A glycosyltransferase mutant and its application in steviol glycoside biosynthesis
By directing the evolution and rational design of glycosyltransferases, and mutating the 9th, 39th, and 393rd amino acid sites, highly efficient glycosyltransferase mutants were obtained, solving the problems of insufficient catalytic activity and stability, and realizing the efficient biosynthesis of steviol glycosides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XITIAN (SHANGHAI) BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-08
- Publication Date
- 2026-06-09
AI Technical Summary
Existing naturally derived glycosyltransferases exhibit insufficient catalytic activity, limited substrate affinity, and poor thermal stability in the conversion of ribobadiol D to ribobadiol M. Furthermore, they are sensitive to fluctuations in the supply of uridine diphosphate glucose, resulting in low conversion efficiency, increased byproducts, and difficulty in achieving efficient industrial-scale preparation.
By directing the evolution and rational design of glycosyltransferases, mutant glycosyltransferases with excellent catalytic performance were obtained by mutating the 9th, 39th and 393rd amino acid sites. Recombinant expression vectors and recombinant strains were constructed and applied to catalyze the conversion of Reb D to Reb M in a multi-enzyme cascade system.
The synthesis yield and process economy of Reb M were significantly improved. The mutant enzyme conversion rate was increased by 7.5% to 24.2% compared with the wild type under different conditions, realizing efficient biomanufacturing of steviol glycosides.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a glycosyltransferase mutant and its application in steviol glycoside biosynthesis. The glycosyltransferase mutant can be produced in various expression hosts such as Pichia pastoris, Escherichia coli, and Bacillus subtilis, and is suitable for targeted biomanufacturing in multi-enzyme cascade systems. Background Technology
[0002] With the increasing global demand for sugar reduction, sugar control, and healthy eating, natural high-intensity sweeteners are gradually replacing traditional sucrose and artificial sweeteners, becoming an important development trend in the food and beverage industry. Stevia glycosides, natural glycoside compounds derived from the stevia rebaudiana plant (Asteraceae family), have attracted widespread attention due to their high sweetness, low calories, and recognized safety properties. The sweetness quality and application value of steviol glycosides are closely related to their degree of glycosylation and glycoside structure. Higher-order glycosides, such as rebaudioside D (Reb D) and rebaudioside M (Reb M), have a taste closer to sucrose and are considered a new generation of natural sweeteners with greater market potential. However, the content of these higher-order glycosides in natural plants is extremely low, and plant extraction alone cannot meet the needs of large-scale production. Therefore, the enzymatic biotransformation synthesis of steviol glycosides has become a key direction for current industry and research.
[0003] In the enzymatic synthesis of steviol glycosides, particularly in the biological process catalyzing the conversion of RebD to the higher-order RebM, uridine diphosphate glucose (UDP-glucose) is an essential glycosyl donor, and its efficient and stable supply is crucial to determining the efficiency of the synthetic pathway. Currently, glycosyltransferases (UGTs) are the core catalytic enzymes for this specific glycosylation step, and their catalytic performance directly affects the yield and purity of the target product, RebM. However, when naturally derived glycosyltransferases are used in in vitro synthetic systems, they often face challenges such as insufficient catalytic activity, limited affinity for the substrate RebD, poor thermal and operational stability, and sensitivity to fluctuations in UDPG supply. During long-term reactions, changes in UDPG supply can easily lead to a sharp drop in catalytic efficiency, ultimately resulting in low conversion efficiency and increased byproducts, thus hindering the efficient industrial preparation of RebM.
[0004] This invention employs a combination of directed enzyme evolution and rational design to systematically modify glycosyltransferases at the molecular level. In the prior art, there are no reports of improving the conversion efficiency of Reb D to Reb M by site-directed mutations at amino acid positions 9, 39, and 393 of glycosyltransferases.
[0005] This invention, through modification of the aforementioned sites, yields a glycosyltransferase mutant with significantly improved performance. When applied to a multi-enzyme cascade system, this mutant enables more efficient and specific glycosyltransfer reactions, significantly improving the synthetic yield and process economy of Reb M. This has significant technical implications and application prospects for the targeted biomanufacturing of high-value steviol glycosides. Summary of the Invention
[0006] The purpose of this invention is to provide a glycosyltransferase mutant, its encoding nucleic acid sequence, expression vector, recombinant strain, and its application in the biosynthesis of steviol glycosides.
[0007] The glycosyltransferase mutant provided by this invention is obtained by molecular modification using a combination of rational design and directed evolution, starting from the wild-type glycosyltransferase sequence. The nucleotide sequence of the wild-type glycosyltransferase is shown in SEQ ID NO.1 (GenBank accession number: AY345974.1), and the nucleic acid sequence encoding the wild-type glycosyltransferase is shown in SEQ ID NO.2.
[0008] This invention first predicts nine potential mutation sites through rational design, and then screens out three core effective mutation sites through experimental verification: positions 9, 39, and 393. Specifically:
[0009] The valine at position 9 is mutated to methionine, denoted as V9M;
[0010] The lysine at position 39 is mutated to arginine, denoted as K39R;
[0011] The lysine at position 393 is mutated to glycine, denoted as K393G.
[0012] The above-mentioned mutation sites can exist alone or in any combination to form glycosyltransferase mutants with excellent catalytic performance.
[0013] The present invention also provides a recombinant expression vector, wherein the vector contains the nucleic acid sequence of the glycosyltransferase mutant.
[0014] The vector is any one of pPIC9K, pPICZA, pET28a, pMOL, pHT43 or a series of derived vectors, or other vectors suitable for eukaryotic or prokaryotic expression systems.
[0015] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the nucleic acid sequence of the glycosyltransferase mutant or the chassis cells of the recombinant expression vector.
[0016] The chassis cells of the recombinant bacteria are selected from any one of Pichia pastoris (such as GS115 or X33), Escherichia coli (such as BL21(DE3)), Bacillus subtilis (such as BS168), or other Gram-positive bacteria, Gram-negative bacteria, or fungal host cells.
[0017] The present invention also provides the glycosyltransferase mutant, its encoding nucleic acid sequence, recombinant expression vector, and recombinant bacteria in the preparation of steviol glycosides, specifically the glycosyltransferase mutant being used to catalyze the conversion of Reb D to Reb M.
[0018] Specifically, the enzymatic conversion method for synthesizing steviol glycosides involves using Reb D as a substrate and an enzyme preparation (including crude enzyme solution, partially purified enzyme, or purified enzyme) containing the glycosyltransferase mutant to efficiently prepare Reb M. The reaction system comprises: Reb D, sucrose, UDPG (uridine diphosphate glucose), buffer solution, and crude enzyme solution. In the reaction system, the amount of Reb D is 0.8 g / L-2 g / L, more preferably 0.8 g / L; the amount of sucrose is 200 g / L-600 g / L, more preferably 600 g / L; the amount of UDPG is 0.5 mM-1 mM, preferably 1 mM; the amount of buffer solution is 50 mM PBS; and the amount of crude enzyme solution is 40 g / L.
[0019] This invention provides glycosyltransferase mutants that significantly improve the conversion efficiency of Reb D. Experiments show that, under reaction conditions of 0.8 g / L Reb D, 600 g / L sucrose, and 1 mM UDPG, the V9M mutant catalyzes a 24.2% higher conversion rate of Reb D to Reb M compared to the wild type; under a substrate condition of 2 g / L Reb D, the V9M mutant achieves a 14.1% higher conversion rate than the wild type, and the K393G mutant achieves a 7.5% higher conversion rate. Attached Figure Description
[0020] Figure 1 shows a map of the plasmid pPIC9K-UGT76G1 carrying the glycosyltransferase.
[0021] Figure 2 shows the catalytic detection results of wild-type glycosyltransferase and mutant with 1 g / L RebD substrate.
[0022] Figure 3 shows the liquid chromatography analysis of RebM synthesized from 1 g / L RebD substrate catalyzed by pPIC9K-UGT76G1.
[0023] Figure 4 shows the liquid chromatography analysis of RebD synthesized from 1 g / L RebD substrate, catalyzed by pPIC9K-UGT76G1-V9M, after the reaction.
[0024] Figure 5 shows the catalytic detection results of wild-type glycosyltransferase and mutant RebD substrate at 2 g / L.
[0025] Figure 6 shows the effect of different sucrose concentrations on glycosyltransferase mutants.
[0026] Figure 7 shows the effect of different UDPG concentrations on glycosyltransferase mutants. Detailed Implementation
[0027] The present invention will be further described below with reference to the embodiments and accompanying drawings.
[0028] The examples used Pichia pastoris as the expression host to verify the function of the glycosyltransferase mutant described in this invention. The methods and equipment used in this invention are conventional methods and equipment in this technical field. Unless otherwise specified, the reagents and consumables used in the following examples are all commercially available products or prepared using conventional methods in this field.
[0029] The culture media and actual ratios involved in the examples are shown in Table 1:
[0030] Table 1
[0031] .
[0032] The detection methods involved in the following embodiments are as follows:
[0033] After the reaction was completed and the cells were inactivated, the solution was diluted with 300 µL of pure acetonitrile, centrifuged at 12000 rpm for 10 min, filtered through a 0.22 µM filter membrane, and the yield of Reb M was analyzed by high performance liquid chromatography (HPLC). The HPLC column used was an Ultimate® XB-C18 column from Yuexu Technology (Shanghai) Co., Ltd., 5 µm, 4.6 × 250 mm.
[0034] Injection volume: 8 µL;
[0035] Column temperature: 40 ℃;
[0036] Detection wavelength: 210 nm;
[0037] Mobile phase A: Sodium phosphate buffer (pH 2.6);
[0038] Mobile phase C: pure acetonitrile;
[0039] Mobile phase D: Ultrapure water;
[0040] Mobile phase flow rate: 0.8 mL / min;
[0041] The procedure is shown in Table 2:
[0042] Table 2
[0043] .
[0044] (1) Obtaining the β-1,3-glycosyltransferase gene and constructing mutants
[0045] The nucleotide sequence of β-1,3-glycosyltransferase from stevia (Stevia rebaudiana) was obtained from GenBank (SEQ ID NO.1, GenBank accession number: AY345974.1). Based on this sequence, a rational design was performed, and nine potential mutation sites were predicted, as follows:
[0046] Position 9: Valine is mutated to methionine (V9M);
[0047] Position 39: Lysine is mutated to arginine (K39R);
[0048] Position 41: Phenylalanine is mutated to histidine (F41H).
[0049] Position 43: Isoleucine is mutated to valine (I43V);
[0050] Position 93: Isoleucine is mutated to phenylalanine (I93F);
[0051] Position 119: Serine is mutated to aspartic acid (S119D).
[0052] Position 221: Serine is mutated to threonine (S221T).
[0053] Position 378: Glycine is mutated to tryptophan (G378W);
[0054] Position 393: Lysine is mutated to glycine (K393G).
[0055] Experimental verification showed that mutants at positions 9, 39, and 393 exhibited the best catalytic performance among the nine potential mutation sites and were identified as core effective mutation sites. Based on the gene sequence of wild-type glycosyltransferase, this invention designed site-directed mutagenesis primers for each of the screened key sites (see Appendix Table 3 for details). Using the vector pPIC9K-UGT76G1 as a template, PCR site-directed mutagenesis technology was used to introduce single-point mutations at each key site. The obtained PCR products were verified by agarose gel electrophoresis. The results showed that all nine mutation sites screened based on sequence analysis could amplify specific bands, and their sizes were consistent with theoretical expectations.
[0056] Table 3
[0057] .
[0058] PCR reaction parameters: Pre-denaturation: 98℃ for 30 s; Denaturation: 98℃ for 10 s; Annealing: 59℃ for 5 s; Extension: 72℃ for 30 s; Complete extension: 72℃ for 1 min;
[0059] Using In-Fusion recombination technology, the purified linearized vector fragments were homologously recombinated with the corresponding mutant gene fragments to construct recombinant expression plasmids of β-1,3-glycosyltransferase mutants, including: V9M, K39R, F41H, I43V, I93F, S119D, S221T, K393G, and G378W.
[0060] Each successfully constructed recombinant plasmid was verified by DNA sequencing. Plasmids with verified correct sequences were then transformed into Pichia pastoris GS115 and X33 hosts, respectively. Positive transformants were obtained using a selection medium, and after sequencing verification, the positive transformants were used for fermentation.
[0061] (2) Preparation of recombinase
[0062] The mutant strain of *Glyceryl* constructed in (1) was streaked onto YPD solid plates and incubated upside down at 30°C for 2-3 days. Single colonies were then picked and inoculated into 5 mL of YPD liquid medium containing the corresponding antibiotic, and cultured with shaking at 30°C and 200 rpm for 12 hours. Then, 2% (v / v) of the culture was inoculated into 100 mL of BMGY medium (initial pH 6.0) and cultured for 24 h. The cells were collected by centrifugation and inoculated into an equal volume of BMMY medium. 500 μL of anhydrous methanol was added every 24 h to induce expression.
[0063] The induced bacterial culture was centrifuged at 8000 rpm for 5 minutes at 4°C, the supernatant was discarded, and the bacterial cells were collected. The wet bacterial culture was resuspended in 50 mM PBS buffer (pH=7.0) to a concentration of 0.1 g / mL. The resuspended bacterial culture was homogenized using an autoclave, and the homogenate was centrifuged again at 10000 rpm for 3 minutes at 4°C. The supernatant obtained was the crude enzyme solution.
[0064] (3) Synthesis of Reb M by 1 g / L Reb D enzymatic method
[0065] Using Reb D as a substrate, the reaction was carried out in a 1 mL reaction system. The catalytic reaction system was as follows: 1 g / L Reb D, 400 g / L sucrose, 1 mM UDP, 50 mM PBS buffer (pH=7.0), and 40 g / L crude glycosyltransferase solution. The reaction was carried out at 35 °C for 17 h. After the reaction, the system was heated at 95 °C for 10 min to terminate enzyme activity. Then, 300 μL of acetonitrile was added, and the mixture was thoroughly mixed and centrifuged at 12000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm organic filter membrane, and transferred to a sample vial for quantitative analysis by HPLC.
[0066] HPLC results showed that different glycosyltransferase mutants exhibited significant differences in the conversion efficiency of Reb D to Reb M (see [link to HPLC results]). Figure 2 Among them, K39R, K393G, and V9M, corresponding to the core mutation sites, were significantly superior to the wild-type (WT) enzymes. HPLC chromatograms of the wild-type (WT) and mutant V9M are shown below. Figure 3 and Figure 4 .
[0067] (4) Synthesis of Reb M by 2 g / L Reb D enzymatic method
[0068] Using Reb D as a substrate, the reaction was carried out in a 2 mL reaction system. The catalytic reaction system was as follows: 2 g / L Reb D, 400 g / L sucrose, 1 mM UDP, 50 mM PBS buffer (pH=7.0), and 40 g / L crude glycosyltransferase solution. The reaction was carried out at 35 °C for 17 h. After the reaction, the system was heated at 95 °C for 10 min to terminate enzyme activity. Then, 300 μL of acetonitrile was added, and the mixture was thoroughly mixed and centrifuged at 12000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm organic filter membrane, and transferred to a sample vial for quantitative analysis by HPLC.
[0069] HPLC results showed that different glycosyltransferase mutants exhibited significant differences in the conversion efficiency of Reb D to Reb M (see [link to HPLC results]). Figure 5 Among them, K39R, V9M, and K393G all showed significantly improved conversion rates compared to wild-type (WT) enzymes; in particular, the V9M mutant exhibited the highest conversion rate, 14.1% higher than the wild-type (WT) enzyme, followed by K393G, whose conversion rate was 7.5% higher than the wild-type.
[0070] (5) Mutant-catalyzed synthesis of Reb M from Reb D under different sucrose concentrations
[0071] Using Reb D as a substrate, the reaction was carried out in a 1 mL reaction system. The catalytic reaction system was as follows: 0.8 g / L Reb D, 200–600 g / L sucrose, 1 mM UDP, 50 mM PBS buffer (pH=7.0), and 40 g / L crude glycosyltransferase solution. The reaction was carried out at 35 °C for 17 h. After the reaction, the system was heated at 95 °C for 10 min to terminate enzyme activity. Then, 300 μL of acetonitrile was added, and the mixture was thoroughly mixed and centrifuged at 12000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm organic filter membrane, and transferred to a sample vial for quantitative analysis by HPLC.
[0072] HPLC results showed that different glycosyltransferase mutants exhibited significant differences in the conversion efficiency of Reb D to Reb M (see [link to HPLC results]). Figure 6 Among them, the mutant showed the highest enzyme conversion rate improvement of 13% compared to the wild-type (WT).
[0073] (6) Mutant-catalyzed Reb D synthesis of Reb M under different UDPG concentrations
[0074] Using Reb D as a substrate, the reaction was carried out in a 1 mL reaction system. The catalytic reaction system was as follows: 0.8 g / L Reb D, 600 g / L sucrose, 0.5–1 mM UDP, 50 mM PBS buffer (pH=7.0), and 40 g / L crude glycosyltransferase solution. The reaction was carried out at 35 °C for 17 h. After the reaction, the system was heated at 95 °C for 10 min to terminate enzyme activity. Then, 300 μL of acetonitrile was added, and the mixture was thoroughly mixed and centrifuged at 12000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm organic filter membrane, and transferred to a sample vial for quantitative analysis by HPLC.
[0075] HPLC results showed that different glycosyltransferase mutants exhibited significant differences in the conversion efficiency of Reb D to Reb M (see [link to HPLC results]). Figure 7 Among them, under 1 mM UDPG conditions, the V9M mutant showed the highest conversion rate, which was 24.2% higher than that of the wild-type (WT) enzyme.
Claims
1. A glycosyltransferase mutant, characterized in that, Compared to the wild-type glycosyltransferase SEQ ID NO.1, it contains a conserved substitution selected from at least one of the following amino acid sites: position 9, position 39, and position 393; wherein the conserved substitution is: valine at position 9 is replaced by methionine, denoted as V9M; lysine at position 39 is replaced by arginine, denoted as K39R; and lysine at position 393 is replaced by glycine, denoted as K393G.
2. A nucleic acid sequence encoding the glycosyltransferase mutant of claim 1, characterized in that, Corresponding to V9M, K39R, and K393G, the nucleic acid sequences are shown as SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.
3. A recombinant expression vector, characterized in that, It contains the nucleic acid sequence corresponding to the glycosyltransferase mutant of claim 1 or the nucleic acid sequence of claim 2.
4. The recombinant expression vector according to claim 3, characterized in that, The vector is any one of pPIC9K, pPICZA, pET28a, pMOL, pHT43 or a series of derived vectors, or other vectors suitable for eukaryotic or prokaryotic expression systems.
5. A recombinant bacterium, characterized in that, The nucleic acid sequence comprising the glycosyltransferase mutant of claim 1, the nucleic acid sequence of claim 2, or the recombinant expression vector of claim 3 or 4.
6. The recombinant bacteria according to claim 5, characterized in that, The chassis cells of the recombinant bacteria are selected from any one of Pichia pastoris, Escherichia coli, Bacillus subtilis, or other Gram-positive bacteria, Gram-negative bacteria, or fungal host cells.
7. The recombinant bacteria according to claim 6, characterized in that, The chassis cells are Pichia pastoris GS115 or X33, Escherichia coli BL21 (DE3), Bacillus subtilis BS168, or their derivative strains.
8. The use of the glycosyltransferase mutant of claim 1, the nucleic acid sequence of claim 2, the recombinant expression vector of any one of claims 3-4, and the recombinant bacteria of any one of claims 5-7 in the preparation of steviol glycosides or as a catalyst for glycosyltransfer reactions.
9. The application according to claim 1, characterized in that, Using Reb D as a substrate, the gene encoding the glycosyltransferase mutant was constructed into a recombinant expression vector, and the recombinant engineered bacteria were obtained by transforming the host bacteria. Reb M was prepared by catalytic transformation using an enzyme preparation containing the glycosyltransferase mutant. The reaction system consisted of Reb D, sucrose, UDPG, buffer solution, and crude enzyme solution.
10. The application according to claim 8, characterized in that, The reaction system contained 0.8 g / L to 2 g / L of Reb D, 200 g / L to 600 g / L of sucrose, 0.5 mM to 1 mM of UDPG, 50 mM PBS as the buffer, and 40 g / L of the enzyme preparation containing the glycosyltransferase mutant.