Method for efficiently synthesizing rebaudioside M by using glycosyl transferase UGT76G1 multi-site mutant

Abstract

The invention discloses a method for efficiently biosynthesizing rebaudioside M by using a glycosyl transferase UGT76G1 mutant, and belongs to the field of enzyme catalysis and biosynthesis. According to the present invention, the amino acid sequence of the glycosyl transferase has an amino acid mutation at the 88th site, the 199th site, the 200th site and the 284th site corresponding to the wild type glycosyl transferase UGT76G1, and the mutant UGT76G1-M88I / I199L / L200A / T284S with high catalytic activity is finally obtained so as to effectively catalyze Reb D to generate Reb M; if the glycosyl transferase mutant provided by the invention is combined with sucrose synthase for use, the use amount of an expensive second substrate UDPG can be reduced, so that the production cost of Reb M is further reduced. The invention provides an efficient way for the production of Reb M.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and enzyme-catalyzed synthesis, and specifically relates to the design of mutants through semi-rational design and molecular simulation based on the structure of glycosyltransferase, and the efficient synthesis of rebaudioside M using multi-site mutants of glycosyltransferase UGT76G1. This method is of great significance for improving the yield of natural sweetener rebaudioside M. Background Art

[0002] Due to the excessive intake of high-calorie sugars, serious diseases such as worldwide severe obesity, diabetes, hypertension, and cardiovascular and cerebrovascular diseases have occurred. In order to reduce the intake of high-calorie sugars, low-calorie sweeteners have emerged. Among them, the natural sweetener steviol glycosides derived from Stevia rebaudiana have received extensive attention due to their stable safety, high sweetness and low calories. The relatively abundant stevioside and rebaudioside A have poor taste, and the bitter aftertaste affects their application and promotion. Rebaudioside M not only has a high sweetness (about 350 times that of sucrose), but also has no bitter aftertaste and excellent taste, and is considered an ideal substitute for high-calorie sugars. However, rebaudioside M only accounts for 0.4%-0.5% of the dry weight of Stevia rebaudiana leaves, which hinders the large-scale application of extracting rebaudioside M from Stevia rebaudiana leaves. At present, it has been found that the glycosyltransferase UGT76G1 derived from Stevia rebaudiana can catalyze the formation of rebaudioside M from rebaudioside D. This method has the advantages of strong specificity, high conversion efficiency, mild reaction conditions, and green and pollution-free, but its low catalytic efficiency is still the bottleneck for the industrial preparation of rebaudioside M. Summary of the Invention

[0003] To solve the problem of low efficiency of glycosyltransferase UGT76G1 in catalyzing the synthesis of rebaudioside M from rebaudioside D, the present invention constructs multi-site mutants by site-directed mutagenesis of glycosyltransferase UGT76G1 based on the semi-rational design of protein structure, and improves its catalytic activity to meet the industrial preparation of rebaudioside M, thereby realizing the efficient biosynthesis of rebaudioside M.

[0004] The first object of the present invention is to provide an efficient glycosyltransferase mutant. By means of genetic engineering, site-directed mutagenesis is carried out on the glycosyltransferase. The mutant is obtained by mutating methionine at position 88 of glycosyltransferase UGT76G1 shown in SEQ ID NO.1 to isoleucine, isoleucine at position 199 to leucine, leucine at position 200 to alanine, and at the same time mutating threonine at position 284 to serine.

[0005] In one embodiment, the nucleotide sequence encoding the glycosyltransferase UGT76G1 is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2; the amino acid sequence of the glycosyltransferase mutant is shown as SEQ ID NO:3.

[0006] The second object of the present invention is to provide a method for characterizing the activity of the glycosyltransferase UGT76G1 mutant by high performance liquid chromatography at the optimal reaction temperature and pH.

[0007] The third object of the present invention is to provide the gene of the above-mentioned glycosyltransferase mutant.

[0008] The fourth object of the present invention is to provide an expression vector carrying the gene encoding the above-mentioned glycosyltransferase mutant.

[0009] The fifth object of the present invention is to provide a method for catalytic synthesis of rebaudioside M, which uses Reb D as a substrate and UDPG as a second substrate, and performs a catalytic reaction in a shaker at pH 8.0 and a temperature of 47 °C using the purified glycosyltransferase UGT76G1 mutant to biosynthesize rebaudioside M.

[0010] In one embodiment, the conditions for the catalytic reaction are as follows: 1 mmol / L Reb D, 1 mmol / L UDPG, 5 mmol / L KPi buffer, and an environment of 47 °C for 30 min.

[0011] The construction method of the mutant includes the following steps:

[0012] (1) Semi-rationally design the structure of the glycosyltransferase UGT76G1 by technical means such as molecular docking and molecular simulation, design mutant primers for site-directed mutagenesis for PCR, and construct a plasmid vector containing the gene encoding the mutant; the mutant plasmid vector is pET32a(+);

[0013] (2) Transform the mutant plasmid into an Escherichia coli host cell.

[0014] (3) Pick monoclonal colonies, further transform them into competent cells C41, culture them in a shake flask for expansion, and extract and purify them by affinity chromatography and ion exchange chromatography to obtain high-purity glycosyltransferase UGT76G1 free of salt ions.

[0015] The present invention also provides the application of the glycosyltransferase mutant in the preparation of rebaudioside M.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The amino acid sequence of glycosyltransferase UGT76G1 of the present invention was subjected to site-directed mutagenesis to obtain

[0018] the optimal mutant UGT76G1-M88I / I199L / L200A / T284S, which significantly improved the efficiency of glycosyltransferase UGT76G1 in catalyzing the biosynthesis of Reb M from Reb D using UDPG as the glycosyl donor, and the kcat / km was increased to 0.44 (s -1 ·mM -1 ), and the relative enzyme activity could reach 6 times that of the wild-type UGT76G1 enzyme. Description of the Drawings

[0019] Figure 1 It is the SDS-PAGE electrophoresis pattern of the glycosyltransferase UGT76G1-M88I / I199L / L200A / T284S mutant protein in Example 2.

[0020] Figure 2 It is the HPLC analysis pattern of the reference standards Reb D and Reb M in Example 2.

[0021] Figure 3 It is the HPLC analysis pattern of the wild-type glycosyltransferase UGT76G1 and the UGT76G1-M88I / I199L / L200A / T284S mutant in catalyzing the synthesis of Reb M from Reb D in Example 3.

[0022] Figure 4 It is the standard concentration curve of Reb M used in Example 5.

[0023] Figure 5 It is the optimal reaction temperature of the glycosyltransferase UGT76G1-M88I / I199L / L200A / T284S in Example 4.

[0024] Figure 6 It is the optimal reaction pH value of the glycosyltransferase UGT76G1-M88I / I199L / L200A / T284S in Example 4. Detailed Embodiments

[0025] The present invention will be further described below in conjunction with the drawings in the specification and specific embodiments.

[0026] Example 1:

[0027] The nucleotide sequence of glycosyltransferase UGT76G1 from Stevia rebaudiana was downloaded from Genbank and ligated to the vector

[0028] Pet-32a(+), and the nucleotide sequence is shown in SEQ ID NO:1:

[0029] ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAG

[0030] CGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGAT

[0031] CGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAA

[0032] CTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGA

[0033] CTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAA

[0034] AGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGC

[0035] ACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTGGTATGAAAGAAACC

[0036] GCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACG

[0037] ACGACAAGGCCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCC

[0038] GCGCGGCAGCCATATGGAAAACAAAACCGAAACCACCGTGCGTCGTCGTCGCCGTATT

[0039] ATTCTGTTTCCGGTTCCGTTTCAGGGTCATATTAATCCGATTCTGCAGCTGGCAAATGT

[0040] GCTGTATAGCAAAGGTTTTAGCATCACCATCTTTCACACCAACTTCAACAAACCGAAA

[0041] ACCAGCAATTATCCGCATTTTACCTTTCGCTTTATCCTGGATAATGATCCGCAGGATGA

[0042] ACGTATTAGCAATCTGCCGACACATGGTCCGCTGGCAGGTATGCGTATTCCGATTATTA

[0043] ACGAACATGGTGCAGATGAACTGCGTCGTGAACTGGAACTGCTGATGCTGGCAAGCGA

[0044] AGAAGATGAAGAAGTTAGCTGTCTGATTACCGATGCACTGTGGTATTTTGCACAGAGC

[0045] GTTGCAGATAGCCTGAATCTGCGTCGCCTGGTTCTGATGACCAGCAGCCTGTTTAACTT

[0046] TCATGCACATGTTAGCCTGCCGCAGTTTGATGAACTGGGTTATCTGGATCCGGATGATA

[0047] AAACCCGTCTGGAAGAACAGGCAAGCGGTTTTCCGATGCTGAAAGTGAAAGATATCAA

[0048] AAGCGCATATAGCAACTGGCAGATCCTGAAAGAAATTCTGGGCAAAATGATTAAACAG

[0049] ACCCGTGCAAGCAGCGGTGTTATTTGGAATAGCTTTAAAGAACTGGAAGAGAGCGAAC

[0050] TGGAAACCGTTATTCGTGAAATTCCGGCACCGAGCTTTCTGATTCCGCTGCCGAAACAT

[0051] CTGACCGCAAGCAGCAGCAGTCTGCTGGATCACGATCGTACCGTTTTTCAGTGGCTGG

[0052] ATCAGCAGCCTCCGAGCAGCGTTCTGTATGTTAGCTTTGGTAGCACCAGCGAAGTTGAT

[0053] GAAAAAGACTTTCTGGAAATTGCCCGTGGTCTGGTTGATAGCAAACAGAGTTTTCTGTG

[0054] GGTTGTTCGTCCGGGTTTTGTTAAAGGTAGCACCTGGGTTGAACCGCTGCCGGATGGTT

[0055] TTCTGGGTGAACGTGGTCGTATTGTTAAATGGGTTCCGCAGCAAGAGGTTCTGGCACAT

[0056] GGTGCCATTGGTGCATTTTGGACCCATAGCGGTTGGAATAGTACCCTGGAAAGCGTTTG

[0057] TGAAGGTGTTCCGATGATTTTTAGCGATTTTGGTCTGGATCAACCGCTGAATGCACGTT

[0058] ATATGAGTGATGTTCTGAAAGTGGGTGTGTATCTGGAAAATGGTTGGGAACGTGGTGA

[0059] AATTGCAAATGCAATTCGTCGTGTTATGGTTGATGAAGAGGGTGAATATATCCGTCAG

[0060] AATGCCCGTGTGCTGAAACAGAAAGCAGATGTGAGCCTGATGAAAGGTGGTAGCAGCT

[0061] ATGAAAGCCTGGAAAGTCTGGTTAGCTATATTAGCAGCCTGTAA

[0062] The amino acid sequence is shown in SEQ ID NO:2:

[0063] MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTRASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL*

[0064] Using plasmid Pet-32a(+)-UGT76G1 as a template, full plasmid PCR was performed successively with primers M88I-F / M88I-R, I199L-F / I199L-R, L200A-F / L200A-R, and T284S-F / T284S-R to mutate methionine at position 88 of UGT76G1 to isoleucine, isoleucine at position 199 to leucine, leucine at position 200 to alanine, and threonine at position 284 to serine, and a mutant recombinant plasmid was constructed.

[0065] Pet-32a(+)-UGT76G1-M88I / I199L / L200A / T284S.

[0066] The obtained plasmid was sequenced and identified and then transformed into competent Escherichia coli DH5α cells. Monoclonal screening was carried out using LB solid medium containing 100 μl / ml ampicillin, and after sequencing and identification, a recombinant strain was finally obtained.

[0067] Table 1 Primer names and primer sequences

[0068]

[0069]

[0070] The amino acid sequence of the mutant UGT76G1-M88I / I199L / L200A / T284S is shown in SEQ ID NO:3:

[0071] MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGIRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQLAKEILGKMIKQTRASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSSSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL*

[0072] Example 2: Characterize the enzymatic activity of the glycosyltransferase mutant described in claim 1 by high performance liquid chromatography at the optimal reaction temperature and pH.

[0073] The reaction system was reacted at different temperatures and pH values to determine the effect of temperature on the glycosyltransferase-catalyzed production of Reb M from Reb D. The selected temperature range was 25-57 °C and the pH range was 6.5-9.0.

[0074] The reaction system was 200 μL, which included 1 mmol / L of Reb D and 1 mmol / L of UDPG. The enzyme concentration of the mutant UGT76G1-M88I / I199L / L200A / T284S was 1.5 μM. The reaction time was 30 min. After the reaction, 200 μL of methanol was added to terminate the reaction. The mixture was centrifuged at 5000 rpm for 10 minutes and filtered through a 0.22 μM filter head, and then detected and analyzed by HPLC. The liquid phase detection method was carried out as described in Example 3. The results showed that the optimal temperature of the mutant UGT76G1-M88I / I199L / L200A / T284S was 47 °C and the optimal pH was 8.0.

[0075] Example 3: Induced Expression of Recombinant Strain and Purification of Target Protein

[0076] The recombinant strain mutant Pet-32a(+)-UGT76G1-M88I / I199L / L200A / T284S constructed in Example 1 was inoculated into 1 L of TB liquid medium containing 100 μl / mL ampicillin and cultured at 170 rpm and 37 °C until the OD 600 reached 0.6 - 0.8. Then the culture temperature was reduced to 18 °C, and isopropyl-β-thiogalactoside (IPTG) with a final concentration of 0.1 mmol / L was added, followed by induced culture for 16 h.

[0077] The induced culture broth was centrifuged (6000 rpm, 15 min, 4 °C) to discard the supernatant, and the cells were collected. The cells were resuspended with buffer and disrupted at 4 °C using a high-pressure homogenizer. Then the disrupted broth was centrifuged (9000 rpm, 25 min), and the supernatant was taken to obtain the crude enzyme solution.

[0078] The crude enzyme solution was purified by affinity chromatography using a Ni + column. After sample loading, the impurity proteins were washed off with Buffer, and the target protein was eluted with elution buffer and collected. The collected target protein was desalted using a desalting column (5 ml Desalting) and concentrated after desalting. The purified protein was identified by 10% SDS-PAGE gel electrophoresis, and the results are shown in Figure 1 , and an enzyme with a clear target band and accurate protein size can be seen.

[0079] Example 4: Glycosylation Reaction of Glycosyltransferase Mutant UGT76G1-M88I / I199L / L200A / T284S Catalyzing the Reaction of Reb D to Synthesize Reb M

[0080] The purified mutant enzyme protein obtained in Example 2 was subjected to a glycosylation reaction. This reaction was carried out in a 200 μL system, and the reaction system was as follows: 1 mmol / L UDPG, 1 mmol / L Reb D, and the concentration of the mutant pure enzyme was 2 μM. The reaction was carried out on a shaker at 42 °C and 170 rpm for 1 h and 3 h, and three parallel repeated experiments were performed. After the reaction, 200 μL of methanol was added to terminate the reaction, and it was centrifuged at 5000 rpm for 10 minutes, filtered with a 0.22 μM filter head, and detected and analyzed by HPLC. The specific HPLC detection conditions are as follows:

[0081]

[0082] Through HPLC analysis, the results are as shown in Figure 3As shown, compared with the Reb D and Reb M standards, there are obvious products identical to Reb M generated in the reaction system, and the peak area is significantly higher than that of the wild type, indicating that the glycosyltransferase UGT76G1-M88I / I199L / L200A / T284S can use UDPG and Reb D as substrates to generate Reb M, and its catalytic activity is significantly improved. This example proves that the glycosyltransferase multi-site mutant UGT76G1-M88I / I199L / T284S / L200A has higher catalytic activity for generating Reb M from Reb D compared with the wild type.

Claims

1. A glycosyltransferase mutant with high catalytic activity constructed based on molecular docking, characterized in that The methionine at position 88 of the glycosyltransferase UGT76G1 shown in SEQ ID NO.1 was mutated to isoleucine, the isoleucine at position 199 was mutated to leucine, the leucine at position 200 was mutated to alanine, and the threonine at position 284 was mutated to serine.

2. Characterize the enzymatic activity of the glycosyltransferase mutant of claim 1 by high performance liquid chromatography at the optimal reaction temperature and pH.

3. A method for constructing and preparing a glycosyltransferase UGT76G1 expression vector, characterized in that Escherichia coli is used as a host and Pet-32a is used as a vector to express the glycosyltransferase mutant according to claim 1.

4. A method for improving the catalytic activity of glycosyltransferase, characterized in that: The methionine at position 88 of the glycosyltransferase UGT76G1 with an amino acid sequence as shown in SEQ ID NO.1 is mutated to isoleucine, the isoleucine at position 199 is mutated to leucine, the leucine at position 200 is mutated to alanine, and the threonine at position 284 is mutated to serine.

5. The method for catalytic synthesis of rebaudioside M according to claim 5, characterized in that: The glycosyltransferase according to claim 4 is added to a reaction system containing 1 mM / L substrate rebaudioside D and 1 mM / L substrate UDPG to prepare rebaudioside M through reaction.

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