Method for biosynthesis of salidroside

Abstract

Provided is a method for biosynthesis of salidroside. The method comprises: under the action of a glycosyl donor, carrying out a catalytic reaction on a substrate 4-hydroxyphenethyl alcohol by using a glycosyltransferase OfT8GT1 having an amino acid sequence as shown in SEQ ID NO: 1, so as to obtain salidroside. The method relates to the technical field of biology, and can solve the problem in the prior art of lack of an efficient method for synthesizing salidroside. The method produces salidroside at a high yield, and is expected to be applied to industrial production of salidroside.

Description

Method for biosynthesis of salidroside

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410638087.9, filed on May 22, 2024, the disclosure of which is incorporated herein in its entirety by reference as part of the present application. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, and in particular, to a method for biosynthesis of salidroside. BACKGROUND

[0004] Salidroside, with a molecular formula of C 14 H 20 O7, has a structural formula of is a phenylethanoid glycoside compound of plant origin. Salidroside is the main active ingredient of medicinal plants of the genus Rhodiola, has strong antioxidant activity, can reduce inflammation, protect cells from oxidative stress, and relieve depression, fatigue, stress, and high altitude reaction, and is widely used in the pharmaceutical and cosmetic industries. Among them, medicine is the main application field of salidroside, in which salidroside is mainly used to manufacture anti-gout, hypoglycemic, anti-tumor and liver damage drugs, and nerve stimulants, etc.

[0005] Natural salidroside is mainly extracted from the plant Rhodiola, but Rhodiola grows in a special high-altitude environment, has a slow growth cycle, and the resource of Rhodiola is scarce, resulting in very limited production of salidroside. In addition, the content of salidroside in Rhodiola is low and the cost of extraction and purification is high, and the efficiency is low, which further deepens the rarity of salidroside. The chemical synthesis of salidroside requires complex hydroxyl protection and deprotection steps and expensive catalysts, which is high in cost, not friendly to the environment, harsh in operating conditions, and has a small amount or trace amount of other toxic chemicals remaining in the product, and poor safety. Therefore, developing a mild and efficient biological method to synthesize salidroside is conducive to reducing production costs and expanding market value.

[0006] Glycosylation refers to the process of adding sugars to proteins or lipids under the control of enzymes. Glycosylation is an important modification of proteins that regulates proteins, helps protein folding, and further affects biological processes such as cell signaling, immune response, and cell recognition. The last step of salidroside biosynthesis is the glycosylation reaction catalyzed by glycosyltransferase, which is a key rate-limiting step. Although a variety of glycosyltransferases have been reported to catalyze the synthesis of salidroside from 4-hydroxyphenethyl alcohol (tyrosol), their catalytic activity and regioselectivity still need to be further improved.

[0007] In summary, developing a highly efficient glycosyltransferase is of great significance for the industrial production of rhodioloside by microbial fermentation, and is a technical problem to be solved.

[0008] SUMMARY

[0009] The main purpose of the present application is to provide a method for biosynthesis of rhodioloside, to solve the problem of lack of efficient synthesis of rhodioloside in the prior art.

[0010] In order to achieve the above-mentioned purpose, according to one aspect of the present application, a method for biosynthesis of rhodioloside is provided, which catalyzes the substrate 4-hydroxyphenethyl alcohol by using the glycosyltransferase OfT8GT1 having the amino acid sequence shown in SEQ ID NO: 1 under the action of a glycosyl donor, to obtain the above-mentioned rhodioloside.

[0011] Further, the above-mentioned glycosyltransferase OfT8GT1 is selected from a crude enzyme solution or a purified enzyme solution containing the above-mentioned glycosyltransferase OfT8GT1.

[0012] Further, the above-mentioned method comprises: inoculating the recombinant cells capable of expressing the above-mentioned glycosyltransferase OfT8GT1 in an overnight culture into a fermentation medium for scale-up culture, culturing to OD600 to 0.6-0.8, adding an inducer for induction fermentation for 18-48h to collect the supernatant product, to obtain the above-mentioned rhodioloside, wherein the above-mentioned 4-hydroxyphenethyl alcohol is added together with the above-mentioned inducer.

[0013] Further, the condition for the above-mentioned scale-up culture is 30℃-37℃, 200rpm-220rpm.

[0014] Further, the condition for the above-mentioned induction fermentation is 28℃-30℃, 200rpm-220rpm.

[0015] Further, the above-mentioned inducer is IPTG.

[0016] Further, the final concentration of the above-mentioned inducer is 0.1mM-1mM.

[0017] Further, the fermentation medium formula comprises: NaCl, tryptone, yeast extract, glucose, 4-hydroxyphenethyl alcohol, antibiotic and MOPS.

[0018] Further, the fermentation medium formula comprises: 10g / L of the above-mentioned NaCl, 10g / L of the above-mentioned tryptone, 5g / L of the above-mentioned yeast extract, 20g / L-30g / L of the above-mentioned glucose, 40g / L.~50g / L MOPS and 50mg / L-100mg / L of kanamycin.

[0019] Further, the sugar donor is UDPG.

[0020] By applying the technical solution of the present application, the sugar transferase OfT8GT1 (having the amino acid sequence shown in SEQ ID NO: 1) of the present application has high catalytic activity, and thus can efficiently catalyze the synthesis of rhodiolin with 4-hydroxyphenethyl alcohol (tyrosol) as the substrate, and the yield of rhodiolin is higher than that of other sugar transferases in the prior art for catalyzing the synthesis of rhodiolin with 4-hydroxyphenethyl alcohol (tyrosol). The present application overcomes the problem of lack of efficient synthesis method of rhodiolin in the prior art, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings constituting a part of the specification of the present application are used to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0022] FIG. 1 shows the SDS-PAGE diagram of the expression of the sugar transferase OfT8GT1 protein according to Embodiment 2 of the present application, wherein lane 1 represents protein Marker; lane 2 represents the total protein of the bacteria after being broken; lane 3 represents the total protein in the supernatant after the bacteria are broken; and lane 4 represents the protein after being purified.

[0023] FIG. 2 shows the HPLC detection diagram of the content of rhodiolin in the fermentation broth of the strain according to Embodiment 4 of the present application, wherein FIG. 2A shows the HPLC diagram of the rhodiolin standard; and FIG. 2B shows the HPLC diagram of the rhodiolin produced by BL21(DE3) / pET28a-OfT8GT1 after being fermented for 48h.

[0024] FIG. 3 shows the LC-MS mass spectrum diagram according to Embodiment 4 of the present application, wherein A shows the mass spectrum diagram of the rhodiolin standard, and B shows the mass spectrum diagram of the rhodiolin produced by BL21(DE3) / pET28a-OfT8GT1 after being fermented for 48h. DETAILED DESCRIPTION

[0025] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0026] As mentioned in the background, salidroside has antioxidant, antidepressant, anti-fatigue and anti-high altitude reaction, etc. However, the natural salidroside is limited by the quantity of the plant Rhodiolae Crenulatae and the chemical synthesis of salidroside has the problems of complicated steps and poor regioselectivity. Therefore, the mild biological method becomes the preferred method for synthesizing salidroside. At present, the glycosyltransferase used for synthesizing salidroside in the prior art has the defect of low activity (for specific comparison data, see Example 3). Therefore, the present application first discovers that the existing glycosyltransferase mutant derived from Osmanthus fragrans, i.e. glycosyltransferase OfT8GT1 (having the amino acid sequence shown in SEQ ID NO: 1) can synthesize salidroside and the yield of salidroside synthesized by the enzyme is higher than that of other glycosyltransferases, which is helpful for the industrial production of salidroside.

[0027] In a typical embodiment of the present application, a method for biosynthesizing salidroside is provided, which utilizes the glycosyltransferase OfT8GT1 having the amino acid sequence shown in SEQ ID NO: 1 to catalyze the substrate 4-hydroxyphenethyl alcohol under the action of a glycosyl donor to obtain salidroside. The glycosyltransferase OfT8GT1 is selected from a crude enzyme solution or a purified enzyme solution containing the glycosyltransferase.

[0028] The amino acid sequence SEQ ID NO: 1 of the glycosyltransferase OfT8GT1 of the present application is as follows:

[0029] The nucleotide sequence SEQ ID NO: 2 of the glycosyltransferase OfT8GT1 of the present application is as follows:

[0030] The method for biosynthesizing salidroside of the present application can be used for in vitro synthesis using a crude enzyme solution or a purified enzyme solution, or in vivo synthesis using a recombinant cell containing the enzyme gene of the present application. In order to make the process of biosynthesizing salidroside more convenient, in a preferred embodiment of the present application, an in vivo synthesis method is adopted, which comprises: inoculating the overnight cultured recombinant cell capable of expressing the glycosyltransferase OfT8GT1 into a fermentation medium for expansion culture, culturing to OD600 of 0.6-0.8, adding an inducer for induction fermentation for 18-48 h to collect the supernatant product to obtain the salidroside, wherein the 4-hydroxyphenethyl alcohol (sterilized by filtering with a sterile membrane) is added together with the inducer. The method is simple and convenient, the yield of the produced salidroside is high, and can be applied to the industrial production of salidroside.

[0031] The recombinant cells need to be expanded after inoculation into the fermentation medium to increase the number of the recombinant cells, and the conditions for the expansion are closely related to the number and growth status of the recombinant cells. When the number of the recombinant cells reaches a certain concentration, the cells need to be induced to fermentation culture. In order to make the recombinant cells produce as much rhodiolin as possible, it is better to culture the recombinant cells under the optimal induction fermentation culture conditions for the production of rhodiolin. In a preferred embodiment of the present application, the conditions for the expansion are 30-37°C and 200-220 rpm; preferably, the conditions for the induction fermentation are 28-30°C and 200-220 rpm.

[0032] The appropriate inducer is selected according to the expression vector. The appropriate concentration of the inducer is selected according to the expression of the target protein in different recombinant cells. In a preferred embodiment of the present application, the inducer is IPTG; more preferably, the final concentration of the inducer is 0.1-1 mM; further preferably, the final concentration of the inducer is 0.1 mM. Under the above preferred conditions, the method of the present application can produce rhodiolin to the maximum extent, improve the yield of rhodiolin, and speed up the production efficiency of rhodiolin.

[0033] The fermentation medium provides appropriate conditions for the growth and metabolism of the recombinant cells. Different recombinant cells have different requirements for the nutritional components in the fermentation medium, and different fermentation media can be selected according to the types of the recombinant cells. In a preferred embodiment of the present application, the formula of the fermentation medium includes NaCl, tryptone, yeast extract, glucose, 4-hydroxyphenethyl alcohol, antibiotics, and MOPS (3-(N-methylpyrazine)-propanesulfonic acid); preferably, 10 g / L of the above NaCl, 10 g / L of the above tryptone, 5 g / L of the above yeast extract, 20-30 g / L of the above glucose, and 0.5 g / L of the above 4-hydroxyphenethyl alcohol; preferably, the antibiotics are kanamycin; more preferably, the concentration of the kanamycin is 50-100 mg / L. The addition of antibiotics is an essential step in the conventional heterologous expression process, which plays a role in killing bacteria, and the type and concentration of the antibiotics can be determined according to the type of the expression vector. Under the above preferred conditions, the method of the present application provides the best nutritional conditions for the recombinant cells, which is an important guarantee for the high production of rhodiolin by the recombinant cells.

[0034] In order to ensure that the reaction of generating salidroside proceeds smoothly, the sugar donor is essential. In a preferred embodiment of the present application, the sugar donor is UDPG. In the in vitro catalytic experiment, the sugar donor can be provided by directly adding UDPG to the catalytic reaction system, and in the in vivo catalytic experiment, the sugar donor can be provided by adding glucose to the fermentation medium, and the glucose is metabolized by the recombinant cells to generate UDPG. Under the action of the sugar donor, the sugar transferase OfT8GT1 can play a role, and finally salidroside is generated. Based on the above preparation method, the sugar transferase OfT8GT1 and the substrate 4-hydroxyphenethyl alcohol (tyrosol) can be used to catalyze 4-hydroxyphenethyl alcohol (tyrosol) to obtain salidroside. The sugar transferase OfT8GT1 has high catalytic activity, high yield of generated salidroside, wide application prospect and high economic value.

[0035] The present application will be further described in detail below in combination with specific examples, which cannot be understood as limiting the scope of the present application. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Example 1 Construction of recombinant E. coli BL21(DE3) / pET28a-OfT8GT1

[0037] The OfT8GT1 gene sequence from Osmanthus fragrans was retrieved through NCBI, and the E. coli codon optimization and plasmid pET28a-OfT8GT1 synthesis were performed by Jiutian Gene Technology (Tianjin) Co., Ltd. The nucleotide sequence is shown as SEQ ID NO: 2. The OfT8GT1 gene fragment was connected to the plasmid pET28a-OfT8GT1 to construct a recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21(DE3) to obtain recombinant E. coli BL21(DE3) / pET28a-OfT8GT1. The specific transformation method is as follows: take the BL21(DE3) competent (1.5 ml EP tube contains 50 μL competent) on ice to thaw, add 50 ng plasmid to the competent, mix well, ice bath for 30 min, heat shock at 42℃ water bath for 90 s, add 600 μL recovery liquid (LB medium), incubate at 37℃ for 50 min, after incubation, spread the whole bacterial liquid on the plate containing 50 mg / L kanamycin (LB medium), and place in a 37℃ incubator for overnight culture.

[0038] Example 2 Protein expression verification of the sugar transferase OfT8GT1

[0039] After overnight culture, the cloned strains were inoculated into LB liquid medium containing 50 mg / L kanamycin, and shaken at 37°C until the OD600 reached 0.8-1. After adding IPTG at a final concentration of 0.15 mM, the culture was induced at 30°C for 16-20 hours. The bacterial cells were collected by centrifugation at 4°C and 5000 rpm, resuspended in PBS buffer, and then broken by ultrasonic instrument in an ice-water mixture at 180W for 3-5 min.

[0040] The supernatant and precipitate of the crude OfT8GT1 enzyme solution were detected by SDS-PAGE, and the results are shown in Figure 1. There is an obvious protein band between 40-55KD, and the molecular weight is close to the molecular weight of his-OfT8GT1, which is 55.4KD. The OfT8GT1 protein expression is clear.

[0041] Example 3: In vitro catalysis of OfT8GT1 to synthesize 4-hydroxyphenethyl alcohol to form rhodiolin

[0042] The purified OfT8GT1 enzyme was obtained by the methods of Examples 1 and 2, and the specific steps are as follows:

[0043] (1) Resuspend the bacterial cells: 8g of bacterial cells were resuspended in 80mL of equilibration buffer, and stirred with a glass rod until there were no granular bacterial clumps.

[0044] (2) Break the bacterial cells: The resuspended solution was broken in a high-pressure homogenizer, with 500bar for 1 pass and 800bar for 2 passes. The cooling circulation water was turned on in advance to reduce the temperature.

[0045] (3) Purification: 2mL Thermo HisPur TM The nickel-NTA resin (model number: 88221) was mixed with 80mL of crude enzyme solution at 4°C for one hour, and then the mixed crude enzyme solution was filtered and purified on a chromatography column.

[0046] (4) Wash the impurities: The impurity proteins were washed with 20mM imidazole buffer, and the Ni filler was washed for 5-10 times the column volume until the protein concentration of the effluent was below 0.1mg / ml, as determined by the Bradford method.

[0047] (5) Elution: The target protein was eluted with 250mM imidazole buffer, and the Ni filler was washed for 5-10 times the column volume until the protein concentration of the effluent was below 0.1mg / ml, as determined by the Bradford method.

[0048] (6) Concentration and buffer exchange: The collected OfT8GT1 purified enzyme was concentrated and buffer exchanged by using 30KD ultrafiltration tube. 8-10ml of equilibration buffer was added to remove the salt. The mixture was centrifuged at 3750rpm for 20min. This step was repeated 3-5 times until the protein volume was concentrated to 0.5-1ml. The final protein concentration was measured.

[0049] (7) Bradford method for measuring protein concentration: Coomassie brilliant blue (CBB) method for measuring protein content belongs to dye binding method. Coomassie brilliant blue is red in free state, with maximum light absorption at 488nm; when it is combined with protein, it becomes cyan, and the protein-pigment combination has maximum light absorption at 595nm wavelength. Its light absorption value is proportional to the protein content, so it can be used for quantitative determination of protein.

[0050] The specific steps are as follows:

[0051] (1) 1.00g of bovine serum albumin (BSA) was weighed with a balance and dissolved in deionized water to make a 100ml solution with a concentration of 10mg / ml. 100ul, 80ul, 60ul, 40ul and 20ul of BSA solution were taken with a pipette and placed in 1.5ml EP tubes, and 900ul, 920ul, 940ul, 960ul and 980ul of phosphate buffer solution (PBS) were added to make a 1ml solution, respectively. Shake to mix the solution evenly. A set of BSA solutions with concentrations of 1.0mg / ml, 0.8mg / ml, 0.6mg / ml, 0.4mg / ml and 0.2mg / ml were obtained.

[0052] (2) 100ul of the prepared BSA solution was taken with a pipette and placed in a 1.5ml EP tube, and 900ul of PBS solution was added to each, and the solution was shaken to mix evenly. A set of BSA solutions with concentrations of 0.10mg / ml, 0.08mg / ml, 0.06mg / ml, 0.04mg / ml and 0.02mg / ml were obtained. In addition, 1ml of PBS solution (BSA solution concentration is 0mg / ml) was taken as a control test.

[0053] (3) 50ul of the prepared BSA solution was taken with a pipette and added dropwise to the well plate, and 200ul of Coomassie brilliant blue (CBB) was added, respectively. After standing for 10min, the absorbance of the BSA solution was measured with an enzyme marker, and the relationship curve between absorbance and BSA concentration was drawn with orgin, and the standard curve was measured: y=0.5421x+0.5047, y=OD595nm, x=protein concentration (mg / mL).

[0054] Sample detection: take 30 μL sample + 150 μL Brandford solution into the enzyme label plate, react for 5 min, measure OD595nm, and calculate the sample protein concentration according to the BSA standard curve.

[0055] Take the above purified and concentrated OfT8GT1 to carry out the following reaction:

[0056] System 1: 1 mM 4-hydroxyphenethyl alcohol, 2 mM UDP-glucose (UDPG), 10 μg OfT8GT1 purified enzyme, 0.05 M Tris-HCl 7.4, 2 mM ascorbic acid, supplemented with deionized water to 100 μL, the reaction system is the same as CN 114317480A;

[0057] System 2: 2 mM 4-hydroxyphenethyl alcohol, 2.5 mM UDP-glucose, 20 μg OfT8GT1 purified enzyme, 0.1 M PBS 7.0, supplemented with deionized water to 100 μL, the reaction system is the same as CN 116716270A;

[0058] System 3: 5 mM 4-hydroxyphenethyl alcohol, 2.5 mM UDP-glucose, 50 μg OfT8GT1 purified enzyme, 0.1 M PBS 7.0, supplemented with deionized water to 100 μL, the reaction system is the same as CN 116716270A.

[0059] After the above systems are placed in a 37°C water bath for 0.5-1 hour, 100 μL of ice methanol is added to each reaction system to terminate the reaction, and the reaction system is cooled on ice for 2 min and centrifuged at 12000 rpm for 5 min. The reaction solution is filtered with a 0.22 μm organic phase filter to prepare a sample, and HPLC analysis is performed to determine the amount of salidroside synthesized in the reaction system. The liquid phase peak area of 4-hydroxyphenethyl alcohol (tyrosol) and the liquid phase peak area of salidroside are detected, and the ratio of the liquid phase peak area of salidroside to the liquid phase peak area of 4-hydroxyphenethyl alcohol (tyrosol) is the conversion rate. The catalytic activity of the glycosyltransferase OfT8GT1 is evaluated according to the conversion rate.

[0060] As shown in Table 1, after the in vitro catalytic reaction is completed, obvious salidroside synthesis can be detected, and the conversion rate of the substrate 4-hydroxyphenethyl alcohol reaches 90% or more. Compared with the glycosyltransferases reported in the prior art patents, OfT8GT1 is more inclined to synthesize salidroside with 4-hydroxyphenethyl alcohol as the substrate when 1 mM 4-hydroxyphenethyl alcohol and 1 mM 3,4-dihydroxyphenethyl alcohol (hydroxytyrosol) are used as substrates, and the highest conversion rate can reach 90%. In addition, by comparing different concentrations of 4-hydroxyphenethyl alcohol and different amounts of different glycosyltransferases, OfT8GT1 of the present application has higher catalytic efficiency and lower enzyme dosage in catalyzing the synthesis of salidroside, which is conducive to achieving higher economic value.

[0061] Table 1 Note: + represents conversion rate of 20-40%, ++ represents conversion rate of 40-60%, +++ represents conversion rate of 60-80%, ++++ represents conversion rate of 80-90%, +++++ represents conversion rate of more than 90%.

[0062] Fermentation culture of recombinant E. coli BL21(DE3) / pET28a-OfT8GT1

[0063] Preparation of seed liquid: pick a colony of PCR-verified correct monoclonal into LB medium containing 50 mg / L kanamycin, 37°C, 200 rpm overnight culture for 18 h.

[0064] Fermentation culture: detect the concentration of seed liquid, calculate the inoculation volume (mL) according to the initial bacterial liquid concentration OD600 of 0.1, transfer to 50 mL fermentation medium (LB+20 g / L glucose+MOPS) containing 80-120 mg / L kanamycin, 35-38°C fermentation culture until OD600 is 0.8-1, then add 0.5 g / L 4-hydroxyphenethyl alcohol (tyrosol) and 0.1 mM IPTG for induction, reduce the temperature to 28-30°C, continue to induce culture at 200 rpm for 18-48 h.

[0065] Example 5. Synthesis of salidroside by recombinant E. coli BL21(DE3) / pET28a-OfT8GT1

[0066] Ferment for 48 h, centrifuge 1 mL sample, take the supernatant for HPLC detection, conditions: chromatographic column Atlantis T3 4.6*100 mm 3 μm, mobile phase H2O+0.1% TFA and ACN+0.1% TFA, flow rate 1.0 mL / min, column temperature 40°C, UV detector, detection wavelength 220 nm, detection time 12.0 min. At the same time, detect organic acids and glucose: chromatographic column Aminex HPX-87H column 300*7.8 mm, mobile phase H2O+5 mM H2SO4, flow rate 0.6 mL / min, column temperature 50°C, RID detector, detection time 25.0 min.

[0067] The results show that when the fermentation is carried out for 48 h, the recombinant strain BL21(DE3) / pET28a-OfT8GT1 can produce 653.6 mg / L of salidroside with 4-hydroxyphenethyl alcohol (tyrosol) as a precursor, and the molar conversion rate reaches 83.1%, as shown in Table 2 below, and the HPLC spectrum is shown in Figure 2B, the retention time Rt of salidroside is 5.1 min, which is consistent with the retention time of the salidroside standard (Figure 2A). The LC-MS spectrum is shown in Figure 3B (see Table 4 for related experimental parameters and results), wherein [M-H]- = 299.3, which is consistent with the molecular weight [M-H]- = 299.2 of the salidroside standard spectrum shown in Figure 3A (see Table 3 for related experimental parameters and results), thus it can be determined that the glycosyltransferase OfT8GT1 can catalyze 4-hydroxyphenethyl alcohol (tyrosol) to synthesize salidroside.

[0068] Table 2

[0069] Table 3 Peak Results / indicates that the data is not provided.

[0070] Table 4 Peak Results / indicates that the data is not provided.

[0071] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: the present application first discovers that the existing glycosyltransferase OfT8GT1 derived from Osmanthus fragrans can efficiently catalyze the reaction of the glycosyl donor UDP-glucose and the substrate 4-hydroxyphenethyl alcohol to generate a single glycosylation product salidroside, wherein 0.5 g / L of 4-hydroxyphenethyl alcohol is added as a substrate by fermentation culture, and the concentration of salidroside in the supernatant can reach 653.6 mg / L. Compared with other glycosyltransferases in the prior art, the glycosyltransferase OfT8GT1 has high activity and high yield of salidroside, has unique advantages, and is expected to be applied to the industrial production of salidroside, has a wide prospect and high value.

[0072] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for biosynthesis of salidroside, characterized in that, The glycosyltransferase OfT8GT1 having the amino acid sequence shown in SEQ ID NO: 1 is used to catalyze the reaction of a substrate 4-hydroxyphenethyl alcohol under the action of a glycosyl donor to obtain the said rhodioside.

2. The method of claim 1, wherein, The glycosyltransferase OfT8GT1 is selected from a crude enzyme solution or a purified enzyme solution containing the glycosyltransferase OfT8GT1.

3. The method of claim 1, wherein, The method comprises: The recombinant cells capable of expressing the glycosyltransferase OfT8GT1 are inoculated into a fermentation medium for scale-up culture, and are cultured to OD600 of 0.6-0.8, and then an inducer is added for induction fermentation for 18-48 h to collect the supernatant product to obtain the said rhodioside, wherein the 4-hydroxyphenethyl alcohol is added together with the inducer.

4. The method of claim 3, wherein, The scale-up culture is carried out at 30-37℃ and 200-220 rpm.

5. The method of claim 3, wherein, The induction fermentation is carried out at 28-30℃ and 200-220 rpm.

6. The method of claim 3, wherein, The inducer is IPTG.

7. The method of claim 3, wherein, The final concentration of the inducer is 0.1-1 mM.

8. The method of claim 3, wherein, The fermentation medium formula comprises NaCl, tryptone, yeast extract, glucose, 4-hydroxyphenethyl alcohol, antibiotics and MOPS.

9. The method of claim 8, wherein, The fermentation medium formula comprises 10 g / L of the NaCl, 10 g / L of the tryptone, 5 g / L of the yeast extract, 20-30 g / L of the glucose, 0.5 g / L of the 4-hydroxyphenethyl alcohol, 40-50 g / L of the MOPS and 50-100 mg / L of kanamycin.

10. The method of claim 1, wherein, The glycosyl donor is UDPG.

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