Ginsenoside CK-producing Chryseobacterium sp. PGOD6479 strain and application thereof

Abstract

The invention provides a Chryseobacterium sp. PGOD6479 strain for producing ginsenoside CK, and the strain is preserved in the China Center for Type Culture Collection in Wuhan University, Wuhan, China, and has the preservation number of CGMCC NO.79. The invention also provides a method for preparing the Chryseobacterium sp. PGOD6479 strain for producing ginsenoside CK. The strain is preserved in China Center for Type Culture Collection (CCTCC), the postal code is 430072, the preservation date is November 11, 2024, and the preservation number is CCTCC NO: M20242515; the 16S rDNA (ribosomal deoxyribonucleic acid) sequence of the strain is as shown in SEQ ID NO. 1. The PGOD6479 strain can be used for producing ginsenoside CK, and the yield is 6.8 g / L. The strain can be used for mass production of ginsenoside CK in a short time, the conversion condition is mild, the yield is relatively high, and the CK can be produced by fermentation of a substrate to the maximum extent. The method for converting and producing ginsenoside CK by using the strain is low in cost and quick in reaction, and has obvious advantages in a large-scale fermentation production process.

Description

Technical Field

[0001] The invention relates to a Chryseobacterium chrysenii strain which can produce ginsenoside CK and belongs to the technical field of microorganisms. Background Art

[0002] Ginseng (Pannax gxinseng CAMeyer), also known as goblin, magic grass, and yellow ginseng, is a perennial herbaceous medicinal plant of the genus Panax in the Araliaceae family. The medicinal parts are the roots and rhizomes. It has magical effects such as replenishing vital energy, restoring pulse and strengthening deficiency, tonifying the spleen and lungs, promoting body fluid and nourishing blood, calming the mind and improving intelligence. Ginseng cultivation has a history of more than 400 years. In recent years, with the further increase in market demand, artificial domestication and cultivation is the only way to meet market demand. However, in recent decades, due to over-exploitation and the destruction of its habitat, the number of wild ginseng resources has sharply decreased. Artificially cultivated ginseng also has problems such as a sharp decline in quality and mixed varieties. Therefore, it is particularly important to find reasonable and effective ways to promote the sustainable development of the ginseng industry.

[0003] Ginsenoside is the main active medicinal ingredient of ginseng. Due to the different types and contents of ginsenoside monomers, the pharmacological effects of various monomer saponins are different. Therefore, isolating the effective monomers and clarifying the pharmacological activities of each monomer have become a research hotspot in this field. At present, more than 60 known ginsenosides have been discovered, and each has unique medicinal functions, mainly divided into two categories: ① Protopanaxadiol (PPD) includes ginsenosides Ra1, Ra2, Rb1, Rb2, Rb3, Rc, Rd, Rg3 and Rh2; ② Protopanaxantriol saponins (PPT) include ginsenosides Re, Rg1, Rg2, Rf and Rh1.

[0004] Ginsenoside CK is a high-value rare ginsenoside that can be obtained by biotransformation of primary ginsenoside Rb1. The theoretical transformation pathway is Rb1→Rd→F2→CK. Rare ginsenoside CK exhibits a variety of important biological activities and pharmacological effects in the field of human health. At present, researchers have screened microbial strains with efficient transformation capabilities, such as specific types of bacteria and fungi, and used the enzymes produced during their fermentation process to transform ginsenosides. A variety of microorganisms have been shown to have the ability to transform ginsenosides into rare ginsenoside CK. However, there are currently no reports on the production of rare ginsenoside CK using other microbial fermentation. Summary of the invention

[0005] The present invention solves the problem in the background technology and provides a Chryseobacterium PGOD6479 strain producing ginsenoside CK. The strain can produce ginsenoside CK with a high fermentation yield of 6.8 g / L.

[0006] The inventors screened a new strain, which was named PGOD6479, a chryseobacterium sp., and was deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, the deposit date is November 11, 2024, and the deposit number is: CCTCC NO: M20242515; the 16S rDNA sequence of the strain is shown in SEQ ID NO.1.

[0007] The above-mentioned Chryseobacterium PGOD6479 strain can be used for fermentation to prepare ginsenoside CK. The microbial fermentation conditions are: culture medium formula: NaCl 12g / L, yeast extract 15g / L, peptone 15g / L, pH 9.0; culture conditions: 35°C, shaking speed 150r / min.

[0008] Compared with the prior art, the present invention has the following advantages: the Chryseobacterium PGOD6479 strain provided by the present invention can produce ginsenoside CK with a yield of 6.8 g / L. The strain can produce ginsenoside CK in large quantities in a short period of time, the conversion conditions are mild, the yield is high, and the substrate fermentation production of CK can be maximized. In the reaction of the strain producing ginsenoside CK, the target product is clear, other impurities have less interference, and it is easier to prepare ginsenoside CK. The cost of strain transformation to produce ginsenoside CK is low, the reaction is fast, and it can have obvious advantages in the large-scale fermentation production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the first HPLC test chart of ginsenoside CK standard;

[0010] Figure 2 This is the first HPLC detection chart of ginsenoside CK in the fermentation broth of Chryseobacterium PGOD6479 strain;

[0011] Figure 3 This is the second HPLC test chart of ginsenoside CK standard;

[0012] Figure 4 This is the second HPLC detection chart of ginsenoside CK in the fermentation broth of Chryseobacterium PGOD6479 strain. DETAILED DESCRIPTION

[0013] The present invention is described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0014] Isolation and cultivation of strains

[0015] In this embodiment, the root tissue of fresh wild ginseng collected from Changbai Mountain was surface disinfected with 75% ethanol for 20 minutes, then washed with sterile water for 3-5 times, placed in a pre-sterilized standby mortar, and ground into a white suspension with 5 mL of sterile water. Take 300 μL of the suspension in each LA plate culture medium, spread it evenly with a sterile coating rod, seal it with a sealing film after air drying, and invert it in a 37°C constant temperature incubator for 3-5 days, observe it in time, and take photos and record it on the 1st, 3rd, and 5th days. If the bacteria grow all over the plate, they will no longer be separated and sterilized. Use a toothpick to pick the bacteria grown on the plate and purify them repeatedly on the LB solid culture medium plate until a single colony is obtained.

[0016] At the same time, wash the stems, remove the necrotic surface hard parts, and then cut them into 1cm 2 The thin slices were soaked in 75% alcohol for 3-5 minutes, then washed with sterile water for 2-3 times to remove the residual alcohol, and then soaked in sterile water for about 5 minutes. After taking out, the surface moisture was absorbed with sterilized filter paper. The processed small pieces were pasted on LA solid plates, 5 pieces were placed on each plate, sealed with sealing film, and cultured in a constant temperature incubator at 37℃ for 3-5 days. Observe and record every day, and purify the grown bacteria repeatedly.

[0017] The purified bacteria on the plate were picked into a 1.5 mL EP tube containing 600 μL LB liquid culture medium, sealed with a sealing film, and then cultured in a 37°C constant temperature shaker at 190 r / min for 1 day, and then an equal volume of 50% glycerol was added, mixed and stored in a -80°C ultra-low temperature refrigerator. At least 6 copies of each strain were stored.

[0018] Screening of strains

[0019] Take 10 μL of the preserved strain and inoculate it into 1000 μL LB liquid medium, and activate it in a 37℃ constant temperature shaker at 180 r / min. After 24 hours, take 1 mL of the activated strain and ferment it in LB liquid medium in a triangular flask, with a liquid volume of 100 mL / 250 mL, and culture it in a 37℃, 180 r / min shaker until OD 600 =0.8, and then switched to purple light culture. After 12 h of fermentation culture, the fermentation broth was collected.

[0020] The first test of ginsenoside CK in the fermentation broth samples of each strain was conducted, and its spectrum and data were compared with the standard product data to roughly determine whether the strain has the ability to produce ginsenoside CK. Then, based on the standard curve of ginsenoside CK, the ginsenoside CK content in the sample and the yield of ginsenoside CK in the fermentation broth of the strain can be calculated. In order to avoid possible interference from other substances, the applicant conducted a second confirmatory qualitative test on the positive strain fermentation broth obtained in the first test after changing the chromatographic detection conditions, including the detection of the standard product and the detection of the sample.

[0021] After the above tests, a strain of ginseng endophyte that can produce ginsenoside CK was screened and named PGOD6479. The yield of ginsenoside CK in the fermentation broth of this strain was 6.8 g / L. The first HPLC test showed Figure 1 and Figure 2 As shown; the second HPLC test is as Figure 3 and Figure 4 shown.

[0022] Extraction and electrophoresis detection of endophytic bacterial DNA

[0023] Primer sequences for 16S rDNA:

[0024] Upstream primer: 515F (5'-GTGYCAGCMGCCGCGGTAA-3')

[0025] Downstream primer: 806R (5'-GGACTACNVGGGTWTCTAAT-3')

[0026] The 16S rDNA PCR product was purified and recovered using the Sigma NA1020 PCR product recovery kit.

[0027] Ligation of 16S rDNA fragment and cloning vector: Ligate the recovered 16S rDNA fragment with the pMD18-T Vector vector, flick to mix, and then ligate at 16°C for 6 hours or 4°C for 12 hours. Transform competent cells with the ligation product.

[0028] Bacterial liquid PCR-clone detection

[0029] Universal primers were used to perform bacterial liquid PCR to determine whether the target fragment was successfully transformed and to screen positive clones.

[0030] Primer sequence: M13R: CAGGAAACAGCTATGACC

[0031] M13F:TGTAAAACGACGGCCAGT

[0032] A conventional amplification reaction system and amplification procedure were used, wherein 2 μL of bacterial solution was used to replace the original 3 μL of template in the reaction system. 10 μL of amplification product was taken for agarose gel electrophoresis detection, and the corresponding positive clones were packaged and preserved.

[0033] Sequencing, sequence alignment and analysis

[0034] The positive clones were sent to a biotechnology company for sequencing, and the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1. The sequencing results were subjected to Blast similarity analysis in the GenBank nucleic acid database. After Blast sequence alignment and phylogenetic tree analysis, the homology of PGOD6479 strain with Chryseobacterium sp. strain BOTRUEML133 was 99%, and the homology with Chryseobacterium aureum strain 17S1E7 was 99%. Combined with the phylogenetic tree, it was determined to be Chryseobacterium sp.

[0035] Deposit of strains:

[0036] The strain was deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, the deposit date is November 11, 2024, and the deposit number is: CCTCC NO: M20242515.

[0037] Optimization of fermentation conditions

[0038] The fermentation medium components of microorganisms must be able to meet the needs of rapid growth and reproduction of the strains, and at the same time, the optimal fermentation culture conditions can ensure the accumulation of a large number of secondary metabolites. The applicant optimized the fermentation medium components of the PGOD6479 strain through single-factor and orthogonal experiments. The optimized optimal medium formula is: NaCl 12g / L, yeast extract 15g / L, peptone 15g / L. After determining the culture medium components and corresponding concentrations, the optimal initial pH value, culture temperature and shaker speed for the strain to transform ginsenosides were further optimized, and the optimal culture conditions for the HGS-487 strain were finally determined to be: initial pH 9.0, culture temperature 31°C, and shaker speed 150r / min.

Claims

1. A chryseobacterium sp. PGOD6479 strain, the deposit number of which is CCTCCNO: M20242515, and the 16S rDNA sequence of which is shown in SEQ ID NO.

1.

2. The use of the chryseobacterium sp. PGOD6479 strain according to claim 1, characterized in that: Used for fermentation to prepare ginsenoside CK.

3. The use according to claim 2, characterized in that: The fermentation conditions were as follows: culture medium formula: NaCl 12 g / L, yeast extract 15 g / L, peptone 15 g / L, pH 9.0; culture conditions: 35°C, shaking speed 150 r / min.

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