Glycosyl transferase mutant and application thereof in synthesis of salidroside

Abstract

The invention belongs to the field of enzyme engineering, and relates to a glycosyl transferase mutant and application thereof in synthesis of salidroside. On the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, iterative saturated mutation is carried out through a molecular modification technology, and the enzymatic activity and catalytic efficiency of the modified glycosyl transferase mutant are greatly improved. The mutation at a specific site can significantly promote the glycosylation activity of a tyrosol substrate. The mutant enzyme is obtained by carrying out induced expression and protein purification on the obtained mutant strain. The substrate conversion rate of the transformed glycosyl transferase mutant for catalyzing the tyrosol glycosylation reaction can reach 95% or above, the yield reaches 55.0 g / L, and an efficient and green new way is provided for salidroside production.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and relates to a glycosyltransferase mutant and application thereof in synthesizing salidroside. Background Art

[0002] Salidroside, also known as Salidroside, has the molecular formula C 14 H 20 O 7 , with a molecular weight of 300.30, is an important active ingredient in the traditional Chinese medicine Rhodiola rosea, with multiple pharmacological activities such as antioxidant, anti-inflammatory, anti-tumor, anti-cardiovascular disease, and anti-aging, and has broad application prospects. Salidroside is a glycoside compound synthesized with tyrosol (p-hydroxyphenylethanol, Tyrosol) as aglycone and glucose as a glycosyl donor.

[0003] At present, the extraction methods of salidroside are mainly divided into plant extraction and chemical synthesis. Extraction from Rhodiola rosea plants faces problems such as low content, high extraction cost, and complex process. The chemical synthesis process requires selective protection of the substrate hydroxyl group, and the various reagents used in it will pollute the environment, which is not in line with the concept of environmental protection and is not conducive to industrial production.

[0004] The preparation of glycoside compounds by biosynthesis is one of the key research directions in recent years. Enzyme-catalyzed biosynthesis has attracted much attention due to its advantages such as simple operation, mild conditions, few by-products, high yield, high efficiency and environmental protection. Salidroside is mainly obtained by glycosylation modification of tyrosol by glycosyltransferase. Through continuous exploration and mining by relevant scientists, glycosyltransferases (UGT73B6, UGT72B14, UGT85A1) that synthesize salidroside using tyrosol as a substrate have been mined and identified, and heterologous expression of these glycosyltransferases and synthesis of salidroside have been achieved by microorganisms such as Escherichia coli. However, the expression level of glycosyltransferases from plants is low, which limits the synthesis of salidroside. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a glycosyltransferase mutant and its application in the synthesis of salidroside, by BS Directed evolution based on protein structure was carried out to successfully obtain a high-efficiency mutant, improve the yield of its bioenzyme-catalyzed tyrosol in preparing salidroside, and provide a method for biosynthesizing salidroside, which has a high conversion rate of salidroside and is suitable for the industrial production of salidroside.

[0006] Our laboratory discovered the glycosyltransferase UGT from Bacillus subtilis 168 BSThe enzyme can be expressed in a soluble and high level in Escherichia coli and has the activity of catalyzing the synthesis of salidroside from tyrosol in the presence of uridine diphosphate glucose (UDPG).

[0007] The enzyme molecule is modified by using directed evolution theory to obtain a glycosyltransferase mutant with high catalytic efficiency. The present invention performs a simulated docking experiment based on the protein structure of the glycosyltransferase and the ligand to obtain a complex model of the enzyme and the ligand. Based on the rational design of the protein structure, the active site is found by analyzing the protein crystal structure and mutating it to obtain a glycosyltransferase mutant that efficiently synthesizes salidroside, which is of great significance for the industrial production of salidroside.

[0008] The present invention uses the glycosyltransferase UGT from Bacillus subtilis 168 excavated in the laboratory in the early stage BS As a template (its amino acid sequence is shown in SEQ ID NO.1), site-directed mutagenesis was performed on it to obtain a mutant UGT BS -M4.

[0009] The present invention provides a glycosyltransferase mutant, wherein the mutant is a glycosyltransferase UGT shown in the amino acid sequence of SEQ ID NO: 1. BS Based on the amino acid sequence of , the 62nd, 129th, 168th and 316th amino acids were mutated simultaneously.

[0010] In one embodiment of the present invention, the mutant is a glycosyltransferase UGT having an amino acid sequence as shown in SEQ ID NO: 1. BS Based on the above, the isoleucine at position 62 was mutated to asparagine, the serine at position 129 was mutated to threonine, the phenylalanine at position 168 was mutated to tryptophan, and the tyrosine at position 316 was mutated to serine, which was named UGT. BS -M4.

[0011] In one embodiment of the present invention, the glycosyltransferase mutant UGT BS -M4 amino acid sequence is shown in SEQ ID NO:2.

[0012] The present invention also provides a method for encoding the above-mentioned glycosyltransferase mutant UGT BS -M4 gene.

[0013] The invention also provides a recombinant vector carrying the gene.

[0014] In one embodiment of the present invention, the recombinant vector is pET-28a(+) as an expression vector.

[0015] The present invention also provides a recombinant cell expressing the glycosyltransferase mutant, or containing the gene, or containing the recombinant vector.

[0016] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0017] The present invention also provides a method for catalytically synthesizing salidroside, wherein the method comprises adding the glycosyltransferase mutant UGT to a reaction system containing tyrosol. BS -M4, or the recombinant cell, react to prepare salidroside.

[0018] In one embodiment of the present invention, the reaction system contains 1-10 mmol / L tyrosol, 2-20 mmol / L UDPG, 1-10% (v / v) DMSO, 5-40% (v / v) crude enzyme solution of glycosyltransferase mutant (enzyme concentration in the reaction system is 0.1-1 mg / ml), and 50-200 mmol / L Tris-HCl buffer.

[0019] In one embodiment of the present invention, the reaction system contains 1-30 mmol / L tyrosol, 0.2-20 mmol / L UDP, 50-200 mmol / L Tris-HCl buffer, 400-1000 mmol / L sucrose, 30-40% (v / v) crude enzyme solution of glycosyltransferase mutant and 15-30% (v / v) crude enzyme solution of sucrose synthase.

[0020] In one embodiment of the present invention, the reaction conditions are: 30-40° C. for 0-50 h.

[0021] The present invention also discloses a catalyst, which contains the aforementioned glycosyltransferase mutant, or the aforementioned gene, or the aforementioned recombinant cell.

[0022] Beneficial effects:

[0023] Compared with the salidroside glycosyltransferase from plants, the salidroside glycosyltransferase from microorganisms discovered by the present invention is easier to express efficiently and soluble in model strains such as Escherichia coli;

[0024] The glycosyltransferase mutant obtained by the invention significantly improves the glycosylation activity of tyrosol. The titer of salidroside synthesized from tyrosol catalyzed by the glycosyltransferase mutant can reach up to 55.0 g / L, the substrate conversion rate is 97.9%, and the space-time yield is 1.3 g / L / h.

[0025] The experimental results confirm that the present invention can promote the industrial production of biosynthetic salidroside, successfully solve the key problem of low glycosyltransferase activity in the enzymatic preparation of salidroside, and the obtained mutant shows great industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 UGT BS -M4 protein electrophoresis, where M is Marker, 1 is unpurified protein, and 2 is purified protein;

[0027] Figure 2 In the single enzyme system, wild-type UGT BS and mutant UGT BS -HPLC spectrum of salidroside produced by tyrosol catalyzed by M4;

[0028] Figure 3 In the dual enzyme system, wild-type UGT BS and mutant UGT BS -Schematic diagram of the catalytic efficiency and regioselectivity of M4. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the methods and devices used in the present invention are conventional methods and devices in the art.

[0030] Unless otherwise specified, the kit materials used in the following examples are all commercially available products.

[0031] The experimental methods described in the present invention are all conventional methods unless otherwise specified.

[0032] The DNA polymerase and DpnⅠ enzyme used in the examples of the present invention were purchased from Takara Biotechnology Co., Ltd., and the recombination cloning kit and plasmid extraction kit were purchased from Axygen Biotechnology Co., Ltd. Primer synthesis and gene sequencing were completed by General Biotechnology (Anhui) Co., Ltd.

[0033] The tyrosol and UDPG used in the present invention were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0034] The expression vector used in the present invention is pET-28a(+), and the host used is Escherichia coli BL21(DE3).

[0035] Example 1 Glycosyltransferase UGT BS Gene cloning

[0036] Using the genome of Bacillus subtilis 168 as a template, F and R primers were designed as shown in Table 1. The F primer is SEQ ID NO: 3 and the R primer is SEQ ID NO: 4. The wild-type glycosyltransferase gene, whose encoded amino acid sequence is shown in SEQ ID NO. 1, was named UGT BS The amplified gene was connected to the pET-28a(+) vector using a one-step cloning kit (ClonExpressⅡOne Step Cloning Kit) to obtain the recombinant plasmid pET28a-UGT BS The recombinant plasmid was transformed into E.coilBL21 (DE3), and the transformation product was spread on LB solid medium containing kanamycin and cultured at 37°C for 12-16h. The transformants were picked and verified by colony PCR. The positive clones were selected and sent for sequencing to obtain the UGT BS Genetically recombinant engineered bacteria.

[0037] Table 1 Cloned UGT BS Primers used

[0038]

[0039] Note: The lowercase bases are the parts that undergo recombination with the vector

[0040] SEQ ID NO.1UGT BS

[0041] MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGE

[0042] ALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQPDLIIYDF

[0043] VALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQ

[0044] EKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEKESLLI

[0045] DKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIP

[0046] ANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELT

[0047] ANRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAG

[0048] GAERAAAEIEAFMKKSAVPQ

[0049] Example 2 Induced expression of recombinant strains and purification of target protein

[0050] The recombinant strain pET28a-UGT constructed in Example 1 was BS Inoculate into 10 mL LB liquid medium containing 50 μg / mL kanamycin, culture at 37°C, 180 rpm overnight, then transfer to fresh 50 ml LB liquid medium containing kanamycin resistance at 2% inoculation, culture at 37°C, 200 rpm. 600 When the cell density reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1mM for induction at 28℃, 200rpm, 24h. After the induction, centrifuge at 4℃, 5000rpm, 20min to obtain cell precipitation, pour out the supernatant and add 25mL Tris-HCl (pH 8.0, 50mM) buffer to suspend evenly, use ultrasonic cell disruptor to disrupt the cells and collect the supernatant by centrifugation to obtain crude enzyme solution. SDS-PAGE analysis was performed to detect protein expression. The results are as follows: Figure 1 As shown, it can be seen that UGT BS It can be expressed efficiently and soluble in E. coli.

[0051] UGT was detected by nickel column affinity chromatography BS Purification: Obtain crude enzyme solution according to the method in Example 2, filter through a 0.45 μm filter membrane, slowly load the sample onto the column to allow the protein to fully bind to the nickel column; wash the impurities with 10 times the volume of lysis buffer, elute the target protein with 10 times the volume of elution buffer, collect the eluate, dialyze it, and store it at -80°C.

[0052] Example 3 Glycosyltransferase activity assay: Detection of enzyme activity by liquid phase analysis of glycosylation reaction results

[0053] The glycosylation reaction was carried out in a 200 μL reaction system, and the reaction system was as follows: 50 mM Tris-HCl (pH 8.0), 2 mM tyrosol, 2% DMSO, 4 mM UDPG and 40 μL crude enzyme solution, 35 ° C, 1000 rpm for 1 hour. The reaction was terminated by adding 4 volumes of methanol, centrifuged at 12000 rpm for 1 min, and analyzed by HPLC after passing through a 0.22 μm filter membrane. HPLC used an EclipsePlus C18 column (4.6 mm × 150 mm, 5 μm diameter), an injection volume of 10 μL, a column temperature of 30 ° C, a detection wavelength of 275 nm, and a mobile phase of A pipeline: water, B pipeline: methanol, and a flow rate of 0.8 mL / min. The specific procedure is shown in Table 2:

[0054] Table 2 HPLC elution program

[0055]

[0056] Enzyme activity definition: the amount of enzyme required to glycosylate 1 μmol tyrosol per hour under standard assay conditions.

[0057] The wild-type glycosyltransferase UGT BS The specific enzyme activity is 3.6U / mg.

[0058] Example 4 Wild-type glycosyltransferase UGT BS Site-directed saturation mutagenesis

[0059] The present invention screens out amino acids near the substrate binding pocket as mutation sites through homology modeling, and these amino acids include G15, I62, V108, S129, L138, F168, F231, M315, Y316, E317, Q318, and L320.

[0060] The present invention designs specific primers (Table 3, sequence numbers are SEQ ID NO: 5-28 from top to bottom) to recombinant plasmid pET28a-UGT BS As a template, a pair of primers with mutation sites were used to perform PCR amplification using Prime Star to obtain a recombinant plasmid with the specified mutation site. The amplified product was digested with DpnⅠ digestion enzyme at 37℃ for 3h to degrade the initial template. The digestion product was transformed into E.coil BL21(DE3), spread on a solid plate containing kanamycin and cultured at 37℃ overnight.

[0061]

[0062]

[0063]

[0064]

[0065] Table 3 UGT BS Primers used for mutants

[0066] Wherein N represents A, C, G or T; M represents A or C; K represents G or T.

[0067] Example 5 Screening of mutant library

[0068] The clones selected from the mutation plate at each site were transferred to a 96-well plate containing kanamycin and 1 mL LB liquid medium for cultivation. 600 When the p-value reaches 0.6-0.8, 1 mM IPTG is used to induce the cells. After the induction, the cell pellet is obtained by centrifugation at 4°C, 5000 rpm, and 20 min. After the supernatant is poured out, Tris-HCl (pH 8.0, 50 mM) buffer is added to suspend evenly, 2 mM tyrosol, 4 mM UDPG, and 2% DMSO are added to react for 12 h, and 4 times the volume of methanol is added to terminate the reaction. The liquid phase method described in Example 3 is used for screening, and the mutant strains with obvious effects are selected for plate transfer, and 24-well plates are used for induction expression, centrifugation, and the precipitate is suspended with 50 mM Tris-HCl, broken, 40 μL of crude enzyme solution is added with 2 mM tyrosol, 4 mM UDPG, 2% DMSO, and 50 mM Tris-HCl (pH 8.0) to react for 12 h, and the upper liquid phase is further used to verify the catalytic effect, and the selected mutant strains are sequenced.

[0069] Example 6 Combination Mutation

[0070] The single mutation sites with significantly improved activity in Example 5 were combined to obtain double mutants with different combinations, and the mutants with significantly improved activity in the double mutation sites were further combined with other high-activity sites for mutation, and then the crude enzyme solution of each mutant combination was reacted with 40 μL crude enzyme solution, 2mM tyrosol, 4mM UDPG, 2% DMSO, 50mM Tris-HCl (pH8.0) in 200 μL reaction system at 35°C and 1000 rpm for 12 hours, and 4 times the volume of methanol was added to terminate the reaction. The liquid phase method described in Example 3 was used for detection.

[0071] Wild-type UGT BS UGT BS -M4 mutation process substrate conversion rate and regioselectivity changes:

[0072] The results are shown in Table 4, and the wild-type glycosyltransferase UGT BS Compared with the mutant UGT BS-M4 has greatly improved catalytic efficiency and regioselectivity.

[0073] Compared with wild-type glycosyltransferase UGT BS , only the 62nd amino acid mutated to N. Compared with the wild-type glycosyltransferase UGT BS , the 62nd amino acid mutated to N, and the 129th amino acid mutated to T. Compared with the wild-type glycosyltransferase UGT BS , the amino acid at position 62 mutated to N, the amino acid at position 129 mutated to T, and the amino acid at position 168 mutated to W.

[0074] Table 4 Substrate conversion rate and regioselectivity of single-point to four-point mutations

[0075]

[0076] The substrate conversion rate refers to: the total substrate concentration entering the reaction system minus the remaining substrate concentration after the reaction is completed, divided by the total substrate concentration entering the reaction system.

[0077] Regioselectivity refers to the molar ratio of salidroside in the two products (salidroside and icariin D2).

[0078] After several rounds of iterative saturation mutagenesis, a glycosyltransferase mutant UGT with significantly improved activity was obtained. BS -M4, the amino acid sequence is shown in SEQ ID NO: 2. Wild-type glycosyltransferase UGT BS and glycosyltransferase mutant UGT BS Liquid chromatography of the product obtained by catalyzing tyrosol with M4 Figure 2 As shown, the results showed that, and wild-type glycosyltransferase UGT BS In contrast, mutant UGT BS -M4 has significantly improved its catalytic activity, substrate conversion rate, and regioselectivity (i.e., the generated byproduct icariin D2 is significantly less, and the mutant UGT BS -M4 has better substrate selectivity for tyrosol than wild-type glycosyltransferase UGT BS ).

[0079] In a single enzyme catalytic system, the glycosyltransferase mutant obtained by the present invention significantly improves the glycosylation activity of tyrosol, and the substrate conversion rate is increased from the wild-type glycosyltransferase UGT BS 44% to mutant UGT BS -M4, and the proportion of salidroside in the two products obtained increased from 82% of the wild type to 95% of the mutant UGT BS -98% of M4, % is the molar concentration.

[0080] In addition, UGT was determined by the method and system in Example 3. BS -Specific enzyme activity of the M4 mutant, mutant UGT BS -M4 has a specific enzyme activity of 7.3 U / mg, which is comparable to wild-type UGT BS Compared with the mutant UGT BS -M4's catalytic activity was significantly improved.

[0081] SEQ ID NO: 2UGT BS -M4

[0082] MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEA

[0083] LIYHTSLNNDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQPDLIIYDFV

[0084] ALAGKLFAEKLNVPVIKLCSTYAQNESFQLGNEDMLKKIREAEAEFKAYLEQE

[0085] KLPAVSWEQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEKESLLI

[0086] DKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIP

[0087] ANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMSEQELTA

[0088] NRVDELGLGVYLPKEEVTVSSLQEAVQAVSSDQELLSRVKNMQKDVKEAGG

[0089] AERAAAEIEAFMKKSAVPQ

[0090] Example 7 Mutant glycosyltransferase UGT BS -M4 preparation of salidroside

[0091] The best mutant UGT obtained in Example 6 BS-M4 strain was subjected to protein expression according to the method of Example 2 to obtain a crude enzyme solution. The sucrose synthase gene was AtSuSy (GenBank ID: NP_001031915.1) from Arabidopsis thaliana, which was constructed into a pET-32a(+) vector to obtain a plasmid, and protein expression was performed according to Example 2 to obtain a crude enzyme solution. The 10 mL catalytic reaction system was: 50 mM Tris-HCl (pH 8.0), 0.5 mM UDP (uridine diphosphate), 800 mM sucrose, 2% DMSO, 30 mM tyrosol, 3.42 mL glycosyltransferase crude enzyme solution and 2.18 ml sucrose synthase crude enzyme solution, and the reaction was carried out at 35°C. 30mM tyrosol was added at 0h and 2h, 20mM tyrosol was added at 6h, 9h, 12h, 15h, 18h, 21h and 24h, 3.42mL of crude glycosyltransferase solution and 2.18ml of crude sucrose synthase solution were added at 7h, 15h, 21h, 30h and 35h respectively. In the presence of UDP, sucrose was catalyzed by sucrose synthase to generate fructose and UDPG, and the generated UDPG was again involved in the synthesis of salidroside.

[0092] The results of the 42h fed-batch reaction are as follows Figure 3 As shown, wild-type glycosyltransferase UGT BS The substrate conversion rate was 50.3%, the final yield of salidroside was 22.0 g / L, the space-time yield was 0.5 g / L / h, the final yield of icariin D2 was 8.2 g / L, the space-time yield was 0.2 g / L / h; mutant glycosyltransferase UGT BS -M4 substrate conversion rate was 97.9%, the final yield of salidroside was 55.0 g / L, the space-time yield was 1.3 g / L / h, and the final yield of icariin D2 was 1.6 g / L, the space-time yield was 0.04 g / L / h.

[0093] The invention uses a molecular modification technology to perform iterative saturation mutation on the basis of the amino acid sequence of the glycosyltransferase shown in SEQ ID NO: 1, and the enzyme activity and catalytic efficiency of the modified glycosyltransferase mutant are greatly improved. The mutation at a specific site can significantly promote the glycosylation activity of the tyrosol substrate. The mutant enzyme is obtained by inducing expression and protein purification of the obtained mutant strain. The substrate conversion rate of the tyrosol glycosylation reaction catalyzed by the modified glycosyltransferase mutant can reach more than 95%, and the yield can reach 55.0 g / L, which provides a new efficient and green way for the production of salidroside.

[0094] The present invention is disclosed as above with the above preferred embodiments, but it is not limited to the implementation methods. For ordinary technicians in this field, various changes and modifications can be made to the above implementation methods without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A glycosyltransferase mutant, characterized in that: The mutant is the glycosyltransferase UGT shown in SEQ ID NO: 1 BS Based on the amino acid sequence, the 62nd, 129th, 168th and 316th amino acids were mutated simultaneously.

2. A glycosyltransferase mutant according to claim 1, characterized in that: The mutant is the glycosyltransferase UGT shown in SEQ ID NO: 1 BS On the basis of , the isoleucine at position 62 is mutated to asparagine, the serine at position 129 is mutated to threonine, the phenylalanine at position 168 is mutated to tryptophan, and the tyrosine at position 316 is mutated to serine.

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

4. A recombinant vector carrying the gene according to claim 3.

5. A recombinant cell expressing the glycosyltransferase mutant according to claim 2, or containing the gene according to claim 3, or containing the recombinant vector according to claim 4.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

7. A method for catalytic synthesis of salidroside, characterized in that: The method comprises adding the glycosyltransferase mutant according to claim 2 or the recombinant cell according to claim 5 or 6 to a reaction system containing tyrosol, and reacting to prepare salidroside.

8. The method for catalytic synthesis of salidroside according to claim 7, characterized in that: The reaction system contains 1-10 mmol / L tyrosol, 2-20 mmol / L UDPG, 1-10% v / v DMSO, 5-40% v / v glycosyltransferase mutant crude enzyme solution, and 50-200 mmol / L Tris-HCl buffer; the enzyme concentration in the reaction system is 0.1-1 mg / ml.

9. Use of the glycosyltransferase mutant according to claim 2, or the gene according to claim 3, or the recombinant cell according to claim 5 or 6 in catalyzing the synthesis of salidroside.

10. A catalyst, characterized in that A method comprising the glycosyltransferase mutant according to claim 2, the gene according to claim 3, or the recombinant cell according to claim 5 or 6.

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