A glycosyltransferase mutant and its application in the synthesis of salidroside

Abstract

The glycosyltransferase mutant provided by the present invention has significantly improved regioselectivity for tyrosol, which is improved from 51% of the wild type to more than 99%, and can generate a single glycosylation product salidroside when used in catalytic synthesis of salidroside; in addition, the microbial glycosyltransferase mutant of the present invention has significantly improved catalytic activity for tyrosol, and the specific enzyme activity is improved from 14 mU / mg of the wild type to 97 mU / mg, and the substrate conversion rate is higher than 99%; the present invention successfully solves the key problems of difficult expression of glycosyltransferase, low regioselectivity and low activity in the preparation process of salidroside, showing a very large industrial application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to a glycosyltransferase mutant and application thereof in catalyzing tyrosol to prepare salidroside. Background Art

[0002] Salidroside is the main medicinal ingredient in Rhodiola rosea. Clinical research results show that salidroside not only has the main functions of anti-hypoxia, anti-cold, anti-fatigue, anti-microwave radiation, anti-virus, anti-tumor, but also has the effects of enhancing attention, improving work efficiency, delaying body aging, and preventing senile diseases. It has very important application value in military medicine, aerospace medicine, sports medicine and health medicine. It is an environmental adaptation drug with great development prospects and has attracted much attention in recent years. Salidroside is non-toxic and harmless, and can be directly used as a natural health food or medicine, and can also be added to almost most foods such as baked products, meat products, flour products, dairy products, jellies, beverages, etc. Salidroside also has a good market prospect in cosmetics and feed additives.

[0003] The current production methods of salidroside are mainly plant extraction and chemical synthesis. Rhodiola rosea grows in high-altitude pollution-free areas, and its wild resources are rare. The content of salidroside is very low. The use of plant extraction methods will destroy the wild plant resources of Rhodiola rosea and is expensive. The use of chemical synthesis methods requires selective protection, activation or the use of expensive metal catalysts, which is costly and easily causes environmental pollution.

[0004] Glycosyltransferases (GT; EC 2.4.xy) are a class of enzymes that can catalyze the transfer of sugar moieties from activated glycosyl donor molecules to specific glycosyl acceptor molecules and form glycosidic bonds. The catalytic glycosylation reaction is a key step in the synthesis of glycoside compounds with complex and diverse structures and physiologically active functions. Compared with chemical methods, the glycosylation reaction mediated by glycosyltransferases does not require cumbersome protection and deprotection, and does not require the use of catalysts such as red phosphorus and bromine that are harmful to the human body and the environment. It is an environmentally friendly biological modification method. Therefore, the synthesis of salidroside by glycosylation of tyrosol by glycosyltransferases has received more and more attention in the field of organic synthesis. Since most natural products with important pharmacological effects are found in plants, plant-derived glycosyltransferases have become the main research object. However, plant-derived glycosyltransferases have the problems of difficult expression and serious inclusion bodies, which seriously limit the application of glycosyltransferases in the synthesis of salidroside. Compared with plant-derived glycosyltransferases, microbial-derived glycosyltransferases are easy to heterologously express using model strains such as Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis, making them easier to achieve industrial production. However, in the face of the non-natural substrate molecule tyrosol, the glycosylation reaction catalyzed by the reported microbial-derived glycosyltransferases not only produces salidroside, but also produces a large amount of byproduct icariin D2. The production of byproducts will not only reduce the yield of salidroside, but also greatly increase the subsequent separation and purification costs. Therefore, improving the regioselectivity of microbial glycosyltransferases for tyrosol through molecular modification is the key to achieving the biosynthesis of salidroside. Summary of the invention

[0005] The present invention aims at the problem of low regional selectivity of microbial glycosyltransferases, and obtains a class of glycosyltransferase mutants with high selectivity for the alcoholic hydroxyl group of tyrosol by performing site-directed saturation mutagenesis on amino acids at the substrate binding pocket, thereby significantly improving the yield of salidroside.

[0006] In order to solve the above technical problems, the first object of the present invention is to provide a glycosyltransferase mutant, which is as follows:

[0007] The methionine at position 112 of the amino acid sequence shown in SEQ ID NO.1 is mutated to leucine, and the amino acid sequence is shown in SEQ ID NO.2; or

[0008] The methionine at position 112 and the isoleucine at position 325 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine and tyrosine, respectively, and the amino acid sequence is shown in SEQ ID NO.3; or

[0009] The methionine at position 112, the isoleucine at position 325, and the leucine at position 70 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, and arginine, respectively, and the amino acid sequence is shown in SEQ ID NO.4; or

[0010] The methionine at position 112, the isoleucine at position 325, the leucine at position 70 and the glutamine at position 136 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, arginine and glutamic acid, respectively, and the amino acid sequence is shown in SEQ ID NO.5; or

[0011] The methionine at position 112, isoleucine at position 325, leucine at position 70, glutamine at position 136 and isoleucine at position 67 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, arginine, glutamic acid and glutamic acid, respectively, and the amino acid sequence is shown in SEQ ID NO.6; or

[0012] The methionine at position 112, the isoleucine at position 325, the leucine at position 70, the glutamine at position 136, the isoleucine at position 67 and the methionine at position 77 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, arginine, glutamic acid, glutamic acid and arginine, respectively, and the amino acid sequence is shown in SEQ ID NO.7.

[0013] The second object of the present invention is to provide a gene encoding the glycosyltransferase mutant according to claim 1.

[0014] The third object of the present invention is to provide an expression vector carrying the gene described in claim 2.

[0015] The fourth object of the present invention is to provide a microorganism expressing the above-mentioned glycosyltransferase mutant, or carrying the above-mentioned gene or the above-mentioned expression vector.

[0016] As a specific example, the microorganism includes but is not limited to genetically engineered bacteria.

[0017] The fifth object of the present invention is to provide a use of the above-mentioned glycosyltransferase mutant, or the above-mentioned gene, or the above-mentioned expression vector, or the above-mentioned microorganism in the synthesis of salidroside.

[0018] As a specific embodiment, the synthesis process of salidroside is as follows:

[0019] In the presence of UDP-glucose provided by the UDP-glucose circulation system, the substrate tyrosol undergoes a glycosylation reaction under the catalysis of the glycosyltransferase mutant to produce salidroside. Specifically, firstly, an enzyme solution of the glycosyltransferase mutant is prepared, the enzyme solution or a genetically engineered bacterium capable of expressing the glycosyltransferase mutant is added to the substrate tyrosol, mixed with the UDP-glucose circulation system, and a glycosylation reaction is carried out in the mixed system to prepare salidroside.

[0020] The reaction temperature is 15-50° C., the pH value of the reaction system is 6-10, and the UDP-glucose concentration is 0-2 mM.

[0021] The enzyme solution can be a crude enzyme solution obtained by disrupting cells of genetically engineered bacteria expressing the glycosyltransferase mutant or a purified enzyme solution.

[0022] The specific cycle process of the UDP-glucose cycle system is as follows: In the presence of UDP, sucrose is catalyzed by sucrose synthase to produce glucose, fructose and UDP-glucose, and the generated UDP-glucose participates in the synthesis of salidroside again. The sucrose concentration is 0-600mM.

[0023] The beneficial technical effects achieved by the present invention are as follows: the glycosyltransferase used in the present invention is of microbial origin, and compared with the glycosyltransferase of plant origin, the glycosyltransferase of the present invention is easier to be efficiently and soluble expressed in model strains such as Escherichia coli; the glycosyltransferase mutant obtained by the present invention has significantly improved regional selectivity for tyrosol, which is increased from 51% of the wild type to more than 99%, and can generate a single glycosylated product salidroside; in addition, the microbial glycosyltransferase mutant of the present invention has significantly improved catalytic activity for tyrosol, and the specific enzyme activity is increased from 14mU / mg of the wild type to 97mU / mg, and the substrate conversion rate is higher than 99%; the present invention successfully solves the key problems of difficult expression of glycosyltransferase, low regional selectivity and low activity in the preparation process of salidroside, and shows a very large industrial application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE analysis of the overexpression effect of glycosyltransferase in Escherichia coli; M is a marker; 1 is before induction; 2 is after induction; 3 is the supernatant after induction;

[0025] Figure 2 Molecular docking of glycosyltransferase BlYjiC and substrate tyrosol and key amino acid residues near the substrate pocket, where the cyan part represents the tyrosol molecule;

[0026] Figure 3The figure is a mutation landscape diagram of the glycosyltransferase BlYjiC regarding regional selectivity; the vertical axis represents the position of the mutated amino acid residue, and the horizontal axis represents the 20 substituted amino acid residues; the wild-type amino acid residue at each position is represented by a thick solid square, and the square containing × represents a mutant showing no enzyme activity;

[0027] Figure 4 Demonstration of the regioselectivity of glycosyltransferase BlYjiC and its mutants; A is the evolutionary process of glycosyltransferase BlYjiC in terms of regioselectivity, regio is regioselectivity, and conv is conversion rate; B is the HPLC chromatogram of glycosyltransferase BlYjiC and its mutants catalyzing tyrosol, Tyrosol is tyrosol; Salidroside is salidroside; Icariside is icariin D2;

[0028] Figure 5 is the specific enzyme activity of wild-type glycosyltransferase BlYjiC and mutants;

[0029] Figure 6 Schematic diagram of the preparation of salidroside by coupling glycosyltransferase mutants and sucrose synthase;

[0030] Figure 7 The time curve of the glycosyltransferase mutant (M112L / I325Y / L70R / Q136E / I67E / M77R) catalyzing tyrosol to produce salidroside. Tyrosol: tyrosol; Salidroside: salidroside; Icariside: icariin D2;

[0031] Figure 8 Salidroside 1 H NMR spectrum;

[0032] Fig. 9 Salidroside 13 C NMR spectrum. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The experimental methods described in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

[0036] The DNA polymerase (2×Phanta Max Master Mix), Dpn I enzyme, recombination cloning kit and plasmid extraction kit used in the examples of the present invention were purchased from Takara Biotechnology Co., Ltd.; the synthesis of genes and primers and gene sequencing were completed by General Biotechnology (Anhui) Co., Ltd., and the use of the above reagents refers to the product instructions.

[0037] The expression vector involved in the present invention is pET-28a(+), and the host used is Escherichia coli BL21(DE3), all of which are preserved in the laboratory of the School of Biological and Pharmaceutical Engineering of Wanxi University where the inventor works.

[0038] All tyrosol, salidroside standard samples, and UDP-glucose used in the present invention were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; other commonly used reagents were purchased from Anage Chemical Reagent Co., Ltd.

[0039] The three-letter or one-letter representation of amino acids used in the present invention adopts the amino acid code specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).

[0040] Example 1 Cloning of glycosyltransferase

[0041] The glycosyltransferase gene was amplified using the genome of Bacillus licheniformis ATCC14580 as a template, F and R as primers, as shown in Table 1, and the amino acid sequence encoded by it was shown in SEQ ID NO.1, named WT-BlYjiC, and the amplified BlYjiC gene was connected to the pET-28a(+) plasmid (with His-tag purification tag at the C-terminus) by a one-step cloning kit (ClonExpress II One Step Cloning Kit) to construct a recombinant plasmid pET28a-BlYjiC. The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells, positive clones were screened and detected, and recombinant engineering bacteria containing the BlYjiC gene were obtained.

[0042] Table 1 Primers used for cloning glycosyltransferases

[0043] Primer name Primer sequences F aagaaggagatatactgcagATGGGACATAAACATATCGCGA R cggagctcgaattcggatccTTATTTTACTCCTGCGGGTGC

[0044] SEQ ID NO.1 WT-BlYJiC

[0045] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQIRELMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDFMAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDE RFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEITARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

[0046] Example 2 Expression and purification of recombinant glycosyltransferase BlYjiC

[0047] The recombinant engineered bacterial strain was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C on an orbital shaker at 200 rpm for 12 hours. 1 mL of the culture was added to a 250 mL Erlenmeyer flask containing 50 mL LB medium and cultured aerobically at 37°C and 200 rpm. When OD 600 When the pH reached about 0.6-0.8, the cells were induced with 0.1 mM IPTG. The cells were harvested after induction at 18°C ​​for 24 hours. The cells were centrifuged, resuspended in 50 mM potassium phosphate buffer, pH 8.0, and disrupted by ultrasound. The cell extract was centrifuged at 12,000 rpm for 20 minutes. The supernatant was the crude enzyme solution. The supernatant and precipitate were analyzed by SDS-PAGE to detect the expression of BlYjiC. The results are shown in Figure 1 As shown, it can be seen that the target protein content in the supernatant is comparable to the total target protein content, proving that BlYjiC can be efficiently and soluble expressed in E. coli, which is better than the expression effect of most plant-derived glycosyltransferases.

[0048] Recombinant glycosyltransferase BlYjiC was purified by nickel column affinity chromatography:

[0049] 1) Sample pretreatment: Resuspend the cells in loading buffer (20 mM pH 8.0 phosphate buffer, 25 mM imidazole, 500 mM NaCl), disrupt by ultrasonication in an ice bath, centrifuge at 4°C, 12000 rpm to remove cell debris, and filter the supernatant with a 0.22 μm filter membrane;

[0050] 2) Loading: Wash the nickel column with 10 column volumes of loading buffer, and slowly load the pretreated sample in (1) onto the column to allow the protein to fully bind to the nickel column;

[0051] 3) Washing: Wash the nickel column with 10 column volumes of washing buffer (20 mM pH 8.0 phosphate buffer, 50 mM imidazole, 500 mM NaCl);

[0052] 4) Elution of target protein: Elution of target protein was performed with elution buffer (20 mM pH 8.0 phosphate buffer, 250 mM imidazole, 500 mM NaCl);

[0053] 5) Ultrafiltration and storage: The collected eluate was transferred to an ultrafiltration tube, and the protein sample was desalted and concentrated by centrifugal ultrafiltration at 4°C; glycerol with a final concentration of 20% was added to the ultrafiltered protein sample, and the sample was quickly frozen in liquid nitrogen and stored at -80°C.

[0054] Example 3 Activity and regioselectivity determination of recombinant glycosyltransferase BlYjiC

[0055] 50mM potassium phosphate buffer (pH 8.0), 2mM tyrosol, 5mM UDP-glucose and an appropriate amount of enzyme were mixed and shaken at 30°C and 1000rpm. The reaction was terminated by adding 4 times the volume of methanol and analyzed by high performance liquid chromatography (HPLC). The unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μM product per minute under standard assay conditions. The regional selectivity was calculated as: molar concentration of product salidroside / molar concentration of total product*100%. HPLC detection used a C18 column (ZORBAX SB-C18, Agilent, USA) to analyze glycosylated products at a flow rate of 1mL / min at 30°C, and the detection wavelength was 275nm.

[0056] Example 4 Site-directed saturation mutagenesis of recombinant glycosyltransferase BlYjiC

[0057] like Figure 2As shown, the present invention discovered through homology modeling and molecular docking that the substrate tyrosol has two spatial orientations in the active pocket. Therefore, the present invention screened out amino acids near the two spatial orientations as subsequent site-directed saturation mutation hotspots, including I62, D63, I67, L70, M77, F111, M112, Q136, F140, M174, F175, M320, P321 and I325.

[0058] The present invention designs specific primers (see Table 2) and utilizes the Quickchange technology (An efficient one step site-directed and site-saturation mutagenesis protocol [J]. Nucleic Acids Research, 2004, 32 (14): e115) to introduce mutations into the wild-type glycosyltransferase BlYjiC gene sequence, thereby constructing a saturation mutation library at sites 62, 63, 67, 70, 77, 111, 112, 136, 140, 174, 175, 320, 321 and 325.

[0059] Specifically, the PCR reaction system is: ddH2O 2μL, DNA polymerase 5μL, upstream primer 1μL, downstream primer 1μL, template 1μL. PCR amplification conditions: 1) pre-denaturation 94℃2min; 2) denaturation 98℃10s, annealing 60℃15s, extension 72℃40s (this step is cycled 30 times); post-extension 72℃5min. The PCR product is digested with Dpn I restriction enzyme at 37℃ for 20 minutes. The digested product is then transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB / Kan solid plates and cultured overnight at 37℃.

[0060] Table 2 Primers used for rational design of glycosyltransferase BlYjiC

[0061]

[0062]

[0063] Note: The bold part is the merged codon after mutation

[0064] Example 5 Screening of mutant library

[0065] 96 clones were selected from the mutation plate for each site and transferred to a 96-well plate containing 50 μg / mL kanamycin and 500 μL LB liquid medium for culture. 600When the pH reaches 0.6-0.8, the cells are induced with 0.1 mM IPTG. The supernatant is removed by centrifugation, and the crude enzyme is used for catalytic reaction after repeated freezing and thawing 3 times. The reaction system contains 2 mM tyrosol, 5 mM UDP-glucose, 1% DMSO and 50 μL crude enzyme solution in 50 mM potassium phosphate buffer (pH 8.0). The reaction is incubated at 30°C, 200 rpm for 6 hours and terminated with 4 times the volume of methanol. The activity and selectivity of the mutants were detected using the detection method described in Example 3. The results are shown in Figure 2. Figure 3 The results showed that mutations at I67, L70, M77, M112, Q136 and I325 sites were more conducive to the production of salidroside, among which M112L showed the highest regional selectivity of 82.3%. The amino acid sequence of BlYJiC-M112L is shown in SEQ ID NO.2.

[0066] SEQ ID NO.2:BlYJiC-M112L

[0067] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQIRELMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDFLAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDE RFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEITARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

[0068] Example 6 Iterative Saturation Mutation

[0069] Using the M112L mutant gene sequence as a template, the site-directed saturation mutagenesis method described in Example 4 was used to perform site-directed saturation mutagenesis on sites 67, 70, 77, 136 and I325, respectively. The mutant library was screened by the method described in Example 5 to obtain a double-site variant M112L / I325Y with effectively improved regional selectivity, and its regional selectivity was significantly improved to 87.3%. Figure 4As shown, the amino acid sequence of the glycosyltransferase mutant BlYJiC-M112L / I325Y is shown in SEQ ID NO.3; using the M112L / I325Y mutant gene sequence as a template, site-directed saturation mutagenesis was performed at sites I67, L70, M77, and Q136 to obtain a triple-point mutant M112L / I325Y / L70R, whose regional selectivity reached 93.1%, as shown in FIG. Figure 4 As shown, the amino acid sequence of the glycosyltransferase mutant BlYJiC-M112L / I325Y / L70R is shown in SEQ ID NO. 4. Subsequently, using M112L / I325Y / L70R as a template, I67, M77, and Q136 were subjected to site-directed saturation mutagenesis to obtain a four-point mutant M112L / I325Y / L70R / Q136E (M4), with a regional selectivity of 97.6%. Figure 4 The amino acid sequence of the glycosyltransferase mutant BlYJiC-M112L / I325Y / L70R / Q136E is shown in SEQ ID NO. 5. Then, M112L / I325Y / L70R / Q136E was used as a template for the next round of mutation, and a five-point mutant M112L / I325Y / L70R / Q136E / I67E was obtained, whose regioselectivity was 98.4%, and the amino acid sequence of the glycosyltransferase mutant BlYJiC-M112L / I325Y / L70R / Q136E / I67E is shown in SEQ ID NO. 6. In addition, based on M112L / I325Y / L70R / Q136E / I67E, site-directed saturation mutagenesis experiments were performed at the I67 and M77 sites to obtain a six-point mutant M112L / I325Y / L70R / Q136E / I67E / M77R. The region selection and enzyme activity were tested using the method described in Example 3. The results are as follows: Figure 4 The amino acid sequence of the glycosyltransferase mutant BlYJiC-M112L / I325Y / L70R / Q136E / I67E / M77R is shown in SEQ ID NO. 7. The results showed that the mutant catalyzed tyrosol regioselectivity of 99.3%, and the specific enzyme activity increased from 14.1mU / mg of the wild type to 97.2mU / mg. Figure 4 and Figure 5 These results show that the present invention successfully achieved strict regional selectivity in BlYjiC catalysis through site-directed mutagenesis, and at the same time improved its catalytic activity, greatly enhancing the application of the enzyme in the synthesis of salidroside. SEQ ID NO.3: BlYJiC-M112L / I325Y

[0070] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPL

[0071] NYRSTLNIDPQQIRELMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLI

[0072] LFDFLAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALK

[0073] NSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKE

[0074] TPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESF

[0075] DDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQE

[0076] YTARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQA

[0077] GGAEKAADEIEAFLAPAGVK

[0078] SEQ ID NO.4:BlYJiC-M112L / I325Y / L70R

[0079] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPL

[0080] NYRSTLNIDPQQIRERMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLI

[0081] LFDFLAMAGKLLAEKFGIEAVRLCSTYAQNEHFTFRSISEEFKIELTPEQEDALK

[0082] NSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKE

[0083] TPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESF

[0084] DDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQE

[0085] YTARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQA

[0086] GGAEKAADEIEAFLAPAGVK

[0087] SEQ ID NO.5:BlYJiC-M112L / I325Y / L70R / Q136E

[0088] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPL

[0089] NYRSTLNIDPQQIRERMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLI

[0090] LFDFLAMAGKLLAEKFGIEAVRLCSTYAENEHFTFRSISEEFKIELTPEQEDALK

[0091] NSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKE

[0092] TPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESF

[0093] DDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQE

[0094] YTARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQA

[0095] GGAEKAADEIEAFLAPAGVK

[0096] SEQ ID NO.6:BlYJiC-M112L / I325Y / L70R / Q136E / I67EMGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQERERMKNKKDMSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDFLAMAGKLLAEKFGIEAVRLCSTYAENEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEYTARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

[0097] SEQ ID NO.7:BlYJiC-M112L / I325Y / L70R / Q136E / I67E / M77R

[0098] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQERERMKNKKDRSQAPLMFIKEMEEVLPQLEALYENDKPDLILFDFLAMAGKLLAEKFGIEAVRLCSTYAENEHFTFRSISEEFKIELTPEQEDALKNSNLPSFNFEDMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKETPIISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPENFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEYTARRVEELGLGKHLQPEDTTAASLREAVSQTDGDPHVLKRIQDMQKHIKQAGGAEKAADEIEAFLAPAGVK

[0099] Example 7 Glycosyltransferase mutant (M112L / I325Y / L70R / Q136E / I67E / M77R) for preparing salidroside

[0100] The preparation of salidroside uses a dual enzyme coupling system for glycosylation of tyrosol and in situ regeneration of UDP-glucose. The preparation process is as follows: Figure 6 As shown, sucrose synthase catalyzes sucrose to produce glucose, fructose and UDP-glucose in the presence of UDP, so that the UDP-glucose produced by the reaction can participate in the synthesis of salidroside again, ensuring the continuous progress of the reaction. Wherein sucrose synthase is derived from soybean (NCBI accession: NP_001237525.1), after codon optimization, it is synthesized into pET28a vector by General Bio (Anhui) Co., Ltd., and transformed into Escherichia coli BL21 (DE3). The preparation and purification of sucrose synthase are the same as the method described in Example 2. Specifically, the reaction mixture (200mL) contains 50mM potassium phosphate buffer (pH 7.5), 2mM tyrosol, 0.5mMUDP, 500mM sucrose, 25mg glycosyltransferase mutant (M112L / I325Y / L70R / Q136E / I67E / M77R) and 50mg sucrose synthase. The reaction is carried out at 30°C with a stirring speed of 800rpm. 2 mM tyrosol was periodically added to the reaction mixture according to the consumption of the substrate. During the reaction, 50 μL of the reactant was drawn at regular intervals, and 150 μL of methanol was added to terminate the reaction. The reaction progress was analyzed using the method described in Example 3.

[0101] An initial concentration of 2 mM tyrosol was used in the cascade reaction, and 2 mM tyrosol was added to the reaction mixture when the substrate was exhausted. Figure 7 As shown. Finally, 11.9 mmol (3.6 g / L) of salidroside were produced, with a molar conversion rate of >99%, a regioselectivity of >99%, and a spatial yield of 10.8 g / L / d, which is the highest level reported so far. These results show that the engineered BlYjiC has great practical application value in the synthesis of salidroside.

[0102] Example 8 Product Identification

[0103] The product obtained in Example 7 was purified by semi-preparative high performance liquid chromatography and C18 semi-preparative column, with 20% methanol as the mobile phase. The obtained salidroside was concentrated by rotary evaporation and freeze-dried, and the obtained powder was dissolved in DMSO-d6 as a solvent and purified by centrifugation. 1 H and 13 C NMR spectroscopy analysis, the results are as follows Figure 8-Figure 9 The results showed that the product was salidroside.

[0104] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A glycosyltransferase mutant, characterized in that: The methionine at position 112 of the amino acid sequence shown in SEQ ID NO.1 is mutated to leucine; or The methionine at position 112 and the isoleucine at position 325 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine and tyrosine, respectively; or The 112th methionine, the 325th isoleucine and the 70th leucine of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine and arginine respectively; or The methionine at position 112, the isoleucine at position 325, the leucine at position 70 and the glutamine at position 136 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, arginine and glutamic acid, respectively; or The methionine at position 112, the isoleucine at position 325, the leucine at position 70, the glutamine at position 136 and the isoleucine at position 67 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, arginine, glutamic acid and glutamic acid, respectively; or The methionine at position 112, isoleucine at position 325, leucine at position 70, glutamine at position 136, isoleucine at position 67 and methionine at position 77 of the amino acid sequence shown in SEQ ID NO.1 are mutated to leucine, tyrosine, arginine, glutamic acid, glutamic acid and arginine, respectively.

2. A gene encoding the glycosyltransferase mutant according to claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A microorganism expressing the glycosyltransferase mutant according to claim 1, or carrying the gene according to claim 2 or the expression vector according to claim 3.

5. The microorganism according to claim 4, characterized in that: The microorganism is a genetically engineered bacterium.

6. Use of the glycosyltransferase mutant according to claim 1, or the gene according to claim 2, or the expression vector according to claim 3, or the microorganism according to any one of claims 4 to 5 in the synthesis of salidroside.

7. The use according to claim 6, characterized in that: In the presence of UDP-glucose provided by the UDP-glucose circulation system, the substrate tyrosol undergoes a glycosylation reaction under the catalysis of a glycosyltransferase mutant to produce salidroside; wherein, the specific circulation process of the UDP-glucose circulation system is as follows: in the presence of UDP, sucrose is catalyzed by sucrose synthase to produce glucose, fructose and UDP-glucose, and the produced UDP-glucose is again involved in the synthesis of salidroside.

8. The use according to claim 7, characterized in that: The reaction temperature was 30°C, the pH value of the reaction system was 8, and the UDP-glucose concentration was 5 mM.

9. The use according to claim 7, characterized in that: The sucrose concentration was 500 mM.

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