A glycosyltransferase reconstructed based on an ancestral sequence and its application

Abstract

The present invention discloses a glycosyltransferase reconstructed based on an ancestral sequence and its application, the glycosyltransferase gene nucleotide sequence is 1485 bases in length, encoding 495 amino acids, and its nucleotide sequence and amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The gene can be efficiently expressed in an Escherichia coli expression system in a soluble manner, and the enzymatic properties of the recombinant enzyme expressed by the gene are as follows: with R'-phenylethanol as a substrate, the enzyme has an optimum reaction temperature of 55 ° C and an optimum pH of 10.5, and has good stability within the range of less than 55 ° C and pH 7-11, and can maintain more than 90% enzyme activity within 8 h. The present invention also provides two mutant strains M1 and M2, whose activities are increased by 4.68 and 5.44 times, respectively. Using the sucrose synthase McSusy mutant S31D coupled with the glycosyltransferase UGTAn85 mutant M2 in a fed-batch reaction, a maximum of 45.8 g / L 2-phenylethanol glycosides and 51.49 g / L salidroside could be produced using tyrosol as a substrate.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a glycosyltransferase reconstructed based on an ancestral sequence and a mutant thereof. Background Art

[0002] Glycosides are secondary metabolites formed during plant growth and development. They are the source of aroma in fruits, flowers, and many commercial crops such as tea and tobacco. In these plant tissues, volatile odorants are synthesized and accumulated in the form of bonded glycosides. Under specific conditions such as dehydration, in vitro detachment, mechanical damage, and pest infestation, the glycosidic bonds are hydrolyzed, releasing the aglycones, which manifest as vital characteristics such as odor and pest resistance. The synthesis and application of glycosides have attracted widespread attention in the chemical, food, agricultural, and pharmaceutical industries. While chemical synthesis of glycosides is relatively mature, its application is significantly limited by the cumbersome reaction steps and the propensity for chemical residues. Enzymatic synthesis of glycosides has attracted considerable attention in recent years due to its mild reaction conditions, high selectivity, and the ability to directly use unprotected free sugars as substrates.

[0003] Phenylethanoid glycosides have broad structural similarities, strong biological activity, and are widely distributed in plants. However, their content is low, their separation and extraction are difficult, and the sample extraction amount is small. Therefore, it is very necessary to conduct synthetic research on phenylethanoid glycosides.

[0004] 2-Phenylethanol (2-PE) is an aromatic alcohol with a rose-like scent, present in many essential oils and fermented foods. Currently, 2-phenylethanol production relies primarily on two chemical methods: the benzene-ethylene oxide method and the styrene oxide hydrogenation method. Both chemical synthesis methods involve toxic reagents and harsh conditions, resulting in the production of byproducts that reduce the quality of the final product. Chemically synthesized 2-PE is inexpensive, while natural 2-PE is expensive. Biotechnology can provide high-purity "natural" 2-PE through an environmentally friendly process, but its cytotoxicity limits its yield. Using glycosyltransferases to convert 2-PE into 2-phenylethanol glycosides can effectively reduce 2-PE's cytotoxicity. Subsequent hydrolysis of the 2-phenylethanol glycosides with hydrolases can significantly increase 2-PE yield.

[0005] Salidroside is a key pharmacological component of the Rhodiola rosea plant, exhibiting remarkable benefits such as hypoxia tolerance, anti-tumor, anti-aging, and cardiovascular protection. The demand for salidroside is increasing, and its biosynthesis pathway has attracted close attention from researchers. The final step in its synthesis is the glycosylation of tyrosol (p-hydroxyphenylethanol), catalyzed by the enzyme uridine diphosphate glucosyltransferase (UGT). Identifying highly active glycosyltransferase genes is crucial for regulating salidroside in vivo or synthesizing it in vitro.

[0006] Ancestral sequence reconstruction (ASR) technology uses existing protein sequences as templates for comparison, and infers the sequences of ancient ancestral proteins through phylogenetic and molecular evolutionary process models that describe the history of protein families, allowing researchers to directly study the effects of sequence evolution on protein structure and function. In addition, the reconstructed ancestral protein sequences can be used to fill in the sequence space, thereby facilitating the inference of remote homology. Existing ancestral sequence reconstruction methods can be divided into two categories: maximum parsimony (MP) and maximum likelihood (ML). The ML method is generally more reliable than the MP method. Ancestral sequence reconstruction technology has gradually become a powerful means to study the relationship between enzyme sequence, structure and function. Summary of the Invention

[0007] The present invention provides a glycosyltransferase and its mutant encoding gene and protein sequence reconstructed based on an ancestral sequence. The glycosyltransferase has a sequence similarity of 79.39% with glycosyltransferase XP_021605632.1 from cassava (Manihot esculenta). The glycosyltransferase was reconstructed from all sequences of the glycosyltransferase UGT85A family (http: / / prodata.swmed.edu / ancescon / ancescon.php).

[0008] Another object of the present invention is to provide the use of the above-mentioned glycosyltransferase and its mutants in catalyzing the conversion of R-phenylethanol into R-phenylethanol glycosides.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A glycosyltransferase UGTAn85 reconstructed based on the ancestral sequence has the following amino acid sequence:

[0011] 1) the amino acid sequence shown in SEQ ID NO. 2;

[0012] 2) An amino acid sequence obtained by conservative variation of the amino acid sequence shown in SEQ ID NO. 2, that is, an amino acid sequence with equivalent function formed by substitution, deletion or addition of amino acids.

[0013] A nucleic acid molecule encoding the glycosyltransferase UGTAn85.

[0014] The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

[0015] An expression vector comprising the above nucleic acid molecule.

[0016] A host cell containing any one of the nucleic acid molecules or expression vectors described above.

[0017] The host cell is selected from bacteria, yeast, plant cells and animal cells.

[0018] The preparation method of the glycosyltransferase UGTAn85 comprises culturing the above host cells in a suitable culture medium, and extracting and purifying the obtained glycosyltransferase.

[0019] The use of the glycosyltransferase UGTAn85, the nucleic acid molecule, the expression vector or the host cell in catalyzing R-phenylethanol to produce R-phenylethanol glycoside.

[0020] The glycosyltransferase UGTAn85 is used in catalyzing R-phenylethanol to generate R-phenylethanol glycoside. UGTAn85 catalyzes the generation of R-phenylethanol glycoside using R-phenylethanol and uridine glucose diphosphate (UDPG) as substrates.

[0021] A sucrose synthase McSusy and its mutant S31D are coupled with the glycosyltransferase UGTAn85 and its mutant to generate R-phenylethanol glycosides using sucrose and R-phenylethanol as substrates.

[0022] A method for catalyzing R-phenylethanol to generate R-phenylethanol glycosides, which uses R-phenylethanol and UDPG as substrates, and uses the glycosyltransferase and its mutant or the host cell to catalyze a transglycosylation reaction to generate R-phenylethanol glycosides.

[0023] The specific reaction system is as follows: using any one of 2mM to 40mM 2-phenylethanol, tyrosol, 3-fluorophenylethanol, o-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol as a substrate, the UDPG concentration is 2mM to 40mM, adding 0.74 to 2μg of purified enzyme solution, reacting at 30 to 55°C under pH = 6 to 11 conditions for 1 to 48 hours, using 1 to 6 times methanol to terminate the reaction, and using HPLC detection after centrifugation to obtain the corresponding product glycoside.

[0024] The glycosyltransferase UGTAn85 can catalyze the conversion of R-phenylethanol to R-phenylethanol glycosides;

[0025] The protein encoded by the glycosyltransferase gene was named UGTAn85;

[0026] A recombinant expression vector, a transgenic cell line or a recombinant microorganism containing the encoding gene.

[0027] The glycosyltransferase and the mutant thereof are used in catalyzing the conversion of R-phenylethanol to R-phenylethanol glycoside.

[0028] Construction of dual-enzyme expression system:

[0029] A method for catalyzing the production of phenylethanol glycosides using sucrose and any one of 2-phenylethanol, tyrosol, 3-fluorophenylethanol, o-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol as substrates, comprising: using 2-phenylethanol, tyrosol, 3-fluorophenylethanol, o-hydroxyphenylethanol, 3,4-dihydroxyphenylethanol, and sucrose as substrates, respectively; and using sucrose synthase McSusy or its mutant S31D coupled with glycosyltransferase UGTAn85 or its mutant, or the host cell, to carry out a catalytic reaction to produce phenylethanol glycosides.

[0030] The specific reaction system is as follows: 2mM to 40mM 2-phenylethanol, tyrosol, 3-fluorophenylethanol, o-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol are used as substrates, the sucrose concentration is 6mM to 400mM, 1-6mg / mL crude enzyme solution is added, the reaction is carried out at 30-55°C and pH=6.5-7.5 for 1-48h, 1-6 times methanol is used to terminate the reaction, and the product R-phenylethanol glycoside is obtained by HPLC detection after centrifugation.

[0031] The nucleic acid sequence of sucrose synthase McSusy is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4. The sucrose synthase McSusy mutant S31D is obtained by mutating serine to aspartic acid at amino acid position 31 of sucrose synthase McSusy, and the nucleic acid sequence is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10.

[0032] The dual enzyme coupling system is used to react with sucrose and 2-phenylethanol, tyrosol, 3-fluorophenylethanol, o-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol as substrates, preferably at 55° C. and pH 7.0.

[0033] The method for obtaining the crude enzyme solution is shown in Example 3.

[0034] Construction of UGTAn85 mutant

[0035] Glycosyltransferase UGTAn85 reconstructed based on the ancestral sequence was screened by alanine scanning and Dock-design virtual point saturation mutagenesis to obtain mutants with higher activity than the wild type. Mutations were made at amino acids 20, 23, 24, 65, 90, 93, 94, 220, 252, 312, and 323 to obtain mutants Y20A / C / D / E / F / G / V / M / L / H, Q23A / D / E / P / H / I / L / S / T / G / V / F / W / N / K / M / C, G24A / E / C / D / F / P / Y / H, N65A / E / D / H / C / S / T / G, I90A / P / H / T / S / K / M / L, L93C / S / E / F / P / G / Y / W / M, and C94A / D / E / F / G / L / M / N / F / Q , F220A / C / S / P / Y / G / D / E / V / M / I / L, T252A / C / S / N / M / P / Q / D / E / G / F, I312A / F / L / M / V / P / R / T / S / Y, and F323A / I / V / M / D / E / S / R / G / W / C / P were higher in activity than the wild type. Based on the above single-point mutations, combined mutagenesis was performed to obtain two mutants with higher activity, UGTAn85-M1 (Y20A / Q23D / G24A / N65D / I90L / F220P / T252S / I312T / F323A, with the amino acid sequence shown in SEQ ID NO. 5 and the nucleic acid sequence shown in SEQ ID NO. 6),

[0036] UGTAn85-M2 (Y20H / Q23E / G24A / N65G / I90A / L93S / C94A / F220D / T252A / F323P, amino acid sequence as shown in SEQ ID NO. 7, nucleic acid sequence as shown in SEQ ID NO. 8), the above mutant strains have enzyme activities increased by 4.68 times and 5.44 times respectively compared with the wild type.

[0037] The present invention obtained the glycosyltransferase UGTAn85 based on ancestral sequence reconstruction: Based on the family classification in the Cazy database (http: / / www.cazy.org / GlycosylTransferase-family), all protein sequences belonging to the UGT85A family were downloaded from the Uniprot database (https: / / www.uniprot.org / ). The protein sequences of this family were aligned, pruned, and uploaded to the ANCESCON website (http: / / prodata.swmed.edu / ancescon / ancescon.php). The website server used the maximum likelihood (ML) method to reconstruct the ancestral sequence of the UGT85A family. The resulting tree contained 82 root sequences. Root sequences 85, 86, and 95 were selected from the output tree file for analysis, from the most distant to the most recent. Root sequence 85 shared a 66.53% identity with the At85A1 sequence reported to catalyze the representative substrates tyrosol and 2-phenylethanol in R-phenylethanol. The sequence was then aligned to the PSPG-BOX region, and a protein model was constructed, followed by molecular docking analysis and soluble expression analysis. Finally, the root sequence No. 85 was selected for synthesis and named UGTAn85 ( Figure 1 ). Enzyme expression, purification, and characterization confirmed that UGTAn85 is a thermostable, alkaline-loving glycosyltransferase. Its nucleotide sequence is 1485 bases long and encodes 495 amino acids. The nucleotide and amino acid sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. This gene can be efficiently expressed in a soluble form in Escherichia coli. The recombinant enzyme expressed from this gene exhibits optimal reaction temperatures of 55°C and pH 10.5 for R-phenylethanol as a substrate. The enzyme exhibits excellent stability below 55°C and within the pH range of 7-11, maintaining over 90% of its activity for 8 hours. Preliminary screening using alanine scanning and Dock-design virtual point saturation screening yielded two mutants, M1 and M2, with increased activity by 4.68- and 5.44-fold, respectively. This demonstrates that the recombinant glycosyltransferase is capable of efficiently converting R-phenylethanol to R-phenylethanol glycosides. Using the sucrose synthase McSusy mutant S31D coupled with the glycosyltransferase UGTAn85 mutant M2 in a fed-batch reaction, a maximum of 45.8 g / L of phenylethanol glycosides could be produced using 2-phenylethanol as a substrate, and 51.49 g / L of salidroside could be produced using tyrosol as a substrate.

[0038] Beneficial effects:

[0039] The glycosyltransferase UGTAn85 of the present invention is a thermophilic, alkaliphilic, highly stable glycosyltransferase that can efficiently catalyze the production of R-phenylethanol glycosides from R-phenylethanol. Compared with most plant-derived glycosyltransferases, it has the characteristics of high temperature resistance, long sustainable catalytic time, and high tolerance to phenylethanol toxicity. It is known that 2-phenylethanol at a concentration of 2-3g / L can inhibit the growth of various bacteria and fungi. The enzyme mutant M2 of the present invention can tolerate 80mM (9.76g / L) of 2-phenylethanol and can be used for multiple fed-batch reactions ( Figure 9 The enzyme of the present invention can react at higher temperatures, thereby improving catalytic efficiency and helping to obtain large amounts of product in a short period of time. Its mutants, UGTAn85 mutant M1 and UGTAn85 mutant M2, have higher enzymatic activity than the wild-type enzyme and can accelerate the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Reconstruction of evolutionary trees using ancestral sequences;

[0041] Figure 2 UGTAn85 crude enzyme and purified enzyme protein bands, where M: DL 180kDa protein molecular standard; 1: crude protein; 2: purified protein;

[0042] Figure 3 UGTAn85 enzyme kinetic curves, a: with 2-PE as the variable; b: with UDPG as the variable;

[0043] Figure 4 Characterization of enzyme properties, a: optimum temperature; b: thermal stability; c: optimum pH; d: pH stability;

[0044] Figure 5 Effects of divalent metal ions on the catalytic activity of UGTAn85;

[0045] Figure 6 Liquid phase diagram of 2-phenylethanol glycoside standard (a) and reaction product (b);

[0046] Figure 7 Liquid phase diagram of 3-fluorophenethanol, o-hydroxyphenethanol, 3,4-dihydroxyphenethanol and reaction products;

[0047] Figure 8 The yield of 2-phenylethanol glycoside produced by the wild type of UGTAn85;

[0048] Figure 9 UGTAn85 wild type and mutant transformation rates;

[0049] Figure 10 The yield of 2-phenylethanol glycoside by mutant M2 using different concentrations of 2-PE as substrate;

[0050] Figure 11 Yields of 2-phenylethanol glycoside (a) and salidroside (b) produced by UGTAn85 mutant M2 in fed-batch reaction;

[0051] Figure 12 Effects of different sucrose synthases and their mutants coupled with UGTAn85 on the production of 2-phenylethanol glycosides. DETAILED DESCRIPTION

[0052] Materials and reagents

[0053] The full UGTAn85 gene was synthesized by GenScript (Suzhou) Biotechnology Co., Ltd., using the pRSF-Duet1 vector and the E. coli BL21 (DE3) expression host strain. Isopropyl-β-D-thiogalactopyranoside (IPTG), kanamycin sulfate, Bradford protein concentration assay kit, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit were purchased from Novation (Nanjing) Co., Ltd.; Protein Marker, Loding Buffer, etc. were purchased from Solebold (Beijing); Ni-NTA chromatography columns were purchased from GenScript (Suzhou) Biotechnology Co., Ltd.; all other reagents were of analytical grade.

[0054] Example 1: Reconstruction of ancestral sequences

[0055] All 121 protein sequences from the UGT85A family were aligned, trimmed, and uploaded to the online ancestral sequence reconstruction server ANCESCON after multiple sequence alignment.

[0056] (http: / / prodata.swmed.edu / ancescon / ancescon.php), ASR reconstruction was performed through this website, and the following parameters were selected: sequence reconstruction for all nodes, edge reconstruction, use of maximum likelihood (ML) ratio factor, and use of alignment-based PI vector.

[0057] 82 root sequences were reconstructed. From the output phylogenetic tree file, three root sequences (85, 86, and 95) closest to the template At85A1 were selected for analysis. Through alignment of the PSPG-BOX region, protein modeling, molecular docking analysis, and solubility prediction, root sequence 85 was ultimately selected for synthetic construction, resulting in the ancestral enzyme protein sequence of the UGT85A family, designated UGTAn85. The corresponding gene sequence was then generated by whole-gene synthesis using codon bias optimization. After enzyme expression, purification, and characterization, a novel glycosyltransferase was obtained that is thermostable, alkaliphilic, and highly efficient at catalyzing the conversion of R-phenylethanol to R-phenylethanol glycosides. The gene sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. The UGT85A family Genebank number used to reconstruct the ancestral sequence is shown in Table 1.

[0058] Table 1 UGT85A family Genebank ID

[0059] F8WKW1 A0A2I0A0N5 A0A1J6KE06 A0A371HE59 A0A438HEE2 F8WLS6 A0A2I0ANA1 A0A1J6KR44 A0A371HE44 A0A438H229 Q9SK82 A0A2I0XH34 A0A1J6IVL1 A0A371EQZ2 A0A438D7Q7 B2XBQ5 A0A896AGQ8 D7KLQ7 A0A371FLI4 A0A438FZL8 Q9LMF0 A0A3G3NBH7 A0A2I0XA06 A0A371F0J5 A0A438D7R8 Q9ZWJ3 A0A2I0W5E8 A0A2I0XA09 A0A371GN84 A0A438G044 Q9LMF1 A0A2I0VVK3 A0A2I0W5E9 A0A371H0K9 A0A438F7P3 Q9LME8 A0A314L2T1 A0A2I0W5G2 A0A371GRV9 A0A438H1I7 Q9M9E7 A0A0K0M736 A0A2I0WVC6 A0A371GH76 A0A3L6FW96 Q6VAB3 A0A1D1Z3V9 B6EWX9 A0A371E0H9 A0A3L6EF14 C4J2R7 A0A3L6EMK7 A0A178WGX1 A0A371E1D8 A0A317YEF2 I2BH98 A0A3L6DXD5 A0A178W9I3 A0A2I2MNG2 A0A3L6EGN4 I2BH99 A0A438FZW8 A0A178WF15 A0A2I0AMQ7 A0A317YH47 A0A2I0AMU8 A0A438FZS2 A0A178W6N7 A0A2I0A0Q3 A0A3L6D8T7 A0A3L6EYE9 A0A438FZU7 B6EWY4 A0A2I2MNC3 A0A3L6F226 A0A2I0X9Z1 A0A438F8M4 A0A2I0XH48 A0A2I2MND5 A0A2Z6GFU0 A0A3L6F150 A0A438KPN2 A0A2I0V6Z7 A0A2H4GSI3 A0A1D1Y4D4 A0A2I7M6C6 A0A438HMG1 B6EWY9 A0A1J6I813 A0A2I0ANC4 A0A1D1XL29 A0A438HFW8 A0A2I0X9Z2 A0A371H0J5 A0A371E1G0 A0A3L6EX12 A0A438FZL4 A0A438H151 A0A371FHP0 A0A2I0AVA2 F4I1C6 A0A1D1XDI7 A0A1D1XL54 A0A178WM93 A0A2I2MND4 A0A1J6KZK7 A0A1D6HBD6 A0A1D1XS86 I2BH96 A0A438FZQ3 A0A371F1K3 A0A438F8F2 A0A438HEF6 A0A1D1ZGB2 A0A371FP41 A0A371HY08 A0A438FZL7 A0A1J6I6D9 I2BH97 A0A2I0WFD9 A0A1D1ZC75

[0060] Example 2: Construction of dual enzyme expression vector

[0061] UGTAn85 was coupled with sucrose synthase McSusy to construct a dual-enzyme system: UGTAn85 was directly cloned between the Nde I and Xho I restriction sites of pRSF-Duet1, and a 6×His tag was added to the N-terminus to obtain the recombinant plasmid pRSFDuet-UGTAn85 (pRSF-Duet1 plasmid was from GenScript). Sucrose synthase McSusy (nucleic acid sequence SEQ ID NO.3, amino acid sequence SEQ ID NO.4, attached to the sequence listing) was then inserted between the Nco I and EcoR I sites of the pRSFDuet-UGTAn85 vector and coupled with UGTAn85. The recombinant plasmid was named pRSFDuet-McSusy-UGTAn85.

[0062] Example 3: Protein expression and purification

[0063] The pRSFDuet-McSusy-UGTAn85 plasmid was transformed into competent E. coli BL21(DE3) cells using the heat shock method. Single colonies were screened to obtain recombinant strains containing the dual-enzyme system. A single colony was inoculated into 5 mL of liquid LB medium containing kanamycin at a final concentration of 50 μg / mL and incubated at 37°C with shaking at 220 rpm for 12 hours. The formulation for 100 mL of solid LB medium is: 0.5 g yeast extract, 1 g peptone, 1 g NaCl, and 2 g agar dissolved in 100 mL of deionized water. Sterilize and pour into a Petri dish. A 2% (v / v) inoculum was transferred to 100 mL of liquid LB medium containing kanamycin resistance. The cells were incubated at 37°C and 200 rpm until the OD reached 0.4-0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and incubated at 25°C for 24 hours to accumulate enzyme production. Then, the cells were collected by centrifugation at 8000 g at 4°C.

[0064] The cells were resuspended in 50 mM PBS buffer (pH 7) and disrupted on ice. The cell fragments were centrifuged at 6500 g for 30 minutes at 4°C in a high-speed refrigerated centrifuge. The resulting supernatant was the crude enzyme solution containing the glycosyltransferase UGTAn85 and sucrose synthase McSusy coupling system.

[0065] Subsequently, the crude enzyme solution was filtered through a 0.45 μm filter membrane and separated and purified using a Ni-NTA affinity chromatography column. The McSusy protein was eluted to obtain the UGTAn85 protein with a 6×His tag. The purified protein was obtained after removing impurities using an ultrafiltration tube.

[0066] The purification buffer was as follows: mobile phase A: Na2HPO4 5.54 g, NaH2PO4 0.7 g, NaCl 29.22 g, glycerol 10% (V / V), and ddH2O diluted to 1 L; mobile phase B: Na2HPO4 5.54 g, NaH2PO4 0.7 g, NaCl 29.22 g, glycerol 10% (V / V), imidazole 17.015 g, and ddH2O diluted to 1 L.

[0067] Example 4: Determination of protein content

[0068] The concentration of the purified protein obtained in Example 3 was determined using the Bradford protein concentration assay kit from Novozymes, and the protein standard curve preparation steps were performed according to the instructions. The molecular weight and purity of the purified protein were determined by SDS-PAGE gel electrophoresis.

[0069] Purified protein bands such as Figure 2The protected enzyme UGTAn85 can be eluted to a single band at a concentration of 250mM to 500mM imidazole, and the protein molecular weight is 57.2kDa.

[0070] Example 5: Determination of enzyme activity

[0071] Enzyme activity (U) is defined as the amount of enzyme required per minute under specific conditions to catalyze the transglycosylation reaction between the substrates 2-phenylethanol and uridine diphosphate glucose (UDPG) to produce 1 μmol / L of phenylethanol glycoside. The enzyme activity assay system consisted of adding a final concentration of 20 mM UDPG, 20 mM 2-phenylethanol, and 2.6 μg of the purified glycosyltransferase UGTAn85 protein from Example 3 to a 1 mL reaction system. The reaction was incubated at 55°C for 20 minutes, followed by inactivation by the addition of 6 volumes of methanol. The specific enzyme activity of UGTAn85 was measured to be 5.3 U / mg.

[0072] Example 6: Kinetic parameter determination

[0073] Using 2-PE as a variable: In a 1 mL system, 10 mM UDPG and different final concentrations of 2-phenylethanol (0.2, 0.3, 0.5, 0.7, 1, 1.5, 2, and 2.5 mM) were used as substrates. 1.74 μg of purified glycosyltransferase UGTAn85 was added and reacted at 45°C for 30 min. Two volumes of methanol containing 0.2% TFA were added and the reaction was rapidly cooled on ice to inactivate. Using UDPG as a variable: In a 1 mL system, 10 mM 2-PE and different final concentrations of UDPG (0.1, 0.5, 1, 1.5, 2, 3, 4, and 5 mM) were used as substrates. 4.35 μg of purified enzyme was added and reacted at 45°C for 30 min. Two volumes of methanol containing 0.2% TFA were added and the reaction was rapidly cooled on ice to inactivate.

[0074] Table 2 Kinetic parameters of UGTAn85

[0075] 2-PE UDPG Km(mM) 0.21±0.17 Km(mM) 0.35±0.02 <![CDATA[Kcat(s -1 )]]> 5.50±0.17 <![CDATA[Kcat(s -1 )]]> 11.40±0.04 <![CDATA[Kcat / Km(mM -1 ·s -1 )]]> 26.03±0.17 <![CDATA[Kcat / Km(mM -1 ·s -1 )]]> 32.59±0.04

[0076] The enzyme kinetic curves of UGTAn85 with 2-PE and UDPG as substrates are shown in Figure 2. Figure 3 a, 3b. The Km and Kcat values ​​of UGTAn85 using 2-PE as substrate are 0.21 mM and 5.50 s -1 , the Km and Kcat values ​​of UDPG as substrate are 0.35mM and 11.4s -1 The results showed that the affinity of UGTAn85 for UDPG was about 1.6 times higher than that for 2-PE. The Kcat / Km values ​​of UGTAn85 for 2-PE and UDPG were 26 mM, respectively. -1 ·s -1 and 32.59mM -1 ·s-1 , indicating that the catalytic efficiency of UGTAn85 for UDPG is 1.25 times that of 2-PE.

[0077] Example 7: Optimum temperature and thermal stability determination

[0078] Optimum temperature: React at 30, 40, 45, 50, 55, 60, 65, 70, and 80°C, respectively. Add 5 mM UDPG and 5 mM 2-PE to 1 mL of the reaction system, add 23 mU / mL of the purified UGTAn85 protein in Example 3, and make up with pH 7.0 PBS buffer. React at the above temperatures for 1 h. Calculate the relative enzyme activity with the maximum enzyme activity as 100%.

[0079] Thermal stability: The purified UGTAn85 protein from Example 3 was incubated at 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 70°C for 0-12 h. The enzyme activity was then measured using the optimal temperature reaction system described above for 1 h. The relative enzyme activity was calculated using the untreated purified protein as a control.

[0080] The results are as follows Figure 4 a. Figure 4 As shown in b. UGTAn85 reached the maximum reaction rate at 70℃, but was inactivated after incubation at 70℃ for 5h and at 55℃. 1 / 2 =10.2h. Considering the reaction time, subsequent experiments were carried out at 55°C.

[0081] Example 8: Optimum pH and pH stability determination

[0082] Optimal pH: The purified UGTAn85 protein from Example 3 was placed in buffer solutions at pH 7, 8, 9, 9.5, 10, 10.5, 11, 11.5, 12, and 13, respectively. 5 mM UDPG and 5 mM 2-PE were added to a 1 mL reaction system. After incubation, 23 mU / mL of the purified protein was added and the solution was topped up with buffer solutions at different pH values. The reaction was incubated at 55°C for 30 min, and the relative enzyme activity was calculated. pH 7-9.5 is PBS buffer, and pH 10-13 is glycine-sodium hydroxide buffer.

[0083] pH stability: The purified UGTAn85 protein in Example 3 was placed in buffer solutions at pH = 9, 10, 10.5, 11, and 11.5 and incubated for 0, 2, 4, 6, 8, and 12 h, respectively. Samples were taken and reacted for 30 min under the same conditions as the optimal pH above to measure the enzyme activity and calculate the relative enzyme activity.

[0084] The results are as follows Figure 4 c, 4d. The optimum pH of UGTAn85 is 10.5.

[0085] Example 9: Metal ion dependence

[0086] A portion of the original enzyme solution was taken as a control, and the remaining enzyme solution was added with 1mM EDTA to chelate the metal ions. The enzyme was dialyzed overnight using a semipermeable membrane with a molecular weight cutoff of 20KDa. The protein concentration of the original enzyme and the dialyzed enzyme was determined separately. The dialyzed enzyme solution was added to a pH 10.5 buffer containing metal ions and incubated for 1 hour. After that, the substrate was added and reacted at 55°C for 1 hour to determine the relative enzyme activity. The relative enzyme activity after EDTA chelation was slightly higher than that of the original enzyme, indicating that the enzyme does not rely on divalent metal ions for reaction ( Figure 5 ).

[0087] Example 10: Product Identification Method

[0088] 2-PE HPLC assay: Agilent TC-C18 column (4.6 mm × 250 mm); mobile phase (A): pure water, (B): methanol; A:B = 50:50. Isocratic elution was performed at a flow rate of 0.7 mL / min, a detection wavelength of 200 nm, and a 10 μL injection volume at a column temperature of 30°C.

[0089] HPLC determination of tyrosol, o-hydroxyphenylethanol, 3-fluorophenylethanol, and 3,4-dihydroxyphenylethanol: Column: Agilent TC-C18 (4.6 mm × 250 mm); Mobile phases: (A): pure water, (B): methanol, (C): acetonitrile; A:B:C = 70:25:5. Isocratic elution was performed at a flow rate of 0.8 mL / min, detection wavelength of 275 nm, injection volume of 10 μL, and column temperature of 30°C.

[0090] 2-phenylethanol glycoside standard and reaction solution HPLC product detection were as follows Figure 6 As shown in a and 6b, the retention time of phenylethanol glycoside is around 5 min.

[0091] The HPLC product detection of o-hydroxyphenylethanol, 3-fluorophenylethanol, 3,4-dihydroxyphenylethanol reaction reference substance (without enzyme solution) and reaction solution (with enzyme solution added) is as follows Figure 7 As shown, the substrates were completely consumed and new product peaks were generated.

[0092] Example 11: Production of 2-phenylethanol glycosides

[0093] Glycosyltransferase-coupled sucrose synthase was used with 40 mM 2-phenylethanol as substrate and 240 mM sucrose concentration. 6 mU / mL of the crude enzyme solution containing UGTAn85 and McSusy coupling system obtained in Example 3 was added. The reaction was carried out at 50°C, pH = 7.0 for 48 h. Samples were taken at different time intervals and the reaction was terminated with methanol. 2-phenylethanol glycosides were obtained by centrifugation. HPLC was used for detection. The above coupling system can catalyze the complete conversion of 40 mM 2-phenylethanol into 2-phenylethanol glycosides ( Figure 8 ).

[0094] Example 12: Construction of mutants

[0095] Using wild-type UGTAn85 as receptor and UDPG and 2-PE as ligands, molecular docking and molecular dynamics (MD) simulation methods were used to study the interaction mode between enzyme and small molecule substrate. Alanine scanning within the range was combined with Dock-design virtual point saturation mutagenesis to predict the site with the best mutation effect. Mutations were performed based on the simulation prediction results. Combination mutagenesis was performed on the basis of single point mutations to obtain two mutants with higher activity, namely mutant M1: UGTAn85 (Y20A / Q23D / G24A / N65D / I90L / F220P / T252S / I312T / F323A) and mutant M2:

[0096] UGTAn85 (Y20H / Q23E / G24A / N65A / I90A / L93S / C94A / F220D / T252A / F323P), the above mutants increased their enzyme activities by 4.68 and 5.44 times ( Figure 9 ).

[0097] Example 13: Yield of 2-phenylethanol glycoside using different concentrations of 2-PE as substrate

[0098] The glycosyltransferase UGTAn85-M2 mutant coupled with sucrose synthase McSusy S31D used 20-70mM 2-phenylethanol as substrate and sucrose concentration was 240mM. 3mU / mL crude enzyme solution of UGTAn85-M2 mutant coupled with sucrose synthase McSusy S31D mutant was added (the crude enzyme solution was obtained by referring to Examples 2 and 3). The reaction was carried out at 50°C and pH = 7.0 for 48h. Samples were taken at different time periods and methanol was used to terminate the reaction. Phenylethanol glycoside was obtained by centrifugation. The reaction products were detected by HPLC to determine the tolerance of the glycosyltransferase UGTAn85 mutant M2 and sucrose synthase McSusy S31D coupling system to 2-PE. The results showed that the above coupling system can tolerate a maximum of 80mM 2-PE. A 2-PE concentration greater than 80mM will greatly reduce the catalytic efficiency of the above system ( Figure 10 ).

[0099] Example 14: Fed-batch reaction to obtain maximum R-phenylethanol glycoside production

[0100] Referring to the method for the crude enzyme solution of the UGTAn85 and McSusy original enzyme coupling system in Example 3, a crude enzyme solution of the coupling system of UGTAn85 mutant M2 and McSusy mutant S31D was obtained, and a fed-batch reaction was carried out.

[0101] Using 2-PE as substrate: Add 40mM 2-PE, 400mM sucrose to a 10mL reaction system, then add 16mU / mL UGTAn85 mutant M2 coupled sucrose synthase McSusy S31D mutant crude enzyme solution, and use PBS buffer at pH = 7.0 to make up the reaction system. React at 50℃ pH = 7.0, and add 40mM 2-PE and 400mM sucrose at 2, 4, 6, 8, 10, and 24h of reaction respectively. Samples were taken at different time periods and methanol was used to terminate the reaction, and the reaction products were detected by HPLC. Finally, a yield of 45.8g / L of 2-phenylethanol glycoside was obtained ( Figure 11 a).

[0102] Using tyrosol as substrate: 80mM tyrosol, 400mM sucrose, and 16mU / mL UGTAn85 mutant M2 coupled sucrose synthase McSusy S31D mutant crude enzyme were added to a 10mL reaction system, and the reaction system was supplemented with PBS buffer at pH = 7.0. The reaction was carried out at 50℃ pH = 7.0, and the feed was added at 2.5, 5, 7, and 9h of reaction. The reaction was terminated by sampling with methanol at different time periods, and the reaction product was detected by HPLC. Finally, a yield of 51.49g / L of salidroside was obtained ( Figure 11 b).

[0103] Example 15: Effects of different sucrose synthases and their mutants coupled with UGTAn85 on the yield of 2-phenylethanol glycosides

[0104] Sucrose synthase AtSusy (Genebank: P49040) from Arabidopsis thaliana, sucrose synthase McSusy (nucleic acid sequence SEQ ID NO.3, protein sequence SEQ ID NO.4) from tropical freshwater microalgae, and McSusy mutant S31D were used to catalyze 2-PE to produce 2-phenylethanol glycoside. Mutant S31D mutated position 31 on the basis of SEQ ID NO.4, so that serine was mutated to aspartic acid, and was named McSusy S31D. The reaction system was: 30mM 2-phenylethanol, 400mM sucrose, 4mU / mL of the above three sucrose synthases coupled with UGTAn85 crude enzyme solution, reacted at 50°C, and samples were taken at different time periods for detection. Figure 12As shown, the catalytic efficiency of AtSusy-coupled UGTAn85 was higher than that of McSusy-coupled UGTAn85. Furthermore, the catalytic efficiency of McSusy S31D-coupled UGTAn85, in which position 31 of sucrose synthase was mutated from serine to aspartic acid, was significantly higher than that of AtSusy-coupled UGTAn85, with complete conversion of 30 mM 2-PE in 3 hours. These results indicate that the McSusy S31D mutant of sucrose synthase significantly improves catalytic efficiency, accelerates reaction progress, and shortens reaction time when coupled to a glycosyltransferase. This coupling system has potential for industrial production of R-phenylethanol substrates.

[0105] Example 16: Conversion of substrates o-hydroxyphenylethanol, 3-fluorophenylethanol, and 3,4-dihydroxyphenylethanol by sucrose synthase McSusy S31D coupled with glycosyltransferase UGTAn85 mutant M2

[0106] 200 mM sucrose, 16 mU / mL crude enzyme solution of UGTAn85 mutant M2 coupled sucrose synthase McSusy S31D, and 20 mM o-hydroxyphenylethanol / 3-fluorophenylethanol / 3,4-dihydroxyphenylethanol were added to 1 mL of the reaction system, and the reaction system was supplemented with PBS buffer at pH 7.0. In addition, the above system was supplemented with PBS without adding crude enzyme solution as a control. After reacting at 50°C pH 7.0 for 3 hours, the reaction product was detected by HPLC ( Figure 11 The results showed that after 3 hours of reaction, the substrate peaks for o-hydroxyphenylethanol, 3-fluorophenylethanol, and 3,4-dihydroxyphenylethanol were completely eliminated, and new product peaks appeared in the reaction sample, indicating that the UGTAn85 mutant M2-coupled sucrose synthase McSusy S31D system can catalyze the conversion of o-hydroxyphenylethanol, 3-fluorophenylethanol, and 3,4-dihydroxyphenylethanol to the corresponding glycosides. The protected enzyme UGTAn85 and its mutants have broad application prospects in catalyzing the conversion of R-phenylethanol to R-phenylethanol glycosides.

Claims

1. A glycosyltransferase UGTAn85 reconstructed based on an ancestral sequence, characterized in that: The amino acid sequence thereof is the amino acid sequence shown in SEQ ID NO.

2.

2. The mutant of glycosyltransferase UGTAn85 according to claim 1, wherein the mutant is UGTAn85-M1, and the amino acid sequence of UGTAn85-M1 is shown in SEQ ID NO.

5.

3. A nucleic acid molecule encoding the glycosyltransferase UGTAn85 according to claim 1.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

1.

5. An expression vector comprising the nucleic acid molecule according to any one of claims 3 or 4.

6. A host cell containing the nucleic acid molecule according to any one of claims 3 or 4 or the expression vector according to claim 5, characterized in that: The host cell is selected from bacteria and yeast.

7. A method for producing the glycosyltransferase UGTAn85 according to claim 1, characterized in that: The host cell according to claim 6 is cultured in a suitable culture medium, and the glycosyltransferase is extracted and purified.

8. Use of the glycosyltransferase UGTAn85 according to claim 1 in catalyzing the production of R-phenylethanol glycosides from R-phenylethanol.

9. A method for catalyzing R-phenylethanol to produce R-phenylethanol glycosides, characterized in that: R-phenylethanol and uridine glucose diphosphate sodium salt are used as substrates, and the glycosyltransferase UGTAn85 according to claim 1 is used to catalyze a transglycosylation reaction to generate R-phenylethanol glycoside.

10. Use of the glycosyltransferase UGTAn85 coupled with sucrose synthase McSusy and its mutant S31D according to claim 1 in catalyzing the reaction of R-phenylethanol to produce R-phenylethanol glycosides, characterized in that: R-phenylethanol and sucrose were used as substrates, and sucrose synthase McSusy and its mutant S31D were coupled with glycosyltransferase UGTAn85 and its mutants to react to produce R-phenylethanol glycosides. The amino acid sequence of sucrose synthase McSusy is shown in SEQ ID NO. 4, and its mutant S31D was obtained by mutating serine to aspartic acid at amino acid position 31 of sucrose synthase McSusy. The amino acid sequence is shown in SEQ ID NO. 10.

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