semi-solid experiment.

[0120] Add 100 μL of P. acnes VB208 bacterial suspension (concentration of 10 8 If it is still liquid, the experimental result is positive (+), otherwise it is negative (-).

[0121] The physiological and biochemical characteristics of P. acnes VB208 are shown in Table 1.

[0122] Table 1 Physiological and biochemical characteristics of P. acnes VB208

[0123]

[0124] The above results show that P. acnes VB208 can produce catalase, can utilize glucose, D-cellobiose and glycerol as carbon sources, and can decompose escin to produce glucose and esculin.

[0125] Example 3: Investigation of conjugation transfer of different strains of P. acnes

[0126] 1. Construction of plasmid p2107-mcherry

[0127] (1) Extraction of genomic DNA of P. acnes VB208

[0128] The bacterial DNA kit D3146-02 from Meiji Bio was used to extract the genomic DNA of P. acnes VB208 according to the extraction steps of the kit. The genomic DNA of P. acnes VB208 was obtained, and the integrity and purity of the DNA were detected by agarose gel electrophoresis.

[0129] (2) PCR amplification of homologous arms

[0130] Upstream and downstream homologous arms (designed based on the common sequence of Propionibacterium acnes VB208) homologous to the genome of Propionibacterium acnes were amplified by PCR for subsequent homologous recombination. After amplification, the homologous arms were obtained and their size and purity were verified by agarose gel electrophoresis.

[0131] (3) Synthesize the erythromycin resistance gene and the Mcherry fluorescent marker gene.

[0132] Based on the codon bias of Propionibacterium acnes VB208, the sequences of the erythromycin resistance gene and the mcherry fluorescent marker gene were optimized and synthesized (commissioned to Nanjing Genscript Biotech Co., Ltd.), and the erythromycin resistance gene and the mcherry fluorescent marker gene were obtained.

[0133] (4) Constructing the recombinant plasmid p2107-mcherry

[0134] Plasmid p2107 contains the gene elements required for conjugation transfer of Gram-positive bacteria. Therefore, plasmid p2107 was selected as the vector, and the homologous arm obtained in step (2), the erythromycin resistance gene obtained in step (3), and the mcherry fluorescent marker gene were connected 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.

[0135] The image of the recombinant plasmid p2107-mcherry can be found in [image / image]. Figure 2 .

[0136] 2. Conjugation transfer experiment

[0137] (1) Selection and treatment of donor strains

[0138] use E. coli S17 and E. coli Using ET12567 as the donor strain, the recombinant plasmid p2107-mcherry obtained in step 1 was transformed into the donor strain via electroporation to obtain the donor strain. E. coli S17' and E. coli ET12567' was cultured in liquid LB medium. After 24 h of culture, donor bacteria were picked separately. E. coli S17' and E. coli Single colonies of ET12567' were inoculated into 50 mL of liquid LB medium (with an additional 400 μg / ml erythromycin antibiotic) and incubated at 37°C for 18 h; then, 2% of each colony was transferred into 50 mL of liquid LB medium (with an additional 400 μg / ml erythromycin antibiotic) and cultured at 37°C for 18 h; E. coli S17' andE. coli OD of ET12567' 600 When reaching 1.0 (about 5 h), 50 mL of the donor bacteria E. coli S17' and E. coli The bacteria were collected by centrifugation of ET12567' at 5000 rpm for 10 min, washed twice with 20 mL of liquid LB medium, centrifuged at 5000 rpm for 10 min to obtain a donor bacteria E. coli S17' and the donor bacteria E. coli ET12567' suspension.

[0139] Liquid LB medium preparation (g / L): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, after preparation, adjust the medium pH to 7.5, sterilize at 121°C for 30 min.

[0140] (2) Selection and treatment of the recipient strain

[0141] The recipient strain was Propionibacterium acnes pa01~pa10, among which pa02 was the preserved strain Propionibacterium acnes VB208, and pa01, pa03~pa10 were other strains obtained from the surface of the face skin of healthy people and numbered in the same batch; the recipient strains pa01~pa10 were inoculated into No. 22 liquid medium and cultured at 37°C for 24 h, then centrifuged and washed twice, and the recipient bacteria pa01~pa10 were resuspended in No. 22 liquid medium to obtain resuspension 1~10 of the recipient bacteria, respectively.

[0142] (3) Conjugation transfer

[0143] The resuspension 1~10 of the recipient bacteria was heat-shocked (metal bath incubation at 40°C) for 1 min, and then mixed with the donor bacteria E. coli S17' suspension or the donor bacteria E. coli ET12567' suspension according to a ratio of 1:10 of the recipient bacteria and the donor bacteria, and the number of the recipient bacteria after mixing was 10 8 CFU, the mixed solution was respectively spotted on dry No. 22 solid medium plates (additional 10 mM MgCl2 was added), and placed in a 37°C anaerobic incubator for a total of 24 h, and then transferred to resistance screening plates for counting the number of transformants, wherein the conjugation transfer frequency = the number of transformants / the number of recipient bacteria.

[0144] The conjugation transfer results of different strains of Propionibacterium acnes are shown in Table 2.

[0145] Table 2 Conjugation transfer results of different strains of Propionibacterium acnes

[0146]

[0147] The results show that: the higher the frequency of conjugation transfer indicates the higher the transformation efficiency, only strain P. acnes pa02 (P. acnes VB208) can be genetically manipulated, and the conjugation transfer efficiency is the highest.

[0148] Example 4: Detection of fluorescent protein expression content of recombinant strain P. acnes VB208'

[0149] P. acnes pa02 (P. acnes VB208) was selected as the recipient strain, E. coli S17 as the donor strain, repeat the conjugation transfer experiment steps in Example 2, and when the culture is 24 h, use 1 mL of sterile water to wash the P. acnes pa02- E. coli S17' mixed bacteria from the plate, inoculate the mixed bacteria on No. 22 solid culture medium plate (additional addition of 10 μg / mL erythromycin and 25 μg / mL naltrexone acid), and incubate at 37°C anaerobically for 6 d to obtain recombinant strain pa02' (P. acnes VB208'), which is observed under iBright™ FL1500 imaging system, and the excitation wavelength is set to 587 nm. Red fluorescence can be observed from the recombinant strain pa02', indicating that the recombinant plasmid p2107-mcherry has been successfully integrated into the target strain P. acnes pa02. Then the fluorescent protein expression content of the recombinant strain P. acnes VB208' is detected, and the specific method is as follows:

[0150] The recombinant strain P. acnes VB208' (experimental group) and the strain P. acnes VB208 (control group) were inoculated into No. 22 liquid medium, respectively. After 6 d of culture, 1 mL of bacterial solution was taken, centrifuged to obtain bacterial precipitate, resuspended with PBS buffer, broken, centrifuged to obtain supernatant, and then diluted 100 times and detected for fluorescent protein content using mCherry ELISA kit (product number: ab221829).

[0151] The results of the fluorescent protein content detection are shown in Table 3.

[0152] Table 3: Results of fluorescent protein content detection

[0153]

[0154] The above results show that the recombinant strain P. acnes VB208' can highly express exogenous proteins.

[0155] Example 5: Investigation of antibiotic sensitivity of different strains of P. acnes

[0156] The P. acnes pa01~pa10 were inoculated into No. 22 liquid medium (additional 10 mg / mL of antibiotic) respectively, wherein pa02 was the preserved strain P. acnes VB208, and P. acnes pa01, pa03~pa10 were other strains screened from the facial skin surface of healthy people and numbered in the same batch; the OD was measured after 37°C culture for 42 h 600 , so as to verify the sensitivity of P. acnes pa01~pa10 to erythromycin, tetracycline and cephalosporin, wherein the OD 600 greater than or equal to the OD of the control group was antibiotic-insensitive (+), that is, the strain could grow normally in the culture medium containing antibiotics, had drug resistance ability, and the OD 600 less than the OD of the control group was antibiotic-sensitive (-), that is, the strain could not grow normally in the culture medium containing antibiotics, and had no drug resistance ability; the control group culture medium was No. 22 liquid medium without adding any antibiotic. 600 600 The control group culture medium was No. 22 liquid medium without adding any antibiotic.

[0157] The results of the investigation of the antibiotic sensitivity of different strains of P. acnes are shown in Table 4.

[0158] Table 4 Investigation results of antibiotic sensitivity of different strains of P. acnes

[0159]

[0160] In Table 4: + is antibiotic-insensitive; - is antibiotic-sensitive

[0161] The above results show that among P. acnes pa01~pa10 screened from the facial skin surface of healthy people and numbered, only the preserved strain P. acnes VB208 (numbered: pa02) does not have drug resistance to conventional antibiotics, and it is more safe when used in skin drugs or care products due to its sensitivity to antibiotics.

[0162] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like 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 application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0163] ​Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that variations, modifications, substitutions and changes can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A strain of Propionibacterium acnes (P. acnes) VB208, characterized in that, Propionibacterium acnes The Propionibacterium acnes was deposited in the China General Microbiological Culture Collection Center on February 20, 2023, and the preservation number is CGMCC No. 26576. ​ 2. A composition characterized in that, The Propionibacterium acnes VB208 of claim 1.

3. A microbial inoculant, characterized in that, The Propionibacterium acnes VB208 of claim 1 or the composition of claim 2.

4. Use of the Propionibacterium acnes VB208 of claim 1, the composition of claim 2, or the microbial inoculum of claim 3 in the preparation of a daily or pharmaceutical product.

5. Application of the Propionibacterium acnes VB208 of claim 1 in genetic engineering or metabolic engineering.

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

7. A method for producing a foreign protein, characterized by, The method comprises: Using the engineered strain expressing the target gene of 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 a gene of interest 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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