Glucosyltransferase mutant catalyzing rebaudioside D to produce rebaudioside M and its application

Abstract

The present invention belongs to the technical field of enzyme engineering, and specifically relates to a glucosyltransferase mutant for catalyzing rebaudioside D to produce rebaudioside M and its application. The glucosyltransferase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to at least one of the following mutations: mutating the phenylalanine at position 84 to tryptophan; and / or mutating the leucine at position 88 to valine; and / or mutating the leucine at position 126 to phenylalanine; and / or mutating the asparagine at position 196 to histidine; and / or mutating the leucine at position 379 to isoleucine. The present invention also provides the application of the mutant in the preparation of rebaudioside M, and the enzyme activity of the mutant is significantly higher than that of the wild type, and the conversion rate of preparing rebaudioside M is high.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a glucosyltransferase mutant for catalyzing rebaudioside D to produce rebaudioside M and an application thereof. Background Art

[0002] Steviosides (SGs) include stevioside, rebaudioside A (RA), rebaudioside B (RB), rebaudioside C (RC), rebaudioside D (RD), rebaudioside E (RE), rebaudioside F (RF), dulcoside A (Dulcoside A), steviolbioside, rebaudioside M (RM) and rebaudioside I (RI). RM accounts for 0.3%-1.0% of stevioside. It has the advantages of low calories and high sweetness of stevioside, and has a pure taste. It is the component of stevioside that tastes closest to sucrose, and its relative sweetness is 200-350 times that of sucrose. RM is sweeter than the mainstream RA in the market, and its bitterness is weaker than RA. Therefore, the stevioside RM component in stevia will have a wider application, suitable for table sweeteners, most foods, beverages, etc., and it does not contain calories or chemicals that are harmful to the human body and the environment. It is an excellent choice for patients with diabetes or other health problems. As a substitute for sucrose, RM has important application value in the food industry, reducing the demand for traditional sweeteners, helping to alleviate the environmental pressures associated with the production of these sweeteners, including land, water resources and greenhouse gas emissions, and helping to maintain the health of ecosystems including the ocean.

[0003] Traditional methods for extracting steviosides mostly use plant extraction methods, such as hot water extraction, solvent extraction, immersion, macroporous resin adsorption, and microwave-assisted extraction. These methods not only have disadvantages such as low efficiency and water waste, but also high cost and the yield is difficult to meet market demand. The biotransformation of stevioside is currently the most economical and effective way to achieve industrial production of stevioside. The biotransformation of stevioside mainly includes: (1) constructing a stevioside synthesis pathway in microorganisms by expressing glycosyltransferase genes and heterologously synthesizing stevioside using glucose as a carbon source; (2) synthesizing stevioside using biocatalysis. Biocatalytic synthesis of stevioside refers to the process of synthesizing stevioside using enzymes or enzyme-producing microorganisms as catalysts.

[0004] At present, the biosynthesis of RM is mainly based on the transformation of UGT76G1 from stevia. Chinese invention patents CN109750071A, CN114214378A, CN114574460A, etc. have studied this gene, but the target proteins available are still limited. The present invention combines the protein structure with the experimental data, and conducts targeted transformation of the protein from the perspective of interaction, providing a mutant with high enzyme activity, and finally applying it to achieve a conversion rate of 95%, which is of great significance to the industrial production of steviol glycoside RM. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a glucosyltransferase mutant that catalyzes rebaudioside D to produce rebaudioside M, and its application in the preparation of RM. The enzyme activity of the mutant is significantly higher than that of the wild type, and the conversion rate of preparing RM is high.

[0006] The technical solution of the present invention is as follows:

[0007] A glucosyltransferase mutant that catalyzes rebaudioside D to produce rebaudioside M, wherein the amino acid sequence of the glucosyltransferase is shown in SEQ ID NO.1, and the glucosyltransferase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to at least one of the following mutations:

[0008] The proline at position 84 was mutated to tryptophan, named P84W;

[0009] and / or mutating the methionine at position 88 to valine, designated M88V;

[0010] and / or mutating the leucine at position 126 to phenylalanine, designated L126F;

[0011] and / or mutating the asparagine at position 196 to histidine, designated as N196H;

[0012] and / or mutating the leucine at position 379 to isoleucine, designated as L379I.

[0013] The glucosyltransferase is from Stevia rebaudiana, and the nucleotide sequence of the glucosyltransferase is shown in SEQ ID NO.2.

[0014] The amino acid sequence of the glucosyltransferase is shown in SEQ ID NO.1:

[0015] MENKTETTVHRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFTQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFPWLDQQPSRSVLYVSFGSTSEVDEEDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVDPLPDGFLGERERMMKWVRQQNVLAHGTIGAFWHHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL。

[0016] The nucleotide sequence of the glucosyltransferase is shown in SEQ ID NO.2:

[0017] atggaaaata aaacggagac caccgttcac cggcgccgga gaataatatt attcccggta

[0018] ccatttcaag gccacattaa cccaattctt cagctagcca atgtgttgta ctccaaagga

[0019] ttcagtatca ccatctttca caccaacttc aacaaaccca aaacatctaa ttaccctcac

[0020] ttcactttca gattcatcct cgacaacgac ccacaagacg aacgcatttc caatctaccg

[0021] actcatggtc cgctcgctgg tatgcggatt ccgattatca acgaacacgg agctgacgaa

[0022] ttacgacgcg aactggaact gttgatgtta gcttctgaag aagatgaaga ggtatcgtgt

[0023] ttaatcaccg atgctctttg gtacttcacg caatctgttg ctgacagtct taacctccga

[0024] cggcttgttt tgatgacaag cagcttgttt aattttcatg cacatgtttc acttcctcag

[0025] tttgatgagc ttggttacct cgatcctgat gacaaaaccc gtttggaaga acaagcgagt

[0026] gggtttccta tgctaaaagt gaaagacatc aagtctgcgt attcgaactg gcaaatactc

[0027] aaagagatat tagggaagat gataaaacaa acaaaagcat cttcaggagt catctggaac

[0028] tcatttaagg aactcgaaga gtctgagctc gaaactgtta tccgtgagat cccggctcca

[0029] agtttcttga taccactccc caagcatttg acagcctctt ccagcagctt actagaccac

[0030] gatcgaaccg tttttccatg gttagaccaa caaccgtcac gttcggtact gtatgttagt

[0031] tttggtagta ctagtgaagt ggatgaggaa gatttcttgg aaatagctcg tgggttggtt

[0032] gatagcaagc agtcgttttt atgggtggtt cgacctgggt ttgtcaaggg ttcgacgtgg

[0033] gtcgacccgt tgccagatgg gttcttgggt gaaagagaac gtatgatgaa atgggttcgg

[0034] cagcagaatg tgctagctca tggaacaata ggcgcattct ggcatcatag cggatggaac

[0035] tctacgttgg aaagcgtttg tgaaggtgtt cctatgattt tctcggattt tgggctcgat

[0036] caaccgttga atgctagata catgagtgat gttttgaagg taggggtgta tttggaaaat

[0037] gggtgggaaa gaggagat agcaaatgca ataagaagag ttatggtgga tgaagaagga

[0038] gaatacatta gacagaatgc aagagttttg aaacaaaagg cagatgtttc tttgatgaag

[0039] ggtggttcgt cttacgaatc attagagtct ctagtttctt acatttcatc gttg.

[0040] The present invention provides the use of the glucosyltransferase mutant in preparing RM.

[0041] The preparation method of the RM comprises the following steps:

[0042] (1) preparing the glucosyltransferase mutant enzyme solution;

[0043] (2) RD is used as a substrate, sucrose and sucrose synthase are added, and then the enzyme solution of step (1) is added to carry out a catalytic reaction to obtain RM. The catalytic reaction conditions are: pH 6-7, temperature 37-38°C. The added amounts of RD, sucrose, sucrose synthase and enzyme solution can be adjusted according to actual needs.

[0044] In step (1), the method for preparing the glucosyltransferase mutant enzyme solution comprises the following steps:

[0045] a. Designing and synthesizing site-directed mutagenesis primers based on the nucleotide sequence of glucosyltransferase, performing site-directed mutagenesis on the glucosyltransferase gene, and constructing a plasmid vector carrying the mutant gene;

[0046] b. Transform the plasmid vector into the host cell;

[0047] c. Select positive clones for cultivation to obtain the glucosyltransferase mutant enzyme solution.

[0048] Preferably, the plasmid vector is a pET vector.

[0049] Preferably, the host cell is a bacterial or fungal cell, more preferably Bacillus subtilis or Escherichia coli.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The glucosyltransferase mutants P84W, M88V, L126F, N196H and L379I provided by the present invention have shake flask fermentation enzyme activities that are 1.12 times, 1.69 times, 1.62 times, 1.63 times and 1.29 times that of the wild type, respectively. The present invention also provides the application of the glucosyltransferase mutants in catalyzing RD to synthesize RM, with a conversion rate of 95%. The glucosyltransferase mutants provided by the present invention increase the application potential of glucosyltransferase in the food, medicine and chemical industries, and are of great significance to the industrial production of glucosyltransferase. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 HPLC chart of the enzymatic activity of the glucosyltransferase mutant M88V;

[0053] Figure 2 This is the HPLC chart of the catalytic activity of the glucosyltransferase mutant M88V enzyme solution. DETAILED DESCRIPTION

[0054] The embodiments of the present invention are only used as further explanations of the present invention and cannot be used as limiting the content or scope of the present invention. For molecular biology experimental methods not specifically described in the present embodiments, reference can be made to the Molecular Cloning Experiment Guide.

[0055] The culture medium involved in the embodiment is as follows:

[0056] LB medium: tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L;

[0057] TB medium: tryptone 12 g / L, yeast powder 24 g / L, glycerol 5 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L.

[0058] Example 1 Construction of wild-type strain of glucosyltransferase

[0059] The UGT76G1 sequence derived from stevia was submitted to a gene company for synthesis and construction of the wild-type strain PET28a-GT. The PET28a-GT bacterial solution was inoculated at a 1% inoculation rate into LB liquid culture medium (containing 50 mg / L kanamycin sulfate) to prepare a seed solution. The seed solution was inoculated at a 1% inoculation rate into TB liquid culture medium. After induction culture and fermentation at 20°C in a shaker for 48 hours, the fermentation liquid was centrifuged at 4°C and 12000 rpm for 15 minutes, the precipitate was taken and crushed, and the supernatant was obtained, which was the enzyme solution of the wild-type glucosyltransferase.

[0060] Example 2 Preparation and expression of glucosyltransferase mutants

[0061] According to the nucleotide sequence of glucosyltransferase, site-directed mutagenesis primers for introducing single mutations were designed and synthesized, and site-directed mutagenesis of the glucosyltransferase gene was performed. The plasmid vector carrying the mutant gene was constructed using seamless cloning technology, and the plasmid vector used was pET28a(+); the vector was introduced into Escherichia coli BL21-DE3 for induced expression to obtain a glucosyltransferase mutant strain. Site-directed mutagenesis: Gene overlapping PCR technology was used to perform site-directed mutagenesis on the target gene, and the linearized fragment was recovered and purified for recombination.

[0062] The nucleotide sequences of the site-directed mutagenesis primers are as follows:

[0063] The site-directed mutagenesis primers for introducing the P84W mutation are:

[0064] Forward primer: 5'-ACCTGCCGACGCATGGTTGGTTAGCGGGTTTGCGTATTC-3', as shown in SEQ ID NO.3;

[0065] Reverse primer: 5'-GAATACGCAAACCCGCTAACCAACCATGCGTCGGCAGGT-3', as shown in SEQ ID NO.4.

[0066] The site-directed mutagenesis primers for introducing the M88V mutation are:

[0067] Forward primer: 5'-CATGGTCCGTTAGCGGGTGTGCGTATTCCGATCATTAACG-3', as shown in SEQ ID NO.5;

[0068] Reverse primer: 5'-CGTTAATGATCGGAATACGCACACCCGCTAACGGACCATG-3', as shown in SEQ ID NO.6.

[0069] The site-directed mutagenesis primers for introducing the L126F mutation were:

[0070] Forward primer: 5'-GCCTGATCACGGATGCGTTCTGGTATTTTGCACAGAGCGTTG-3', as shown in SEQ ID NO.7;

[0071] Reverse primer: 5′-CAACGCTCTGTGCAAAATACCAGAACGCATCCGTGATCAGGC-3′, as shown in SEQ ID NO.8.

[0072] The site-directed mutagenesis primers for introducing the N196H mutation are:

[0073] Forward primer: 5'-CATTAAAAGCGCATATAGCCACTGGCAGATTGCCAAAGAGAT-3', as shown in SEQ ID NO.9,

[0074] Reverse primer: 5′-ATCTCTTTGGCAATCTGCCAGTGGCTATATGCGCTTTTAATG-3′, as shown in SEQ ID NO.10.

[0075] The site-directed mutagenesis primers for introducing the L379I mutation are:

[0076] Forward primer: 5'-GATCTTTAGCGACTTCGGCATCGACCAGCCGTTGAATGCGCG-3', as shown in SEQ ID NO.11;

[0077] Reverse primer: 5'-CGCGCATTCAACGGCTGGTCGATGCCGAAGTCGCTAAAGATC-3', as shown in SEQ ID NO.12.

[0078] PCR reaction system: 1 μL each of 10 μM forward primer and reverse primer, 25 μL PrimeStar polymerase, 1 μL template, and 50 μL ddH2O added to make up.

[0079] PCR reaction program: pre-denaturation at 94°C for 4 min; followed by 25 cycles (94°C for 10 s, 55°C for 5 s, 72°C for 7 min 50 s); extension at 72°C for 10 min; and finally incubation at 4°C.

[0080] The PCR products were detected by 1% agarose gel electrophoresis.

[0081] The above-verified PCR product was seamlessly cloned and transformed into Escherichia coli TOP10 competent cells. The transformation product was spread on LB plates containing 50 mg / L kanamycin sulfate and cultured at 37°C overnight. Five single colonies were picked from the plates and inoculated into LB liquid culture medium. The bacterial liquid was PCR verified and sequenced, and the results were correct.

[0082] The correctly sequenced plasmid was transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli expressing the mutant.

[0083] Expression of mutants:

[0084] The prepared recombinant Escherichia coli were inoculated into LB liquid culture medium for 16 hours to obtain seed solution, and the seed solution was inoculated into TB liquid culture medium at a 1% inoculation amount. After the Escherichia coli was cultured and fermented in a shaking incubator at 20°C for 48 hours, the fermentation liquid was centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant was crushed to obtain the glucosyltransferase mutant enzyme solution.

[0085] Example 3 Detection of enzymatic activity of glucosyltransferase mutants

[0086] The enzyme solution obtained in Example 2 was subjected to activity assay. The enzyme activity assay method was as follows: 1 mL 1.2 mMRD, 1 mL UDPG, and 1 mL wild-type or mutant enzyme solution of glucosyltransferase were reacted at 37° C. and 200 rpm for 1 h, and then HPLC assay was performed to obtain the concentration changes of substrate RD and product RM. The enzyme activity results of wild-type and mutant glucosyltransferase are shown in Table 1 (based on the wild-type enzyme activity as 100%). The HPLC chart of the enzyme activity of mutant M88V is shown in Table 1. Figure 1 .

[0087] The specific method parameters of HPLC determination are as follows: add 200 μL of chromatographic acetonitrile, shake and mix, let stand for 10 minutes, centrifuge at 12000 rpm for 10 minutes at room temperature, and analyze the supernatant after passing through a 0.22 μm organic membrane. HPLC uses a C18 reverse-phase bonded silica gel separation column (4.6 mm×250 mm, 5 μm), 25% acetonitrile as the mobile phase, a flow rate of 1 mL / min, a column temperature of 40°C, a UV detector PDA, a PDA detector wavelength of 210 nm, and an injection volume of 10 μL.

[0088] Table 1 Enzyme biopsy results of wild-type and mutant glucosyltransferases

[0089]

[0090] As can be seen from Table 1, the enzyme activities of the glucosyltransferase mutants provided by the present invention are all higher than that of the wild type. The shake flask fermentation enzyme activities of the mutants P84W, M88V, L126F, N196H, and L379I are 1.12 times, 1.69 times, 1.62 times, 1.63 times, and 1.29 times that of the wild type, respectively.

[0091] Example 4 Conversion of Glucosyltransferase

[0092] The mutant M88V enzyme solution obtained in Example 2 was used for catalytic reaction: water was used as solvent, 30 g / L RD, 30 g / L sucrose, 5 g / L sucrose synthase were added, and then 8 g / L mutant M88V enzyme solution was added. The reaction was catalyzed at 37°C and pH 7 for 24 h. After the reaction was completed, HPLC detection was performed. The results were as follows: Figure 2 The conversion rate after 24 hours of reaction was 95%.

Claims

1. A glucosyltransferase mutant that catalyzes the production of rebaudioside M from rebaudioside D, characterized in that: The amino acid sequence of the glucosyltransferase is shown in SEQ ID NO.1, and the glucosyltransferase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to the following mutations: The asparagine at position 196 was mutated to histidine.

2. The glucosyltransferase mutant that catalyzes the production of rebaudioside M from rebaudioside D as claimed in claim 1, characterized in that: The glucosyltransferase is from Stevia rebaudiana, and the nucleotide sequence of the glucosyltransferase is shown in SEQ ID NO.

2.

3. Use of the glucosyltransferase mutant that catalyzes the production of rebaudioside M from rebaudioside D as claimed in claim 1 in the preparation of rebaudioside M.

4. The use according to claim 3, characterized in that: The preparation method of rebaudioside M comprises the following steps: (1) preparing the glucosyltransferase mutant enzyme solution; (2) Using rebaudioside D as a substrate, adding sucrose and sucrose synthase, and then adding the enzyme solution of step (1) to carry out a catalytic reaction to obtain rebaudioside M.

5. The use according to claim 4, characterized in that: The method for preparing the glucosyltransferase mutant enzyme solution comprises the following steps: a. Designing and synthesizing site-directed mutagenesis primers based on the nucleotide sequence of glucosyltransferase, performing site-directed mutagenesis on the glucosyltransferase gene, and constructing a plasmid vector carrying the mutant gene; b. Transform the plasmid vector into the host cell; c. Select positive clones for cultivation to obtain the glucosyltransferase mutant enzyme solution.

6. The use according to claim 5, characterized in that: The plasmid vector is a pET vector.

7. The use according to claim 5, characterized in that: The host cell is a bacterial or fungal cell.

8. The use according to claim 7, characterized in that: The host cell is Bacillus subtilis or Escherichia coli.

9. The use according to claim 4, characterized in that: The catalytic reaction conditions in step (2) are: pH 6-7, temperature 37-38°C.

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