Method for preparing rare ginsenoside CK by using combined transformation of genes and application thereof

Abstract

The application discloses a method for preparing rare ginsenoside CK by gene combination transformation and application. Bacillus subtilis str. 168)and bifidobacterium ( Bifidobacterium breve 689b)can efficiently transform ginsenoside Rc into rare ginsenoside CK in vitro by using the glycosidase produced by the genes of the bacteria.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a method for preparing rare ginsenoside CK by gene combination transformation and application, in particular to a method for preparing rare ginsenoside CK by transforming ginsenoside Rc using Escherichia coli co-expressing Bacillus subtilis alpha-L-arabinofuranosidase and Bifidobacterium beta-glucosidase. BACKGROUND

[0002] Ginsenosides are the most important active ingredients in ginseng, and belong to tetracyclic triterpenoids. According to the types and numbers of glycosidic bonds at C-3, C-6 and C-20 positions of the aglycone, ginsenosides can be divided into dammarane-type and oleanolic acid-type saponins. Dammarane-type saponins can be further divided into protopanaxadiol-type saponins (PPD-type saponins, mainly including Rb1, Rb2, Rc and Rd, etc.) and protopanaxatriol-type saponins (PPT-type saponins, including Re and Rg1, etc.). According to the content of ginsenosides in plants, ginsenosides can be divided into major ginsenosides (such as Rb1, Rb2, Rc, Rd, Re and Rg1, etc.) and rare ginsenosides (such as Rg3, Rh1, Rh2, F1, F2, CK, C-O, CY and C-Mc, etc.). So far, more than 100 kinds of ginsenosides have been found in ginseng plants. However, Rb1, Rb2, Rc, Rd, Re and Rg1 account for more than 80% of the total saponins. These major ginsenosides usually contain 3-4 glycosyl groups, and it is difficult for them to be directly absorbed by the human body due to the high number of glycosyl groups, resulting in a generally lower physiological activity than that of rare ginsenosides with fewer glycosyl groups.

[0003] Among the currently known ginsenosides, low-glycosyl rare ginsenosides such as F2, Rg3, Rh2 and CK have a high absorption rate in the human body. These rare ginsenosides have important application values in anticancer, antitumor, antithrombus, immune enhancement, anti-inflammatory, anti-aging, anti-diabetes and anti-anxiety, prevention and relief of senile dementia, etc. Studies have shown that ginsenoside CK has unique advantages in anti-cell mutation, inhibition of tumor cell metastasis, induction of tumor cell apoptosis, reversal of tumor cell drug resistance and anti-tumor induced angiogenesis, which cannot be replaced by other ginsenosides. In combination with radiotherapy and chemotherapy, it can enhance the effect of radiotherapy and chemotherapy. In addition, ginsenoside CK also has anti-allergic and anti-inflammatory activities, and can play a neuroprotective role, an anti-diabetic role and an anti-skin aging role. The pharmacological activity of ginsenoside CK has the characteristics of multi-target, high activity and low toxicity.

[0004] In order to obtain high activity of rare ginsenosides with low sugar content, there are a large number of research reports on the use of intestinal bacteria, microorganisms and cloned enzymes transformation. It is found that ginsenoside CK is the main metabolic product of protopanaxadiol saponins in the human intestinal tract after taking. And most of the protopanaxadiol saponins can only be absorbed by the human body after being metabolized into CK, so ginsenoside CK is the real molecule of protopanaxadiol saponins in the human body to play a pharmacological role, and other diol-type ginsenosides are only drug precursors. Although ginsenoside CK plays a very important pharmacological activity in the human body, but due to their extremely rare content in nature, it is very difficult to separate them from plants such as ginseng by traditional methods, which has greatly limited the use of rare ginsenoside CK.

[0005] According to the structure of the main ginsenosides, the main difference between rare ginsenosides is the type and position of the sugar group. At present, the research on the preparation of rare ginsenosides mainly adopts chemical hydrolysis method and glycosidase biotransformation method. We know that the chemical hydrolysis method has poor selectivity, low yield, is not easy to purify, and is easy to cause environmental pollution. The use of specific types of glycosidases produced by organisms to transform and prepare rare ginsenosides has the advantages of good selectivity, high yield, less by-products, no pollution and easy to scale production. Although the method of biological transformation needs to go through a relatively complex way, but rare ginsenosides such as CK which are difficult to obtain by chemical method can be obtained.

[0006] A large number of studies have been conducted on the production of rare ginsenosides by using glycosidases produced by organisms. At present, the studies mainly focus on the use of strains or enzymes screened from soil microorganisms, fungi and other microorganisms to convert major ginsenosides, and the research and optimization of conversion conditions to achieve a high level of preparation of rare ginsenosides. Hong et al. obtained a fungus Monascus pilosus KMU103 through screening, and used it to convert ginsenosides in red ginseng, obtaining a series of rare ginsenosides such as Rh1, Rh2, Rg3, etc. Cheng et al. screened Caulobacter leidyia GP45 from soil, and found that the β-glucosidase thereof can convert Rb1 into CK. Liu et al. found that the ginsenosidase in Aspergillus niger belongs to a kind of glycosidase, which can convert PPD-type major ginsenosides Rb1, Rb2, Rc and Rd into C-O, F2, C-Mc, CY and CK and other rare ginsenosides through different pathways. Bae et al. used intestinal lactic acid bacteria to convert PPD-type ginsenosides to prepare ginsenoside CK, and found that the co-fermentation of B. minimum KK-1 and B. choerinum KK-2 has the best effect on the conversion of Rb1, and the conversion rate can reach about 41%. Although there have been related reports on the preparation of ginsenoside CK from ginsenoside Rb1 by biological conversion, the conversion efficiency is still low, and it still cannot meet the production requirements. Rc is also one of the major ginsenosides with a relatively high content, and how to specifically and efficiently convert ginsenoside Rc to prepare adult ginsenoside CK is still one of the difficult problems to be solved. There is no report on the use of glycosidases produced by Bifidobacterium breve 689b and Bacillus subtilis to convert ginsenoside Rc into rare ginsenoside CK in vitro. This method has the advantages of high specificity, low cost, mild reaction conditions, high selectivity and good safety, and is a feasible method for producing rare ginsenoside CK. SUMMARY

[0007] The present application aims to overcome the technical defects and deficiencies of the prior art natural enzymes, such as difficult to obtain, low single enzyme activity, high selectivity to ginsenosides and other glycosides, and complex operation, and provides a method for converting ginsenoside Rc to prepare rare ginsenoside CK by co-expressing codon-optimized Bacillus subtilis alpha-L-arabinofuranosidase and Bifidobacterium beta-glucosidase in Escherichia coli, which is simple, efficient and environmentally friendly.

[0008] In order to achieve the above-mentioned goal, the technical scheme provided by the present application is as follows:

[0009] The method for converting and preparing rare ginsenoside CK by using gene combination comprises the following steps:

[0010] (1) codon-optimizing the α-L-arabinofuranosidase gene BsAbfA from Bacillus subtilis str. 168 to obtain a BsAbfA-op gene; codon-optimizing the β-glucosidase gene BbBgl2 from Bifidobacterium breve 689b to obtain a BbBgl2-op gene; and inserting an Internal ribosomal-binding site (IRBS) sequence between the BsAbfA-op gene and the BbBgl2-op gene; wherein the sequence of the BsAbfA gene is shown as SEQ ID NO. 1, the sequence of the BsAbfA-op gene is shown as SEQ ID NO. 2, the sequence of the BbBgl2 gene is shown as SEQ ID NO. 3, the sequence of the BbBgl2-op gene is shown as SEQ ID NO. 4, and the sequence of the IRBS is shown as SEQ ID NO. 5;

[0011] (2) connecting the gene sequence after the insertion of the IRBS sequence between the BsAbfA-op gene and the BbBgl2-op gene to the BamH I and EcoR I enzyme cutting sites of a pET28a vector to obtain a recombinant expression vector containing the BsAbfA-op and BbBgl2-op genes, named pET-BsAbfA-BbBgl2-op, and the sequence of the recombinant expression vector pET-BsAbfA-BbBgl2-op is shown as SEQ ID NO. 6;

[0012] (3) transforming the recombinant expression vector pET-BsAbfA-BbBgl2-op to Escherichia coli BL21, and then preparing a recombinant bacterium containing the BsAbfA-op and BbBgl2-op genes through kanamycin screening, PCR and sequencing detection;

[0013] (4) culturing the recombinant bacterium obtained in step (3) at 37°C until the OD600 is 0.4-0.6, and then inducing with 0.1-1.0 mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG) for 2-10 hours to obtain a recombinant bacterium liquid;

[0014] (5) Ginsenoside Rc is dissolved in methanol and mixed with acetic acid-sodium acetate buffer at pH 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 1-50 g / L. The ginsenoside Rc substrate solution is then mixed with the recombinant bacterial solution induced by IPTG at a volume ratio of 1:(1-10), reacted at pH 4-6 and 30-50°C for 12-24 hours, inactivated in a water bath at 70-80°C, preferably 80°C for 10-20 minutes, preferably 20 minutes, and then centrifuged at room temperature (preferably 25°C) for 10-15 minutes, preferably at 12000 rpm for 10-15 minutes, and the supernatant is collected to obtain rare ginsenoside CK.

[0015] Preferably, in step (2), the BsAbfA-op and BbBgl2-op genes are sequentially located downstream of the T7 promoter of the pET28a vector.

[0016] Preferably, in step (4), the concentration of fresh cells in the recombinant bacterial solution is 10-40 g / L, preferably 20 g / L.

[0017] The present invention also provides a recombinant expression vector for preparing rare ginsenoside CK. The gene sequence of the recombinant expression vector is shown in SEQ ID NO. 6 and is named pET-BsAbfA-BbBgl2-op.

[0018] The present invention also provides a recombinant bacterium for producing rare ginsenoside CK. The recombinant bacterium is prepared by transforming the recombinant expression vector pET-BsAbfA-BbBgl2-op described in claim 4 into Escherichia coli BL21. The recombinant bacterium is then screened with kanamycin, tested by PCR, and sequenced. The enzyme expressed by the recombinant bacterium is more active than the enzymes of the individual components. Enzyme property analysis shows that the co-expressed enzyme has an optimal reaction pH of 5.0 and an optimal reaction temperature of 40°C. It maintains high activity within the pH range of 5-7. The co-expressed enzyme exhibits high temperature stability within the range of 30-50°C, maintaining an enzyme activity of over 85%.

[0019] The present invention will be further described below:

[0020] The α-L-arabinofuranosidase (BsAbfA) gene used in the present invention is from Bacillus subtilis str. 168, and the β-glucosidase (BbBgl2) gene is from Bifidobacterium breve 689b. Based on the codon preference of Escherichia coli, the structural characteristics of ginsenosides, and the enzyme-substrate binding simulation data ( Figure 1 and Figure 2), and the results showed that BsAbfA had good binding ability with ginsenoside Rc and good selectivity. The BsAbfA and BbBgl2 genes were codon optimized, and the optimized gene sequences BsAbfA-op (SEQ ID NO.2) and BbBgl2-op (SEQ ID NO.4) were obtained by whole gene synthesis. An IRBS sequence was added between the two genes, and BamH I and EcoR I restriction site sequences were added upstream and downstream of the concatenated gene sequences, respectively. The recombinant plasmids (pET-BsAbfA-BbBgl2-op) were transformed into Escherichia coli TOP10 by enzyme digestion and ligation, and the calcium chloride method. After kanamycin selection, enzyme digestion, and sequencing identification, the recombinant plasmids (pET-BsAbfA-BbBgl2-op) were extracted and transformed into Escherichia coli BL21 by the calcium chloride method. After kanamycin selection, enzyme digestion, and sequencing, Escherichia coli capable of efficiently expressing the recombinant enzymes (β-glucosidase and α-L-arabinofuranosidase) were obtained. Recombinant bacteria containing the BsAbfA-op and BbBgl2-op genes are cultured at 37°C to an OD600 of 0.4-0.6, and then induced with 0.1-1.0 mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG) for 2-10 hours to obtain a recombinant bacterial solution with relatively high activity of both enzyme proteins. The two enzymes produced in this recombinant bacteria are expressed in the same bacterium using the codon-optimized BsAbfA-op and BbBgl2-op genes, and exhibit high enzymatic activity, exceeding that of enzymes expressed by co-expressing unoptimized genes (BsAbfA and BbBgl2) or by optimizing a single gene (BsAbfA-op or BbBgl2-op).

[0021] Both β-glucosidase and α-L-arabinofuranosidase are glycosidases, and both are abundant in nature. However, the functions of β-glucosidase or α-L-arabinofuranosidase from different sources, and even those from the same source and with similar structures, can differ significantly. The glycosidic bond type and spatial structure of the substrate can affect their substrate conversion ability (Biotechnol Lett (2016) 38:1775–1780. Microbiology (2009), 155, 2739–2749. Process Biochemistry (2010), 45, 1226–1235). Furthermore, different enzymatic reaction conditions can significantly affect enzyme activity and substrate conversion ability. Therefore, it is difficult to infer the functions of other glycosidases based on the activity of some existing glycosidases. Even if some glycosidases may have some conversion ability in native cells, their biological activities may differ significantly when heterologously expressed in other microorganisms. On the one hand, it is affected by the expression level of heterologous cells. On the other hand, the activity of heterologously expressed enzymes may not reach the optimal state and often fails to exert their specific functions.

[0022] In the research of producing specific ginsenosides by glycosidase conversion, it is found that there are few glycosidases with the ability to convert ginsenosides, and even fewer can selectively convert ginsenoside Rc to CK. Even the few glycosidases with the ability to convert ginsenosides are difficult to be applied due to low activity. Before the present application, no research on the conversion of ginsenoside Rc by exogenous BsAbfA gene and BbBgl2 has been found. Through screening and gene structure optimization, we confirmed that the proteins encoded by the codon-optimized BsAbfA gene and BbBgl2 gene have high activity in converting ginsenoside Rc, and we found that the co-expression of BsAbfA-op and BbBgl2-op genes in E. coli cells can produce two enzymes, which can efficiently convert ginsenoside Rc to Rd, and finally convert ginsenoside Rd to the rare ginsenoside CK. The recombinant bacteria containing BsAbfA-op and BbBgl2-op genes induced by IPTG used in the present application can convert ginsenoside Rc to the rare ginsenoside CK with a conversion efficiency of more than 76% within 12 hours, and a conversion efficiency of more than 85% within 24 hours. Since the cell has the advantage of easy absorption of the conversion product ginsenoside CK, it has unique pharmacological effects and important medicinal value in clinical practice. The method of the present application can improve the application value of the ginsenoside Rc-containing pharmaceutical ingredients in the medical market.

[0023] In summary, the glycosidases produced by Bifidobacterium breve 689b and Bacillus subtilis can efficiently convert ginsenoside Rc to the rare ginsenoside CK in vitro. The present application has the advantages of high specificity, low cost, mild reaction conditions, short conversion period, high catalytic efficiency, good selectivity, good stability, good safety, few by-products, and easy industrial production, etc. It is a feasible method for producing rare ginsenosides. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the molecular docking result of ginsenoside Rc and BsAbfA protein;

[0025] Figure 2 is the main site of the molecular combination of ginsenoside Rc and BsAbfA protein;

[0026] Figure 3 is the result of polyacrylamide gel electrophoresis analysis of the crude enzyme solution extracted from the recombinant bacteria and the purified enzyme under the conditions of Example 1; in the figure, 1 represents the extracted crude enzyme solution, 2 and 3 represent the BsAbfA and BbBgl2 enzymes purified by nickel column affinity method; M is the protein molecular weight standard;

[0027] Figure 4The results of thin layer chromatography of ginsenoside Rc transformed by recombinant bacteria under the conditions of Example 10 and Example 14 are shown; in the figure, 0h represents the reaction of the recombinant bacteria with ginsenoside Rc for 0 hour, 12h represents the reaction of the recombinant bacteria with ginsenoside Rc for 12 hours, 24h represents the reaction of the recombinant bacteria with ginsenoside Rc for 24 hours, and S represents ginsenoside Rc, Rd, F2, and CK reference substances;

[0028] Figure 5 These are the high performance liquid chromatography (HPLC) results of the transformation of ginsenoside Rc by the recombinant bacteria under the conditions of Example 10 and Example 14; in the figure, 12h indicates that the recombinant bacteria reacted with ginsenoside Rc for 12 hours, 24h indicates that the recombinant bacteria reacted with ginsenoside Rc for 24 hours, and S indicates ginsenoside Rc, Rd, F2, and CK reference substances. DETAILED DESCRIPTION

[0029] Example 1

[0030] 1. The recombinant bacteria were cultured in Luria-Bertani (LB) liquid medium. The LB medium was prepared as follows: 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl were dissolved in an appropriate amount of water. After the solutes were dissolved, the pH was adjusted to 7.0 with 5 mol / L sodium hydroxide (NaOH), and the volume was adjusted to 1 L with deionized water. The medium was sterilized under high pressure at 121°C for 20 min.

[0031] 2. The specific method for fermentation culture of the recombinant bacteria is as follows: the recombinant bacteria are inoculated into solid LB medium containing 50 μg / mL kanamycin, cultured overnight (8-10 hours) at 37°C, and then a single colony is picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C until the absorbance OD600 is 0.4-0.6, and 0.1-1 mmol / L IPTG is added for induction for 2-10 hours to obtain the recombinant bacterial fermentation broth.

[0032] 3. Centrifuge the recombinant bacterial fermentation broth at 4°C and 8000rpm for 10 minutes, discard the supernatant, collect the bacterial cells, and divide the collected cells into two parts. Add fresh liquid LB culture medium to one part until the cell concentration is 20g / L for later use. Wash the other part of the cells twice with pH 6.5 PBS buffer; use an ultrasonic cell disruptor to disrupt on ice for 30 minutes. The disruption conditions are: power 200W, ultrasonic time 2s, interval time 3s, and working times 30 times. After the ultrasonic disruption is completed, centrifuge at 12000rpm and 4°C for 20 minutes, and take the supernatant to obtain the crude enzyme solution. The crude enzyme solution is purified by nickel column, and the eluent is pH 6.8 phosphate buffer. Repeat this process several times to separate and obtain the purified recombinant enzyme. The molecular weight is analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of BsAbfA-op and BbBgl2-op are approximately 57kDa and 81kDa, respectively (see Figure 3 ).

[0033] Figure 3 Figure 1 is the result of polyacrylamide gel electrophoresis analysis of the crude enzyme solution and the purified enzyme obtained from the recombinant bacteria under the conditions of Example 1; in the figure, 1 represents the extracted crude enzyme solution, 2 and 3 represent the BsAbfA and BbBgl2 enzymes separated and purified by nickel column affinity method; M is a protein molecular weight standard; comparison with the molecular weights of the predicted proteins of BsAbfA (sequence numbers AL009126.3, 2938330-2939832 in the GenBank database) and BbBgl2 (sequence numbers CP006715.1, 1716905-1719178 in the GenBank database) showed that the molecular weights of the purified BsAbfA and BbBgl2 were consistent with the theoretical values ​​(57 kDa and 81 kDa), respectively.

[0034] 4. Determination of recombinase activity

[0035] The Folin-phenol method was used to determine the content of the isolated and purified protein, and then the activity of the two recombinant enzymes was determined. The specific method for determining the activity of the BsAbfA-op recombinant enzyme was as follows: using p-nitrophenyl-α-L arabinofuranoside (pNPA) as a substrate and p-nitrophenol (pNP) as a product to determine the activity of the enzyme. Take 200 μL of recombinant enzyme, add 200 μL of pNPA (10 mM), pH 6.5, 55 ° C, incubate for 2 hours, add an equal volume of 200 mM Na2CO3, and measure pNP at 405 nm. 1 unit of enzyme activity (U) is defined as the release of 1 μmol of pNP in 1 min. The results showed that α-L-arabinofuranosidase K m The value is 3.45×10 -3 mol / L, α-L-arabinofuranosidase has strong catalytic activity.

[0036] The specific method for determining the activity of BbBgl2-op recombinase is as follows: the activity of the enzyme is determined by using p-nitrophenyl-β-D-glucoside (pNPG) as the substrate and p-nitrophenol (pNP) as the product. 200 μL of the recombinase is taken, 200 μL of pNPG (10 mM) is added, and the mixture is incubated at 55°C and pH 6.5 for 2 hours. Then, an equal volume of 200 mM Na2CO3 is added, and the pNP is determined at 405 nm. One enzyme activity unit (U) is defined as the release of 1 μmol of pNP per minute. The results show that the β-glucosidase K m value is 1.48 x 10 -3 mol / L, the β-glucosidase has strong catalytic activity.

[0037] Example 2

[0038] The recombinant BL21 bacterial liquid containing the BsAbfA (SEQ ID NO. 1) and BbBgl2 (SEQ ID NO. 3) genes (the codons are not optimized) and the internal ribosomal-binding site (IRBS) sequence (SEQ ID NO. 5) added between the two genes is prepared by the culture method in Example 1, and is formulated into a bacterial liquid with a cell concentration of 40 g / L. Ginsenoside Rc is dissolved in methanol, mixed with an acetic acid-sodium acetate buffer at pH 4.0-6.0, and formulated into a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution is mixed with the recombinant bacterial liquid at a volume ratio of 1:2, the pH is 5.0, and the mixture is reacted at 40°C for 24 hours. Then, the mixture is inactivated in a water bath at 80°C for 20 min, centrifuged at 10000 rpm at 25°C for 10 min, and the supernatant is dried at 60°C to obtain dilute ginsenoside CK.

[0039] Example 3

[0040] The recombinant BL21 bacterial liquid containing only the BsAbfA (SEQ ID NO. 1) gene (the codons are not optimized) is prepared by the culture method in Example 1, and is formulated into a bacterial liquid with a cell concentration of 40 g / L. Ginsenoside Rc is dissolved in methanol, mixed with an acetic acid-sodium acetate buffer at pH 4.0-6.0, and formulated into a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution is mixed with the recombinant bacterial liquid at a volume ratio of 1:2, the pH is 5.0, and the mixture is reacted at 40°C for 24 hours. Then, the mixture is inactivated in a water bath at 80°C for 20 min, centrifuged at 10000 rpm at 25°C for 10 min, and the supernatant is dried at 60°C to obtain dilute ginsenoside CK.

[0041] Example 4

[0042] The recombinant BL21 bacterial liquid containing only BbBgl2 (SEQ ID NO. 3) gene (codon not optimized) was prepared by the culture method in Example 1, and was prepared into a bacterial liquid with a cell concentration of 40 g / L; Ginsenoside Rc was dissolved in methanol, mixed with acetic acid-sodium acetate buffer with pH 4.0-6.0, and prepared into a ginsenoside Rc substrate solution with a concentration of 40 g / L; then the ginsenoside Rc solution was mixed with the recombinant bacterial liquid at a volume ratio of 1:2, pH 5.0, and reacted at 40°C for 24 hours, and then was placed in a water bath at 80°C for 20 min, centrifuged at 10000 rpm at 25°C for 10 min, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0043] Example 5

[0044] The recombinant BL21 bacterial liquid containing only BsAbfA-op (SEQ ID NO. 2) gene (codon optimized) was prepared by the culture method in Example 1, and was prepared into a bacterial liquid with a cell concentration of 40 g / L; Ginsenoside Rc was dissolved in methanol, mixed with acetic acid-sodium acetate buffer with pH 4.0-6.0, and prepared into a ginsenoside Rc substrate solution with a concentration of 40 g / L; then the ginsenoside Rc solution was mixed with the recombinant bacterial liquid at a volume ratio of 1:2, pH 5.0, and reacted at 40°C for 24 hours, and then was placed in a water bath at 80°C for 20 min, centrifuged at 10000 rpm at 25°C for 10 min, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0045] Example 6

[0046] The recombinant BL21 bacterial liquid containing only BbBgl2-op (SEQ ID NO. 4) gene (codon optimized) was prepared by the culture method in Example 1, and was prepared into a bacterial liquid with a cell concentration of 40 g / L; Ginsenoside Rc was dissolved in methanol, mixed with acetic acid-sodium acetate buffer with pH 4.0-6.0, and prepared into a ginsenoside Rc substrate solution with a concentration of 40 g / L; then the ginsenoside Rc solution was mixed with the recombinant bacterial liquid at a volume ratio of 1:2, pH 5.0, and reacted at 40°C for 24 hours, and then was placed in a water bath at 80°C for 20 min, centrifuged at 10000 rpm at 25°C for 10 min, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0047] Example 7

[0048] A recombinant BL21 bacterial solution containing BsAbfA and BbBgl2 proteins was prepared using the culture methods of Examples 3 and 4, and a bacterial solution with a cell concentration of 40 g / L was prepared (the mass ratio of the two bacterial solutions was 1:1); ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution having a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution having a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:2, the pH was 5.0, and the mixture was reacted at 40°C for 24 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes, centrifuged at 10,000 rpm for 10 minutes at 25°C, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0049] Example 8

[0050] A recombinant BL21 bacterial solution containing BsAbfA-op and BbBgl2-op proteins was prepared using the culture methods of Examples 5 and 6, and a bacterial solution with a cell concentration of 40 g / L was prepared (the mass ratio of the two bacterial solutions was 1:1); ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution having a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution having a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:2, the pH was 5.0, and the mixture was reacted at 40°C for 24 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes, centrifuged at 10,000 rpm for 10 minutes at 25°C, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0051] Example 9

[0052] A recombinant BL21 bacterial solution containing the pET-BsAbfA-BbBgl2-op vector was prepared using the culture method in Example 1 to prepare a bacterial solution with a cell concentration of 40 g / L; ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution at a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:1, the pH was 5.0, and the mixture was reacted at 40°C for 12 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes and centrifuged at 10,000 rpm for 10 minutes at 25°C. The supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0053] Example 10

[0054] The recombinant BL21 bacterial liquid containing pET-BsAbfA-BbBgl2-op vector was prepared by the culture method in Example 1, and was prepared into a bacterial liquid with a cell concentration of 40 g / L. Ginsenoside Rc was dissolved in methanol, mixed with an acetic acid-sodium acetate buffer with a pH of 4.0-6.0, and prepared into a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution and the recombinant bacterial liquid were mixed in a volume ratio of 1:2, with a pH of 5.0, and reacted at 40°C for 12 hours. Then, the mixture was placed in a water bath at 80°C for 20 min, centrifuged at 10,000 rpm at 25°C for 10 min, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0055] Example 11

[0056] The recombinant BL21 bacterial liquid containing pET-BsAbfA-BbBgl2-op vector was prepared by the culture method in Example 1, and was prepared into a bacterial liquid with a cell concentration of 40 g / L. Ginsenoside Rc was dissolved in methanol, mixed with an acetic acid-sodium acetate buffer with a pH of 4.0-6.0, and prepared into a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution and the recombinant bacterial liquid were mixed in a volume ratio of 1:3, with a pH of 5.0, and reacted at 40°C for 12 hours. Then, the mixture was placed in a water bath at 80°C for 20 min, centrifuged at 10,000 rpm at 25°C for 10 min, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0057] Example 12

[0058] The recombinant BL21 bacterial liquid containing pET-BsAbfA-BbBgl2-op vector was prepared by the culture method in Example 1, and was prepared into a bacterial liquid with a cell concentration of 40 g / L. Ginsenoside Rc was dissolved in methanol, mixed with an acetic acid-sodium acetate buffer with a pH of 4.0-6.0, and prepared into a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution and the recombinant bacterial liquid were mixed in a volume ratio of 1:4, with a pH of 5.0, and reacted at 40°C for 12 hours. Then, the mixture was placed in a water bath at 80°C for 20 min, centrifuged at 10,000 rpm at 25°C for 10 min, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0059] Example 13

[0060] A recombinant BL21 bacterial solution containing the pET-BsAbfA-BbBgl2-op vector was prepared using the culture method in Example 1 to prepare a bacterial solution with a cell concentration of 40 g / L; ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution at a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:1, the pH was 5.0, and the mixture was reacted at 40°C for 24 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes, centrifuged at 10,000 rpm for 10 minutes at 25°C, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0061] Example 14

[0062] A recombinant BL21 bacterial solution containing the pET-BsAbfA-BbBgl2-op vector was prepared using the culture method in Example 1 to prepare a bacterial solution with a cell concentration of 40 g / L; ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution at a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:2, the pH was 5.0, and the mixture was reacted at 40°C for 24 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes and centrifuged at 10,000 rpm for 10 minutes at 25°C. The supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0063] Example 15

[0064] A recombinant BL21 bacterial solution containing the pET-BsAbfA-BbBgl2-op vector was prepared using the culture method in Example 1 to prepare a bacterial solution with a cell concentration of 40 g / L; ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution at a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:3, the pH was 5.0, and the mixture was reacted at 40°C for 24 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes, centrifuged at 10,000 rpm for 10 minutes at 25°C, and the supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0065] Example 16

[0066] A recombinant BL21 bacterial solution containing the pET-BsAbfA-BbBgl2-op vector was prepared using the culture method in Example 1 to prepare a bacterial solution with a cell concentration of 40 g / L; ginsenoside Rc was dissolved in methanol and mixed with an acetic acid-sodium acetate buffer solution at a pH of 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 40 g / L. The ginsenoside Rc solution was then mixed with the recombinant bacterial solution in a volume ratio of 1:4, the pH was 5.0, and the mixture was reacted at 40°C for 24 hours. The mixture was then inactivated in an 80°C water bath for 20 minutes and centrifuged at 10,000 rpm for 10 minutes at 25°C. The supernatant was dried at 60°C to obtain rare ginsenoside CK.

[0067] Example 17

[0068] HPLC determination method

[0069] The induced bacteria-substrate reaction solution from Example 2-17 and the substrate reaction solution without bacteria were centrifuged at 8000 rpm for 5 minutes at 25°C. The supernatant was then dried at 60°C and ultrasonically dissolved with an appropriate amount of methanol. After filtering through a 0.45 μm filter, the volume was fixed and the content was determined by HPLC. The conversion rate of ginsenoside Rc was calculated based on the molar ratio of the reaction substrate to the product. The results are shown in Table 1.

[0070] The HPLC assay conditions were as follows: a Diamonsil C18 column (150 mm × 4.6 mm, 5 μm); a mobile phase consisting of acetonitrile-50 mmol / L potassium dihydrogen phosphate / potassium dihydrogen phosphate (10:90, v / v) (pH adjusted to 4.0 with phosphoric acid); a flow rate of 1.0 mL / min; a column temperature of 30°C; and a detection wavelength of 203 nm.

[0071] In Examples 10 and 14, the TLC results of the conversion of ginsenoside Rc to CK are shown in Figure 4 , Figure 4 Here, 0h indicates the reaction of the recombinant bacteria with ginsenoside Rc for 0 hours, 12h indicates the reaction of the recombinant bacteria with ginsenoside Rc for 12 hours, 24h indicates the reaction of the recombinant bacteria with ginsenoside Rc for 24 hours, and S indicates ginsenoside Rc, Rd, F2 and CK reference substances. The results showed that after 12 hours of reaction, most of the ginsenoside Rc was converted into F2 and CK, and after 24 hours of reaction, the conversion of ginsenoside Rc into CK was further improved.

[0072] In Examples 10 and 14, the HPLC results of the conversion of ginsenoside Rc to CK are shown in Figure 5 , Figure 5Here, 12h indicates that the recombinant bacteria reacted with ginsenoside Rc for 12 hours, 24h indicates that the recombinant bacteria reacted with ginsenoside Rc for 24 hours, and S indicates ginsenoside Rc, Rd, F2 and CK reference substances. The results showed that after 12 hours of reaction, ginsenosides were mainly converted into ginsenoside Rd, F2 and CK, and after 24 hours of reaction, most of the ginsenosides were converted into CK, indicating that the recombinant bacteria in the invention can convert ginsenoside Rc into CK through the Rd and F2 conversion pathways.

[0073] The results of the above examples show that the conversion rate of ginsenoside Rc to CK by recombinant bacteria containing two unoptimized genes used in the present invention was only 32.4% (Example 2); recombinant bacteria containing either unoptimized or optimized single genes were unable to convert ginsenoside Rc to CK (Examples 3-6); the conversion rate of ginsenoside Rc by a combination of recombinant bacterial solutions containing both BsAbfA and BbBgl2 proteins was very low (39.6% in Example 7); the conversion rate of ginsenoside Rc by a combination of recombinant bacterial solutions containing both BsAbfA-op and BbBgl2-op proteins was significantly improved (54.1% in Example 8); and the recombinant bacteria co-expressing the codon-optimized BsAbfA-op and BbBgl2-op genes in the present invention could convert ginsenoside Rc to the rare ginsenoside CK with a conversion rate of 70-85%. The method adopted is simple to operate, low in cost, high in yield, and environmentally friendly. This method can realize the industrial preparation of rare ginsenoside CK, can meet the market needs of the pharmaceutical and food industries, and has high market application value.

[0074] Table 1 Analysis of the conversion rate of ginsenoside Rc to CK by recombinant bacteria

[0075] CK conversion (%) Example 2 32.4 Example 3 0 Example 4 0 Example 5 0 Example 6 0 Example 7 39.6 Example 8 54.1 Example 9 73.2 Example 10 76.5 Example 11 71.4 Example 12 70.3 Example 13 82.4 Example 14 85.3 Example 15 84.4 Example 16 80.2 Sequence Listing <110> Hunan Institute of Technology <120> A method for preparing rare ginsenoside CK by gene combination transformation and its application <141> 2020-12-10 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1488 <212> DNA <213> null <400> 1 atgtctgaac atcaagcagt gattcaaaca gatatcgcaa aaggaaccat taacaaaaat 60 atatacggtc attttgctga gcatttagga agagggattt atgaggggat ctgggtcgga 120 acggactcag acatccccaa tatcaacggg atacgaaagg acgtgctgga ggcgctcaaa 180 cagctgcaca ttcctgtcct taggtggccg ggcgggtgtt ttgcggacga ataccattgg 240 gcaaacggtg tcggtgaccg taagacaatg ctgaacactc actggggcgg tacaattgaa 300 tcaaatgaat tcggaacgca tgaatttatg atgctttgcg agctgcttga atgcgagcca 360 tatatttgcg gcaatgtcgg aagcggaacc gttcaggaaa tgtcggagtg gattgagtat 420 atgacatttg aagaaggcac gccgatgtca gactggagaa agcaaaatgg aagagaagag 480 ccttggaagc tgaaatattt cggcgtgggc aatgaaaact ggggctgcgg cggcaacatg 540 catcccgaat actacgcaga tctgtaccgg cgttttcaga cttatgtccg caattacagc 600 gggaatgaca tttataaaat tgcaggcggc gcaaatgtgg atgattttaa ttggacggac 660 gtgcttatga aaaaagccgc tggcctgatg gacgggttga gtcttcatta ttacacgatt 720 ccgggggatt tctggaaggg caaaggatca gccacagaat tcacggaaga tgaatggttt 780 attacgatga aaaaagccaa atacatcgat gaattgattc aaaaacacgg cacgattatg 840 gaccggtacg atccggagca gcgggtcggg ctgattattg atgaatgggg cacgtggttt 900 gatcccgagc caggcacgaa tcccggtttc ttatatcagc aaaacaccat tcgtgatgca 960 ctggtggcgg cttctcattt ccacattttc catcagcatt gccgccgggt gcaaatggcc 1020 aacatcgccc aaacagtaaa tgttcttcaa gcgatgattt tgactgaggg agagcggatg 1080 cttttgacac cgacgtacca tgtattcaat atgtttaagg tgcaccagga cgcttctctt 1140 ttagccacag agacaatgtc tgccgactat gaatggaacg gtgaaacgct tccgcaaatc 1200 agcatttcag cgtcgaaaca agctgaaggc gatatcaata tcacaatttg caacatcgat 1260 caccaaaaca aagcagaggc ggaaatcgag ctgaggggcc tacacaaggc agcggaccat 1320 tccggagtca ttcttacggc agaaaaaatg aatgcgcata acacgtttga cgatcctcat 1380 catgtcaaac cggaatcctt cagacaatac acgctcagca aaaacaaact gaaagtaaaa 1440 ctcccgccaa tgtcagtcgt cttacttacg ctgcgtgctg attcttaa 1488 <210> 2 <211> 1503 <212> DNA <213> null <400> 2 atgaaaaaag cgcgaatgat tgtagacaaa gaatataaaa tcggtgaagt agataaacgg 60 attatggct cgtttatcga acatatgggt cgtgcggtat atgaaggcat atacgagcct 120 gatcaccctg aagcggatga agatggattt agaaaagatg tccagtcgct gatcaaagaa 180 ttacaggttc ccatcatccg ctatccgggc ggaaactttt tatccggata caactgggag 240 gacggtgtcg gaccagtcga aaaccgcccg agacggcttg acttggcatg gcaaacgaca 300 gaaaccaatg aagtgggaac aaatgaattt ttatcttggg caaaaaaggt gaacactgag 360 gtcaatatgg ccgtcaacct tggcacaaga ggcatagatg ccgcccgtaa tctcgttgaa 420 tattgcaacc atccgaaagg ctcttactgg agtgatttaa gaagatcgca tggctatgaa 480 cagccgtatg gcatcaaaac atggtgctta ggaaacgaaa tggatggacc atggcagatc 540 ggccacaaaa cagctgatga atacggacgg cttgccgcag agacagcaaa ggtcatgaag 600 tgggttgacc catcaattga actcgttgcc tgcggcagct caaacagcgg tatgccgacc 660 tttatcgatt gggaagcgaa ggtgcttgag catacgtatg agcatgtcga ctatatctct 720 cttcacactt actacggaaa ccgggataac aatctgccaa actacttggc acgttctatg 780 gatttggatc attttatcaa atcagtcgct gcgacctgtg actatgtaaa agcaaaaaca 840 cgcagcaaga aaactatcaa tctctctctg gatgaatgga acgtctggta ccactcaaat 900 gaggctgata aaaaagtcga gccgtggatc actgcgcgtc cgattttaga ggatatttac 960 aattttgaag atgccttatt agtcggctct ctgctcatta cgatgctgca gcacgcagac 1020 cgtgtgaaaa ttgcgtgtct tgcacagctt gttaatgtca tcgcgccgat catgacggaa 1080 aaaggcggag aagcatggag acagccgatt ttctatccat acatgcatgc ttctgtttac 1140 ggaaggggcg agtcactgaa accgcttatt tcttctccta agtacgattg ttctgatttc 1200 actgatgtgc catatgttga tgctgctgtt gtgtactctg aagaggaaga aacactcact 1260 atttttgcgg taaacaaggc tgaggatcag atggagacgg agatttcgct cagaggcttt 1320 gaatcctacc aaatcgcaga gcacatcgta cttgagcatc aggatatcaa agcacaaac cage gaaaaaatgt cgttccgcat tccaacggat catcgtctgt cageaaac ggcttaactg ctcatttcac gccgctttcc tggaatgtga tccgcctgaa aaaacagtca taa 1503 <210> 3 <211> 2274 <212> DNA <213> null <400> 3 atgagcgagt ccacctaccc gtccgtcaag gatctgaccc tggagaga ggcatccctc acctccggcg gcgacgcctg gcatctgcag ggcgtggagt ccaaaggcat cccaggctac 120 atgatcactg acggtcctca cggtctaagg aagtccctgg cctccagcgc cggtgagacc 180 240. gaccttgacg actccgtgcc cgccacctgc ttcccgccgg ccgccggctt gtccagctct tggaatcctg agctcattca caaggttggt gaggcgatgg ctgaggagtg cattcaggag aaggtggctg tgattcttgg ccccggcgtc aatatcaagc gcaacccgct cggcggtcgc 360 tgcttcgaat actggtccga agacccgtac ttggccggtc atgaggccat cggcattgtt 420 gaaggcgtgc agtccaaggg cgtgggcacg tcgctcaagc acttcgccgc caacaatcag 480 gaaaccgacc gactgcgcgt cgatgcccgc atcagcccgc gcgccctgcg cgagatctac 540 ttcccggcct tcgaacacat cgtcaagaag gcccagccgt ggaccatcat gtgctcctac 600 aaccgcatca acggcgtgca ttccgcacaa aaccattggc tgctgaccga cgtgctgcgc 660 gacgaatggg gcttcgatgg catcgtcatg tccgactggg gcgccgatca tgaccgtggg 720 gcctccctga atgcaggcct gaatctggaa atgccgccga gctacaccga cgaccagatt 780 gtctacgccg tccgcgacgg cctaatcacc cccgcccagc tcgatcgtat ggctcagggc 840 atgattgact tggtaaacaa gacccgcgct gcaatgagta tcgataatta ccgtttcgat 900 gtggacgccc acgatgaagt cgcccatcag gccgctattg aatccattgt gatgctcaag 960 aacgatgatg cgattctgcc gctgaacgcc ggtccagtcg ccaatccgtc cgccacgccc 1020 cagaagatcg ccgttatcgg cgaattcgcc cgcaccccgc gctaccaggg cggcggctcc 1080 tcccacatca cccccaccaa gatgaccagc ttcctcgaca cgctcgccga gcgcggcatc 1140 aaggccgact tcgcccccgg cttcacgctc gatctggaac cggccgaccc ggccctcgaa 1200 tccgaagccg tggaaaccgc caagaacgcc gacgttgtcc tcatgttcct gggcctgccg 1260 gaagccgcgg aatccgaagg cttcgaccgc gacaccctcg acatgcccgc caagcagatc 1320 accctactcg aacaggtcgc cgccgcgaac cagaacgtcg tggtcgtact gtccaacggt 1380 tccgtgatca ccgtggcccc gtgggccaag aacgccaagg gcatcctcga atcctggttg 1440 ctcggccagt ccggtggccc ggcgctcgcc gatgtgatct tcggccaggt cagcccgtcc 1500 ggcaagctcg cccagtcgat tccgctggac atcaacgatg acccgagcat gctgaactgg 1560 ccgggcgagg aaggccacgt cgactacggc gagggcgtgt tcgtcggtta ccgctactac 1620 gacacctacg gcaaggctgt cgactacccg ttcggctacg gcctgagcta cgccacgttc 1680 gagatcaccg gtgtcgccgt tgccaagacc ggcgcgaaca ccgccaccgt gaatgccact 1740 gtgaccaaca cctccgatgt ggacgctgcc gaaaccgtgc aggtgtacgt tgtgccgggc 1800 aaggccgacg tggctcgccc gaagcacgag ctcaagggct tcaccaaggt gttcctcaag 1860 tccggtgagt ccaagaccgt gaccatcgac ctcgacgagc gcgcgttcgc ctactggtcc 1920 gaaaagtaca acgactggca cgtggaggcc ggcgaatatg ccatcgaagt gggcgtgagc 1980 tcccgcgaca ttgccgacac cgttgccgtg gccctcgatg gcgacggcaa gacccagccg 2040 ctcaccgaat ggtccaccta cggcgagtgg gaggccgatc cgttcggcgc caagatcgtg 2100 gccgccgtgg ccgccgccgg cgaggccggc gagctgccga agcttccgga taatgcgatg 2160 atgcgcatgt tcctcaactc catgcccatc aactcgctgc ccaccctgct gggcgaaggc 2220 ggcaagaaga tcgcccagtt catggttgac gagtacgcca agctgagcaa gtaa 2274 <210> 4 <211> 2274 <212> DNA <213> null <400> 4 atgagcgaaa gcacctatcc gagcgtgaag gacctgaccc tggaggaaaa agcgagcctg 60 accagcggtg gcgatgcgtg gcacctgcaa ggtgttgaga gcaagggcat cccgggttac 120 atgattaccg acggcccgca cggtctgcgt aaaagcctgg cgagcagcgc gggtgaaacc 180 gatctggacg atagcgttcc ggcgacctgc ttcccgccgg cggcgggtct gagcagcagc 240 tggaacccgg agctgatcca caaagttggc gaagcgatgg cggaggaatg catccaggaa 300 aaggtggcgg ttattctggg cccgggtgtt aacatcaaac gtaacccgct gggtggccgt 360 tgcttcgagt attggagcga agacccgtac ctggcgggtc atgaggcgat cggcattgtg 420 gaaggtgttc aaagcaaggg cgtgggtacc agcctgaaac actttgcggc gaacaaccag 480 gagaccgacc gtctgcgtgt tgatgcgcgt attagcccgc gtgcgctgcg tgagatctat 540 ttcccggcgt ttgaacacat tgtgaagaaa gcgcaaccgt ggaccattat gtgcagctac 600 aaccgtatca acggtgttca cagcgcgcag aaccactggc tgctgaccga cgtgctgcgt 660 gatgagtggg gcttcgacgg tatcgttatg agcgattggg gcgcggacca tgatcgtggt 720 gcgagcctga acgcgggcct gaacctggaa atgccgccaa gctataccga cgatcaaatc 780 gtgtacgcgg ttcgtgacgg tctgattacc ccggcgcagc tggaccgtat ggcgcaaggc 840 atgatcgatc tggtgaacaa aacccgtgcg gcgatgagca ttgacaacta tcgttttgac 900 gtggatgcgc atgatgaggt tgcgcaccag gcggcgatcg aaagcattgt tatgctgaag 960 aacgacgatg cgatcctgcc gctgaacgcg ggtccggtgg cgaacccgag cgcgaccccg 1020 caaaaaatcg cggttattgg cgagttcgcg cgtaccccgc gttaccaggg tggcggtagc 1080 agccacatca ccccgaccaa gatgaccagc tttctggaca ccctggcgga acgtggtatt 1140 aaagcggatt ttgcgccggg ttttaccctg gacctggagc cggcggaccc ggcgctgggag 1200 agcgaagcgg tggaaaccgc gaagaacgc gacgtggttc tgatgtttct gggtctgccg 1260 gaggcggcgg agagcgaagg ctttgaccgt gataccctgg atatgccggc gaaacagatc 1320 accctgctgg aacaagttgc ggcggcgaac cagaacgtgg ttgtggttct gagcaacggt 1380 agcgtgatca ccgttgcgcc gtgggcgaag aacgcgaaag gtattctgga gagctggctg 1440 ctgggtcaaa gcggcggtcc ggcgctggcg gacgtgattt tcggtcaagt tagcccgagc 1500 ggcaagctgg cgcagagcat cccgctggac attaacgacg atccgagcat gctgaactgg 1560 ccgggcgagg aaggtcacgt ggattatggc gagggtgtgt ttgttggtta tcgttactat 1620 gacacctacg gcaaggcggt tgattacccg ttcggctatg gtctgagcta cgcgaccttt 1680 gaaatcaccg gtgtggcggt tgcgaaaacc ggtgcgaaca ccgcgaccgt gaacgcgacc 1740 gttaccaaca ccagcgatgt ggatgcggcg gagaccgtgc aggtttacgt ggttccgggc 1800 aaggcggacg ttgcgcgtcc gaagcacgag ctgaaaggtt tcaccaaggt gtttctgaaa 1860 agcggcgaaa gcaagaccgt taccattgac ctggatgagc gtgcgttcgc gtattggagc 1920 gaaaaataca acgattggca cgtggaggcg ggtgaatatg cgatcgaagt gggcgttagc 1980 agccgtgaca ttgcggatac cgtggcggtt gcgctggatg gtgatggtaa aacccaaccg 2040 ctgaccgaat ggagcaccta cggcgagtgg gaagcggacc cgtttggcgc gaagatcgtt 2100 gcggcggttg cggcggcggg tgaagcgggt gaactgccga aactgccgga taacgcgatg 2160 atgcgtatgt tcctgaacag catgccgatc aacagcctgc cgaccctgct gggcgagggc 2220 ggtaagaaaa ttgcgcagtt tatggttgac gaatacgcga agctgagcaa ataa 2274 <210> 5 <211> 36 <212> DNA <213> null <400> 5 aataatttg tttaacttta agaaggagat atacat 36 <210> 6 <211> 9182 <212> DNA <213> null <400> 6 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg cagcgccct agcgcccgct cctttcgct tcttccttc 120 cttctcgcc acgtcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt tttcgccct ttgacgttgg agtccacgtt 300 cttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa ccctatttg tttatttttc taatacatt caaatatgta 540 tccgctcatg aattaattct tagaaaact catcgagcat caaatgaaac tgcaatttat 600 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctgta tcggtctgcg attccgactc 720 gtccaacatc atacaacct attaatttcc cctcgtcaaa ataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gatccggtg agaatggcaa aagtttagc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaccaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 attackacaac aggaatcgaa tgcaccggc gcaggacc tgccagcgca tcacaat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgtttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt agccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaac aactctggcg catcggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccattatata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgttttcccg ttgaatatgg ctcatacac 1380 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100 gccttttc ggttcctggc cttttgctgg cttttgctc acatgttctt tcctgcgtta 2160 tccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggag cggaagagcg cctgatgcgg 2280. tattttctcc ttacgcatct gtgcggtatt tcacaccgca fatherggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatga catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgctgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaaaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagaga tcgagatctc gatcccggaga 4980 aattaacg actcactata ggggaattgt gagcggataa cattcccct ctagaataa 5040 ttttgttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100 agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaatg 5160 ggtcgcggat ccatgaaaaa agcgcgaatg attgtagaca aagaatataa atcggtgaa 5220 gtagataac ggatttatgg ctcgtttatc gaacatatgg gtcgtgcggt attgaaggc 5280 atatacgagc ctgatcaccc tgaagcggat gagatggat ttagaaaga tgtccagtcg 5340 ctgatcaaag aattacaggt tcccatc cgctatccgg gcggaactt ttatccgga 5400 tacaactggg aggacggtgt cggaccagtc gaaaaccgcc cgagacggct tgacttggca 5460 tggcaaacga cagaaaccaa tgaagtggga acaatgaat ttttactg gggcaaaaag 5520 gtgaacactg aggtcaat gggccgtcaac cttggcacaa gaggcataga tgccgcccgt 5580 aatctcgttg atattgcaa ccatccgaaa ggctcttact ggagtgattt aagagatcg 5640 catggctatg aacagccgta tggcatcaa acatggtgct taggaacga aatggatgga 5700 ccatggcaga tcggccacaa aacagctgat gaatacggac ggcttgccgc agagacagca 5760 aaggtcatga agtgggttga cccatcaatt gaactcgttg cctgcggcag ctcaaacagc 5820 ggtatgccga cctttatcga ttgggaagcg aaggtgcttg agcatacgta tgagcatgtc 5880 gactatatct ctcttcacac ttactacgga aaccgggata acaatctgcc aaactacttg 5940 gcacgttcta tggatttgga tcattttatc aaatcagtcg ctgcgacctg tgactatgta 6000 aaagcaaaaa cacgcagcaa gaaaactatc aatctctctc tggatgaatg gaacgtctgg 6060 taccactcaa atgaggctga taaaaaagtc gagccgtgga tcactgcgcg tccgatttta 6120 gaggatattt acaatttga agatgcctta tagtcggct ctctgctcat tacgatgctg 6180 cagcacgcag accgtgtgaa aattgcgtgt cttgcacagc ttgttaatgt catcgcgccg 6240 atcatgacgg aaaaaggcgg agaagcatgg agacagccga ttttctatcc atacatgcat 6300 gcttctgttt acggaagggg cgagtcactg aaaccgctta tttcttctcc taagtacgat 6360 tgttctgatt tcactgatgt gccatatgtt gatgctgctg ttgtgtactc tgaagaggaa 6420 gaaacactca ctattttgc ggtaaacaag gctgaggatc agatggagac ggagatttcg ctcagaggct ttgaatccta ccaaatcgca gagcacatcg tacttgagca tcaggatatc aaagcaacaa accagcataa cagaaaaaat gtcgttccgc attccaacgg atcatcgtct gtcagcgaaa acggcttaac tgctcatttc acgccgcttt cctggaatgt gatccgcctg 6660 aaaaaacagt cataaata ttttgtttaa ctttaagaag gagatataca tatgagcgaa agcacctatc cgagcgtga ggacctgacc ctggagga aagcgagcct gaccagcggt ggcgatgcgt ggcacctgca aggtgttgag agcaagggca tcccgggtta catgattacc 6840. gacggcccgc acggtctgcg taaaagcctg gcgagcagcg cgggtgaaac cgatctggac 6900. gatagcgttc cggcgacctg cttcccgccg gcggcgggtc tgagcagcag ctggaacccg 6960. 7020. gagctgatcc acaaagttgg cgaagcgatg gcggaggat gcatccagga aaaggtggcg gttattctgg gcccgggtgt taacatcaaa cgtaacccgc tgggtggccg ttgcttcgag tattggagcg aagacccgta cctggcgggt catgaggcga tcggcattgt ggaaggtgtt caaagcaagg gcgtgggtac cagcctgaaa cactttgcgg cgaacaacca ggagaccgac 7200 cgtctgcgtg ttgatgcgcg tattagcccg cgtgcgctgc gtgagatcta tttcccggcg 7260 tttgaacaca ttgtgaagaa agcgcaaccg tggaccatta tgtgcagcta caaccgtatc 7320 aacggtgttc acagcgcgca gaaccactgg ctgctgaccg acgtgctgcg tgatgagtgg 7380 ggcttcgcg gtatcgttat gagcgattgg ggcgcggacc atgatcgtgg tgcgagcctg 7440 aacgcgggcc tgaacctgga aatgccgcca agctataccg acgatcaaat cgtgtacgcg 7500 gttcgtgacg gtctgattac cccggcgcag ctggaccgta tggcgcaagg catgatcgat 7560 ctggtgaaca aaacccgtgc ggcgatgagc attgacaact atcgttttga cgtggatgcg 7620 catgatgagg ttgcgcacca ggcggcgatc gaaagcattg ttatgctgaa gaacgacgat 7680 gcgatcctgc cgctgaacgc gggtccggtg gcgaacccga gcgcgacccc gcaaaaaatc 7740 gcggttattg gcgagttcgc gcgtaccccg cgttaccagg gtggcggtag cagccacatc 7800 accccgacca agatgaccag ctttctggac accctggcgg aacgtggtat taaagcggat 7860 tttgcgccgg gttttaccct ggacctggag ccggcggacc cggcgctgga gagcgaagcg 7920 gtggaaaccg cgaagaacgc ggacgtggtt ctgatgtttc tgggtctgcc ggaggcggcg 7980 gagagcgaag gctttgaccg tgataccctg gatatgccgg cgaaacagat caccctgctg 8040 gaacaagttg cggcggcgaa ccagaacgtg gttgtggttc tgagcaacgg tagcgtgatc 8100 accgttgcgc cgtgggcgaa gaacgcgaaa ggtattctgg agagctggct gctgggtcaa 8160 agcggcggtc cggcgctggc ggacgtgatt ttcggtcaag ttagcccgag cggcaagctg 8220 gcgcagagca tcccgctgga cattaacgac gatccgagca tgctgaactg gccgggcgag 8280 gaaggtcacg tggattatgg cgagggtgtg tttgttggtt atcgttacta tgacacctac 8340 ggcaaggcgg ttgattaccc gttcggctat ggtctgagct acgcgacctt tgaaatcacc 8400 ggtgtggcgg ttgcgaaaac cggtgcgaac accgcgaccg tgaacgcgac cgttaccaac 8460 accagcgatg tggatgcggc ggagaccgtg caggtttacg tggttccggg caaggcggac 8520 gttgcgcgtc cgaagcacga gctgaaaggt ttcaccaagg tgtttctgaa aagcggcgaa 8580 agcaagaccg ttaccattga cctggatgag cgtgcgttcg cgtattggag cgaaaaatac 8640 aacgattggc acgtggaggc gggtgaatat gcgatcgaag tgggcgttag cagccgtgac 8700 attgcggata ccgtggcggt tgcgctggat ggtgatggta aaacccaacc gctgaccgaa 8760 tggagcacct acggcgagtg ggaagcggac ccgtttggcg cgaagatcgt tgcggcggtt 8820 gcggcggcgg gtgaagcggg tgaactgccg aaactgccgg ataacgcgat gatgcgtatg 8880 ttcctgaaca gcatgccgat caacagcctg ccgaccctgc tgggcgaggg cggtaagaaa 8940 attgcgcagt ttatggttga cgaatacgcg aagctgagca aataagaatt cgagctccgt 9000 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa 9060 caaagcccga aaagctg agttggctgc tgccaccgct gagcaataac tagcataacc 9120 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagggaa ctatatccgg 9180 at 9182

Claims

1. A method for preparing rare ginsenoside CK by using gene combination transformation, characterized in that: The method comprises the following steps: (1) The α-L-arabinofuranosidase gene BsAbfA from Bacillus subtilis str.168 was codon-optimized to obtain the BsAbfA-op gene; the β-glucosidase gene BbBgl2 from Bifidobacterium breve689b was codon-optimized to obtain the BbBgl2-op gene; and an IRBS sequence was inserted between the BsAbfA-op gene and the BbBgl2-op gene; wherein the BsAbfA gene sequence is shown in SEQ ID NO.1, the BsAbfA-op gene sequence is shown in SEQ ID NO.2, the BbBgl2 gene sequence is shown in SEQ ID NO.3, the BbBgl2-op gene sequence is shown in SEQ ID NO.4, and the IRBS sequence is shown in SEQ ID NO.5; (2) The gene sequence after inserting the IRBS sequence between the BsAbfA-op gene and the BbBgl2-op gene was ligated between the BamH I and EcoR I restriction sites of the pET28a vector to obtain a recombinant expression vector containing the BsAbfA-op and BbBgl2-op genes, which was named pET-BsAbfA-BbBgl2-op. The gene sequence of the recombinant expression vector pET-BsAbfA-BbBgl2-op is shown in SEQ ID NO. 6; the BsAbfA-op and BbBgl2-op genes were sequentially located downstream of the T7 promoter of the pET28a vector; (3) The recombinant expression vector pET-BsAbfA-BbBgl2-op was transformed into Escherichia coli BL21, and then selected by kanamycin, PCR and sequencing were performed to prepare recombinant bacteria containing the BsAbfA-op and BbBgl2-op genes; (4) The recombinant bacteria obtained in step (3) were cultured at 37°C until the OD600 reached 0.4-0.6, induced with 0.1-1.0 mmol / L IPTG for 2-10 hours, centrifuged, and then added with fresh LB medium to prepare a recombinant bacterial solution; (5) Ginsenoside Rc was dissolved in methanol and mixed with acetic acid-sodium acetate buffer at pH 4.0-6.0 to prepare a ginsenoside Rc substrate solution with a concentration of 1-50 g / L. The ginsenoside Rc substrate solution was then mixed with the recombinant bacterial solution induced by IPTG at a volume ratio of 1:(1-10), reacted at pH 5 and 40°C for 24 hours, inactivated in a 70-80°C water bath for 10-20 minutes, and centrifuged at room temperature for 10-15 minutes. The supernatant was collected to obtain rare ginsenoside CK.

2. The method for preparing rare ginsenoside CK by utilizing gene combination transformation according to claim 1, characterized in that: In the step (4), the concentration of fresh cells in the recombinant bacterial solution is 10-40 g / L.

3. A recombinant expression vector, characterized in that: The gene sequence of the recombinant expression vector is shown in SEQ ID NO. 6, and is named pET-BsAbfA-BbBgl2-op.

4. A recombinant bacterium, characterized in that The recombinant bacteria are prepared by transforming the recombinant expression vector pET-BsAbfA-BbBgl2-op described in claim 3 into Escherichia coli BL21, and then screening with kanamycin, PCR and sequencing to detect the recombinant bacteria containing BsAbfA-op and BbBgl2-op genes.

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