Glycosyl transferase mutant for synthesizing rebaudioside D as well as preparation method, product and application of glycosyl transferase mutant

Abstract

The invention belongs to the technical field of biological enzymes, and particularly relates to a glycosyl transferase mutant for synthesizing rebaudioside D as well as a preparation method, a product and application of the glycosyl transferase mutant. The glycosyltransferase mutant comprises at least one substitution mutation selected from the following positions on the basis of an amino acid sequence as shown in SEQ ID NO: 1: the 158th amino acid is mutated from S to F or the 93rd amino acid is mutated from H to Q. The mutant can efficiently catalyze a conversion reaction from RebA to RebD, so that the reaction process is greatly accelerated, and the generation rate and yield of a product are improved. The achievement effectively overcomes the ubiquitous problems of low activity, low reaction efficiency, insufficient stability and the like when glycosyl transferase catalyzes the reaction in the prior art, provides powerful technical support for industrial efficient synthesis of RebD, and has important practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological enzymes, and particularly relates to a glycosyltransferase mutant for synthesizing rebaudioside D, a preparation method, products and applications thereof. BACKGROUND

[0002] The natural sweetener in stevia rebaudiana, i.e., steviol diterpene glycoside, is commonly referred to as steviol glycosides (SGs). Steviol glycosides are a mixture of multiple components, which have the characteristics of high sweetness, low heat, stable properties, easy solubility in water and alcohols (such as methanol and ethanol), etc.

[0003] The main components of steviol glycosides include stevioside, rebaudioside A~O, dulcoside A, rubusoside and steviolbioside, etc. Among them, stevioside and rebaudioside A (RA) have the highest content, accounting for more than 80% of the total amount of steviol glycosides, and are the main components determining the sweet taste characteristics. They have the characteristics of slow release of sweetness, long duration and close to sucrose in taste; however, they will present a bitter taste at high concentrations, which limits their application range.

[0004] Recent studies have found that increasing the content of rebaudioside D (RD) can effectively improve the bitter taste of steviol glycosides, and its taste is also close to sucrose. Therefore, rebaudioside D is hailed as the "next generation of steviol" and is increasingly widely used.

[0005] At present, the production process of rebaudioside D mainly includes extraction method and biological catalysis method. The extraction method is to directly extract, separate and purify rebaudioside D from stevia rebaudiana; but due to its extremely low content (<0.5%) in raw materials, large-scale production is limited. The biological catalysis method uses enzymes or microbial cells as biological catalysts to synthesize rebaudioside D. Yang Yufeng, Fei Liwen and Wang et al. expressed UGT11 in Escherichia coli, realized RebA catalytic synthesis of RebD, and the conversion rate could reach more than 90%. Compared with the extraction method, this method can obtain higher yield of rebaudioside D, and thus is more widely used.

[0006] However, the UDP-glucose transferase used in the biological catalysis method generally has the problems of low enzyme activity and low catalytic efficiency, which leads to high cost of industrial large-scale production of rebaudioside D. Therefore, it is urgent to modify the enzyme to improve the biological catalytic synthesis efficiency of rebaudioside D. SUMMARY

[0007] In view of the above shortcomings, the present application provides a glycosyltransferase UGT11 mutant modified by mutation technology, which improves the enzyme activity of glycosyltransferase UGT11 and can realize efficient catalytic synthesis of RA to RD.

[0008] The technical scheme of the present application is: In one aspect, the present application provides a glycosyltransferase mutant for synthesizing rebaudioside D, which is based on the amino acid sequence shown in SEQ ID NO: 1 and comprises at least one substitution mutation selected from the following positions: the 158th amino acid is mutated from S to F or the 93rd amino acid is mutated from H to Q.

[0009] Specifically, the amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0010] In another aspect, the present application provides a nucleic acid encoding the aforementioned glycosyltransferase mutant, and the sequence of the nucleic acid is shown in SEQ ID NO: 7 or SEQ ID NO: 9.

[0011] In another aspect, the present application provides an expression vector comprising the aforementioned nucleic acid.

[0012] Specifically, the expression vector is selected from a plasmid, a bacteriophage, a virus, or an artificial chromosome.

[0013] Preferably, the expression vector is a plasmid.

[0014] Preferably, the plasmid is selected from the pET series, the pQE series, the pGEX series, or the pMAL series.

[0015] Preferably, the plasmid is pET-30a.

[0016] In another aspect, the present application provides a host cell comprising the aforementioned nucleic acid or expression vector.

[0017] Specifically, the host cell can be a prokaryotic cell or a eukaryotic cell.

[0018] Preferably, the host cell includes, but is not limited to, Escherichia coli, yeast, Bacillus, or Lactobacillus.

[0019] Preferably, the host cell has a preservation number of CGMCC No. 34492.

[0020] In another aspect, the present application provides a method for preparing the aforementioned glycosyltransferase mutant, comprising the following steps: S1, expressing the nucleic acid encoding the glycosyltransferase mutant in a host cell; S2, culturing the host cell and inducing protein expression; S3, isolating and purifying the glycosyltransferase mutant.

[0021] In another aspect, the present application provides a cell culture or extract obtained by culturing the aforementioned host cell.

[0022] In another aspect, the present application provides an enzyme preparation comprising the aforementioned glycosyltransferase mutant or cell culture or extract.

[0023] Specifically, the enzyme preparation further comprises a pharmaceutically or industrially acceptable excipient.

[0024] Preferably, the excipient includes but is not limited to a buffer, a stabilizer, a preservative or a lyophilizing agent.

[0025] In another aspect, the present application provides the aforementioned glycosyltransferase mutant or nucleic acid or expression vector or host cell or enzyme preparation for use in the synthesis of rebaudioside D.

[0026] The present application has the following beneficial effects: The present application significantly improves the enzyme activity and thermal stability by mutating the glycosyltransferase UGT11, enabling it to more efficiently catalyze the generation of RebD from RebA, overcoming the problem of low activity in the prior art.

[0027] Deposit information: Biological material R1; Classification name: Escherichia coli (E. coli) Escherichia coli ); Deposit number: CGMCC No. 34492; Date of deposit: May 09, 2025; Depositing unit: China General Microbiological Culture Collection Center; Abbreviation of the depositing unit: CGMCC; Address of deposit: No. 3, Beichen West Road, Haidian District, Beijing. DETAILED DESCRIPTION

[0028] The present application will be further clarified by the following examples, which are only a part of the embodiments of the present application and are not intended to limit the present application. The experimental methods used in the following examples are conventional experiments, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0029] Basic examples The detection method of steviol glycosides (STV, RA, RD, RM) is: HPLC detection, specifically: Chromatographic column: ShimNex UP C18 (4.6*150mm, 5µm); Mobile phase: A: water, B: acetonitrile; UV detector; Detection wavelength: 210 nm; Column temperature: 40℃; Flow rate: 1.0 ml / min; Drift tube temperature: 40℃; Injection volume: 10 μL; Gradient elution program: T / min (B%): 0 (20), 20 (50), 20.01 (20), 30 (20).

[0030] Elution program: 30% acetonitrile (0-30 min).

[0031] Molar conversion rate calculation formula: Molar conversion rate = (molar amount of product generated / molar amount of initial substrate added) x 100%.

[0032] Construction and identification of recombinant E. coli in Example 1 1. Plasmid construction E. coli BL21 (DE3) (Cat. No. B528414), DH5ɑ competent cells (Cat. No. A338951) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Plasmid pET-30a (+) (Cat. No. B540185) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; All genes, primer synthesis and sequencing services were provided by Suzhou Jinyuzhi Biotechnology Co., Ltd.; 2x Phanta Flash Master Mix high-fidelity DNA polymerase (Cat. No. P510-01) was purchased from Nanjing Novozyme Bio-tech Co., Ltd.; Restriction endonucleases NdeI (Code No. 1621), XhoI (Code No. 1635) were purchased from Baori Biotech (Beijing) Co., Ltd. (1) Gene acquisition UGT11 gene was synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd.

[0033] The amplification primers are shown in Table 1: Table 1 Primer Sequences

[0034] Note: italicized bold font CATATG is the NdeI restriction site, CTCGAG is the XhoI restriction site.

[0035] The pcr amplification program is: the target fragment is amplified by 2x Phanta Flash Master Mix high-fidelity DNA polymerase, and the specific reaction system and amplification program are shown in Table 2.

[0036] Table 2 PCR reaction system and amplification procedure of Phanta DNA polymerase

[0037] The pcr amplification product was purified and recovered using a DNA gel recovery purification kit (Zymo Research; D4008), and the purified AtSUS gene fragment was obtained.

[0038] (2) Enzymatic digestion and ligation The amplified DNA fragments or plasmids were digested by restriction endonucleases. QuickCut™ series restriction endonucleases were used in this study, and the reaction system was as shown in Table 3. The reaction was performed at 37℃ for 5 min.

[0039] Table 3 Enzymatic digestion reaction system

[0040] After the digestion of the fragments, T4 DNA ligase (Novagen; C301-01) was mixed with the UGT11 gene digestion and purification fragments and the pET30a(+) plasmid digestion and purification fragments, and placed in a 22℃ metal bath for 2.5 h for ligation. The reaction was completed. The ligation reaction system is shown in Table 4.

[0041] Table 4 Ligation reaction system

[0042] (3) Transformation verification The ligation product was transformed (conventional chemical transformation method) into E. coli DH5a competent cells, and the single colonies growing on the selection plate (50µg / mL kanamycin) were sent to Jinweizhi for sequencing. After sequencing, the positive plasmid pET30a(+) -UGT11 was returned.

[0043] Amino acid sequence SEQ ID NO: 1 UGT11: MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH Nucleotide sequence of SEQ ID NO: 6 UGT11:

[0044] (4) Mutant expression plasmid construction UGT11 mutant positive plasmids pET30a (+) -UGT11 (S158F), pET30a (+) -UGT11 (T193P), pET30a (+) -UGT11 (H93Q), pET30a (+) -UGT11 (F186S) were returned after sequencing.

[0045] Table 5 Mutant primer list

[0046] Amino acid sequence SEQ ID NO: 2 UGT11 (S158F): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIAFIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Amino acid sequence SEQ ID NO: 3 UGT11 (T196P): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRPKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH Amino acid sequence of SEQ ID NO: 4 UGT11 (H93Q): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPQDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH Amino acid sequence SEQ ID NO: 5 UGT11 (F186S): MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTSEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDHHHHHH* Nucleotide sequence SEQ ID NO: 7 UGT11 (S158F): Nucleotide sequence of SEQ ID NO: 8 UGT11 (T196P): Nucleotide sequence of SEQ ID NO: 9 UGT11 (H93Q): Nucleotide sequence of SEQ ID NO: 10 UGT11 (F186S):

[0047] 1.5 Construction of recombinant strain The plasmid constructed above was transformed (routine chemical transformation method) into E. coli BL21 (DE3) competent cells, and overnight culture, and the primers (upstream primer test-pET-F (SEQ ID NO: 11): 5'-CATCGGTGATGTCGGCGATATAG -3', downstream primer test-pET-R (SEQ ID NO: 12): 5'-CCGGATATAGTTCCTCCTTTCAGCA -3') were designed to verify the single colonies grown in the screening plate (50 μg / mL kanamycin) by colony PCR, and the corresponding recombinant strain was constructed.

[0048] Example 2 Induction expression of recombinant E. coli and preparation of enzyme solution 1. Induction expression The single colony of the engineering bacteria was inoculated into 2YT medium (2YT medium formula: 16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 6 h; 1% (v / v) inoculum was transferred to 150 ml 2YT medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm until OD600 was 0.6-0.8, then 0.3 mM IPTG was added, and the culture was induced at 16°C for 20 h, then centrifuged at 4°C and 6000 rpm for 15 min, and the supernatant was discarded, and the precipitate was reserved.

[0049] 2. Preparation of crude enzyme solution 10% (m / v) UGT11 mutant bacterial solution was prepared using Tris (50 mM pH 7.5) solution, and the target protein was released by high-pressure homogenization (900 bar) of the bacterial cells, then centrifuged at 4°C and 6000 rpm for 15 min, and the supernatant enzyme solution was collected to obtain UGT11 mutant crude enzyme solution.

[0050] 3. Protein purification (1) Nickel gravity column pretreatment: HisSep Ni-NTA Agarose Resin was loaded into a suitable purification column by gravity, and the chromatography column was washed with 2 column volumes of deionized water, and the chromatography column was equilibrated with 2 column volumes of Tris; (2) Sample loading: the crude enzyme solution prepared above was added dropwise to the purification column, and the sample loading speed was controlled to ensure that the target protein contacted with Ni 2+ sufficiently to improve the yield of purification; (3) Washing: 2 column volumes of Wash Buffer were used for washing; (4) Elution: Elute with 30 mL Elution Buffer, collect the eluate as the target protein solution; (5) Column regeneration: 1 M imidazole for 1 column volume, and deionized water for 2 column volumes; (6) Preservation: Finally, the resin is preserved in deionized water at 4℃; (7) Ultrafiltration: Add 50 mM Tris buffer (pH 7.5) into the ultrafiltration tube, centrifuge at 5000 rpm for 10 min at 4℃, and discard the supernatant; add an appropriate amount of eluted target protein solution, centrifuge at 5000 rpm for 10 min at 4℃ until the volume is about 1 mL; add an equal volume of 50 mM Tris buffer (pH 7.5), centrifuge at 5000 rpm for 10 min at 4℃ until the volume is about 1 mL, and repeat this step once to remove high-concentration imidazole, and obtain the protein purification solution.

[0051] Example 3 Enzymatic activity determination of recombinant E. coli (UGT11 mutant) UGT11 mutant (RA→RD) enzyme activity determination: Definition: the amount of enzyme required to generate 1 µmol of RD in 1 minute at a reaction temperature of 37℃ is defined as 1 enzyme activity unit (U).

[0052] (1) Enzymatic reaction Add 1 mM UDPG, 1 mM RA, 50 mM Tris (pH 7.5), and 0.1 mg / mL purified protein to the reaction system in a 96-well plate, and react at 37℃ for 30 min. After mixing with an equal volume of methanol, centrifuge through a 0.22 µm filter membrane, and detect the amount of RD generated.

[0053] (2) Enzyme activity calculation Specific activity (U / g) = RD generation amount (µmol) ÷ reaction time (min) ÷ protein mass (g) According to the above enzyme activity determination method, the detection results are shown in Table 6: Table 6

[0054] As shown in Table 6, the specific activity of the recombinant E. coli induced expression of the glycosyltransferase UGT11 mutant UGT11 (S158F) is significantly improved, which is 403.47 U / g, and the enzyme activity is increased by 9.89 times compared with UGT11.

[0055] Example 4 Enzymatic activity thermal stability determination of recombinant E. coli (UGT11 mutant) After placing the above mutant in the buffer system in a 55℃ water bath for 30 min, the enzyme activity determination method shown in Example 3 is used to detect the results, as shown in Table 7: Table 7

[0056] As shown in Table 7, the recombinant E. coli induced expression of glycosyltransferase AtSUS mutant AtSUS (P94N) still retained 93.66% of the enzyme activity after 30 min incubation at 55°C, and under the same treatment conditions, the wild-type enzyme activity was retained at 42.89%. The mutant UGT11 (S158F) expression strain was named R1 (CGMCC No. 34492).

[0057] Example 5 Whole-cell catalytic synthesis of RebD According to the induction expression method shown in Example 2, the wet bacteria of the engineering bacteria R1 (UGT11 (S158F)) were obtained, and the AtSUS induction expression wet bacteria were prepared by the same method. In the reaction vessel, 0.1 mmol / L of UDP was added, and the final concentration of sucrose was 0.5 M, the final concentration of Reb A was 20 g / L, and the volume was made to 100 mL with pH 7.5 50 mmol / L Tris-HCl buffer, and the amount of UGT11 (S158F) and AtSUS expression bacteria wet bacteria was 100 U, and the reaction was stirred at 220 rpm for 12 hours. After the reaction was completed, 100 μL of the reaction solution was added to 900 μL of anhydrous methanol for dilution, and after high-speed centrifugation at 10000 rpm, 3 min, 4°C, the supernatant was filtered and detected by HPLC. The results showed that Reb A was basically converted, and the conversion rate of Reb D was 96.49%.

[0058] The above detailed description is a specific description of one of the feasible embodiments of the present application, and this embodiment is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A glycosyltransferase mutant for use in the synthesis of rebaudioside D, characterized in that, The glycosyltransferase mutant comprises at least one substitution mutation selected from the following positions based on the amino acid sequence set forth in SEQ ID NO: 1: the amino acid at position 158 is mutated from S to F or the amino acid at position 93 is mutated from H to Q.

2. The glycosyltransferase mutant of claim 1, wherein, The amino acid sequence of the glycosyltransferase mutant is set forth in SEQ ID NO: 2 or SEQ ID NO:

4.

3. A nucleic acid encoding the glycosyltransferase mutant of claim 1 or 2. The nucleic acid has a sequence set forth in SEQ ID NO: 7 or SEQ ID NO:

9.

4. An expression vector comprising the nucleic acid of claim 3.

5. The expression vector of claim 4, wherein, The expression vector is selected from a plasmid, a bacteriophage, a virus, or an artificial chromosome.

6. The expression vector of claim 5, wherein, The expression vector is a plasmid.

7. The expression vector of claim 6, wherein, The plasmid is selected from the pET series, the pQE series, the pGEX series, or the pMAL series.

8. The expression vector of claim 6, wherein, The plasmid is pET-30a.

9. A host cell comprising the nucleic acid of claim 3 or the expression vector of any one of claims 4-8.

10. The host cell of claim 9, wherein, The host cell is a prokaryotic cell or a eukaryotic cell.

11. The host cell of claim 10, wherein, The host cell is Escherichia coli, Saccharomyces, Bacillus, or Lactobacillus.

12. The host cell of claim 11, wherein, The host cell has a deposit number of CGMCC No. 34492.

13. A method of producing the glycosyltransferase mutant of claim 1 or 2, comprising, comprising the following steps: S1, expressing the nucleic acid encoding the mutant in a host cell; S2, culturing the host cell and inducing protein expression; S3, isolating and purifying the glycosyltransferase mutant.

14. A cell culture or an extract obtained by culturing the host cell of any one of claims 9-12.

15. An enzyme preparation, characterized in that, comprising the glycosyltransferase mutant of claim 1 or 2 or the cell culture or the extract of claim 14.

16. Use of the glycosyltransferase mutant of claim 1 or 2 or the nucleic acid of claim 3 or the expression vector of any one of claims 4-8 or the host cell of any one of claims 9-12 or the enzyme preparation of claim 15 in the synthesis of rebaudioside D.

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