A glycosyltransferase mutant and its application in synthesis of steviol glycosides

Abstract

The application belongs to the technical field of enzyme engineering, and particularly relates to a glycosyltransferase mutant and application thereof in synthesis of steviol glycoside. The glycosyltransferase mutant is reformed on the basis of an amino acid sequence SEQ ID NO. 1, and key mutation sites include: the 77th methionine is mutated into tyrosine M77Y and the 142th methionine is mutated into tryptophan M142W; the application also discloses a nucleic acid sequence coding the mutant, an expression vector, a recombinant strain, and application thereof in industrialized production of steviol glycoside, and realizes efficient synthesis of rebaudioside D; experimental results show that the efficiency of catalyzing Reb A to synthesize Reb D is compared with that of the wild-type glycosyltransferase, the catalytic efficiency of M77Y is the highest, the conversion rate is more than 70%, which is more than 5 times of the wild type; the conversion rates of M142W and M142W / M77Y are more than 40% and more than 29% respectively, which are 3 times and more than 2 times of the wild type respectively.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to glycosyltransferase mutants and their applications. Background Technology

[0002] Steviol glycosides (SGs) are natural, high-sweetness, low-calorie sweeteners extracted from the leaves of the stevia (Steviarebaudiana Bertoni), a plant in the Asteraceae family. Their main active components are steviol glycoside (STV) and rebaudioside A (Reb A), with contents of 4%-10% and 2%-5% respectively in dried leaves. Steviol glycosides are approximately 200-300 times sweeter than sucrose, while their caloric value is only 1 / 300th that of sucrose. This sweetener can be widely used in various food and pharmaceutical fields, including ice cream, soft drinks, tea, dairy products, and pharmaceuticals, to provide sweetness and enhance flavor. Furthermore, research indicates its potential to regulate physiological functions (such as aiding in lowering blood pressure and blood sugar), making it valuable for meeting the dietary sweetness needs of specific populations (such as diabetics, children, and pregnant women).

[0003] The sweetness characteristics of steviol glycosides are closely related to the number and type of sugar groups linked to C-13 and C-19 sites in their molecular structure. For example, steviol glycosides (STV) and rebaudioside A (Reb A), which are abundant in natural extracts, are often accompanied by unpleasant tastes such as bitterness and astringency. In contrast, rebaudioside D (Reb D) exhibits a sweetness more similar to sucrose, characterized by a purer sweetness, longer sweetness persistence, and no obvious bitterness or unpleasant aftertaste, making it a potential advantage in sweetener applications. However, the content of Reb D in natural stevia (Stevia rebaudiana Bertoni) leaves is generally low (usually only 0.4-0.5% of dry weight), which severely restricts the feasibility of large-scale economical acquisition through traditional leaf extraction methods. In addition, the traditional leaf extraction process for steviol glycosides faces multiple bottlenecks: (1) Raw material quality constraints: Affected by factors such as germplasm degradation, the content of target glycosides (especially high-value components such as Reb D) in raw leaf materials shows a downward trend; (2) Insufficient extraction efficiency and selectivity: Current extraction methods (such as solvent extraction and column chromatography) generally suffer from poor selectivity and low separation precision, resulting in limited purity of the obtained product and the potential for residues or the generation of undesirable flavor substances (such as post-bitter components); (3) Weak production process control: Insufficient control of the production environment (such as temperature, humidity, and cleanliness) makes it difficult to consistently meet the stringent hygiene and safety standards in the food additive field; (4) Resource consumption and environmental pressure: The process consumes a large amount of water resources and chemical raw materials (such as organic solvents) and generates organic wastewater with high chemical oxygen demand (COD) and high biological oxygen demand (BOD), resulting in a heavy environmental treatment burden. Based on the above limitations, it is difficult to achieve efficient and large-scale industrial production of high-quality steviol glycosides (especially high-value Reb D) by relying on the traditional leaf extraction route. In comparison, the enzymatic conversion synthesis method exhibits significant technical advantages: the process is mature and stable, has high production efficiency, and can produce high-purity target steviol glycoside components (such as Reb D) in a targeted and efficient manner, effectively overcoming the limitations of natural sources.

[0004] Enzymes, as biocatalysts, play a vital role in transformation due to their high efficiency and substrate specificity. However, natural enzymes often face challenges in industrial applications, such as insufficient catalytic activity, poor stability, and unsatisfactory substrate / product selectivity. The development of synthetic biology and artificial intelligence technologies has provided new methods and perspectives for in-depth analysis of enzyme catalytic mechanisms, optimization of enzyme properties (such as activity, stability, and selectivity), and design of novel functional enzymes. Currently, techniques such as directed evolution based on experimental screening, rational / semi-rational design based on structure and mechanism, and de novo enzyme design based on computational simulation have become core strategies in the field of enzyme engineering to improve the industrial applicability of enzymes. Summary of the Invention

[0005] The purpose of this invention is to provide a glycosyltransferase mutant and its application in the biosynthesis of steviol glycosides.

[0006] The glycosyltransferase mutant provided by this invention contains one of the following amino acid mutations relative to the wild-type glycosyltransferase (accession number: WP_004399256.1): methionine at position 77 is mutated to tyrosine, denoted as M77Y, and methionine at position 142 is mutated to tryptophan, denoted as M142W.

[0007] The nucleic acid sequence encoding the glycosyltransferase is shown in SEQ ID NO.1.

[0008] The present invention also provides a recombinant expression vector, wherein the vector contains the nucleic acid sequence of the glycosyltransferase mutant.

[0009] The carrier can be any carrier or derivative carrier carrying the MBP solubilization tag, such as the pMAL series carriers or the pET28a-MBP derivative carrier.

[0010] 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.

[0011] The chassis cells are Escherichia coli, such as E. coli BL21(DE3) and E. coli JM109.

[0012] The present invention also provides the application of the glycosyltransferase mutant in the preparation of steviol glycosides.

[0013] Specifically, steviol glycosides are synthesized by enzymatic conversion; Reb A is used as a substrate, and crude enzyme solution of the recombinant bacteria is used to efficiently prepare Reb D.

[0014] The reaction system consists of: rebaudioside A, sucrose, UDPG (uridine diphosphate glucose), buffer solution, and crude enzyme solution.

[0015] In the reaction system, the amount of substrate rebaudioside A is preferably 0.5 g / L-3 g / L, more preferably 0.5 g / L; the amount of sucrose is preferably 200 g / L-400 g / L, more preferably 400 g / L; the amount of UDPG is preferably 1 mM-3 mM, more preferably 3 mM; the amount of buffer is 10 mM-50 mM PBS, more preferably 50 mM; and the amount of crude enzyme solution is 25 g / L-50 g / L, more preferably 50 g / L.

[0016] This invention provides glycosyltransferase mutants that can significantly improve the conversion efficiency of Reb A. Experiments show that, compared with the efficiency of wild-type glycosyltransferases, the mutant M77Y has the highest catalytic efficiency in synthesizing Reb D from Reb A, with a conversion rate of over 70%, which is more than 5 times that of the wild type; the conversion rates of M142W and M142W / M77Y also reach over 40% and over 29%, respectively, which are more than 3 times and 2 times that of the wild type. Attached Figure Description

[0017] Figure 1 A map showing the plasmid pET28a-MBP-Yojk carrying the glycosyltransferase.

[0018] Figure 2 SDS-PAGE gel electrophoresis patterns of E. coli BL21(DE3)pET28a-MBP-Yojk, E. coli BL21(DE3)pET28a-MBP-Yojk-M77Y and E. coli BL21(DE3)pET28a-MBP-Yojk-M142W.

[0019] Figure 3 The spectrum for Reb A was retained for 25.485 min.

[0020] Figure 4 The spectrum for Reb D was retained for 17.072 min.

[0021] Figure 5 This is a liquid chromatography analysis of the reaction of Reb A to Reb D catalyzed by the glycosyltransferase pET28a-MBP-Yojk in Example 3.

[0022] Figure 6 Liquid chromatography analysis of Reb A to Reb D catalyzed by the glycosyltransferase mutant pET28a-MBP-Yojk-M77Y.

[0023] Figure 7 Liquid chromatography analysis of Reb A to Reb D catalyzed by the glycosyltransferase mutant pET28a-MBP-Yojk-M142W. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the embodiments do not limit the present invention in any way. The methods and equipment used in the present invention are conventional methods and equipment in this technical field. Unless otherwise specified, the reagents and consumables used in the following embodiments are all commercially available products or prepared by conventional methods in the art.

[0025] The culture media involved in the examples are as follows: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder.

[0026] The detection methods involved in the following embodiments are as follows:

[0027] After the reaction was completed and the cells were inactivated, the solution was diluted with 300 μL of pure acetonitrile, centrifuged at 12000 × g for 10 min, filtered through a 0.22 μM filter membrane, and the yield of Reb D was analyzed by high-performance liquid chromatography (HPLC). HPLC was performed using a column from Yuexu Technology (Shanghai) Co., Ltd. XB-C18, 5μm, 4.6x250 mm.

[0028] Injection volume: 8 μL;

[0029] Column temperature: 40℃;

[0030] Detection wavelength: 210nm;

[0031] Mobile phase A: Sodium phosphate buffer (pH 2.6);

[0032] Mobile phase C: pure acetonitrile;

[0033] Mobile phase D: Ultrapure water;

[0034] Mobile phase flow rate: 0.8 mL / min;

[0035] The procedure is shown in Table 1:

[0036] Table 1

[0037] Time / min Sodium phosphate buffer / % Acetonitrile / % 0 75 25 13 75 25 15 68 32 40 68 32 42 75 25 45 75 25 .

[0038] (1) Obtaining glycosyltransferase genes and constructing mutants

[0039] The amino acid sequence of the glycosyltransferase Yojk from Bacillus subtilis (accession number: WP_004399256.1) was downloaded from Genebank, and the gene was synthesized and codon optimized by Beijing Qingke Biotechnology Co., Ltd. and ligated into the multiple cloning site of the vector pET28a-MBP to obtain the recombinant plasmid pET28a-MBP-Yojk.

[0040] Using the recombinant plasmid pET28a-MBP-Yojk as a template, the PCR amplification system was established using the primers in Table 2 as follows: 2×Phanta Flash Master Mix 25μL, upstream primer 2μL, downstream primer 2μL, ddH2O 20.5μL, and template 0.5μL.

[0041] Table 2: Primer Names and Primer Sequences

[0042]

[0043]

[0044] PCR reaction parameters: Pre-denaturation: 98℃ for 30s; Denaturation: 98℃ for 10s; Annealing: 59℃ for 5s; Extension: 72℃ for 10s; Complete extension: 72℃ for 10min;

[0045] After purification, the fragments were obtained, and recombinant plasmids carrying glycosyltransferase mutants, namely M77K, M77R, M77K, M77N, M142W, M142W / M77R, M142W / M77Y, and M142W / M77N, were constructed using the In-Fusion method.

[0046] The above plasmids were sequenced and verified. The plasmids with correct sequencing were transformed into E. coli BL21(DE3) competent cells and recombinant strains carrying the corresponding plasmids were obtained by screening with resistant solid plates.

[0047] (2) Preparation of recombinant strains

[0048] The recombinant strains of E. coli BL21(DE3) M77K, M77R, M77K, M77N, M142W, M142W / M77R, M142W / M77Y, and M142W / M77N carrying glycosyltransferase mutants constructed in (1) were inoculated into 3 mL of LB medium containing the corresponding antibiotics and cultured at 37°C and 200 rpm for 6 h. They were then transferred to 100 mL of LB medium containing the corresponding antibiotics at a 1% (v / v) inoculation rate and cultured at 37°C and 200 rpm until OD... 600The concentration was 0.6–0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and expression was induced at 25 °C and 200 rpm for 12 h.

[0049] Centrifuge the induced bacterial culture (4℃, 8000 rpm, 5 min) and discard the supernatant. Weigh the collected bacterial cells, resuspend them in 50 mM PBS at a ratio of 1:10 (M / V, g / mL), and sonicate them. Centrifuge the lysed bacterial culture (4℃, 8000 rpm, 5 min), and the supernatant is the crude enzyme solution.

[0050] (3) Glycosyltransferase mutants are used to catalyze the synthesis of Reb D from Reb A.

[0051] Using Reb A as a substrate, the reaction was carried out in a 1 mL reaction system. The catalytic reaction system was as follows: 0.5 g / L–3 g / L Reb A, 400 g / L sucrose, 1 mM–3 mM UDPG, 50 mM PBS buffer, and 50 g / L crude glycosyltransferase mutant enzyme solution. The reaction was carried out at 35 °C for 21 h. After the reaction, the enzyme was inactivated at 95 °C for 10 min, 300 μL of acetonitrile was added, and the mixture was centrifuged (12000 rpm, 10 min). The supernatant was filtered through a 0.22 μM organic filter into a sample vial for HPLC analysis. The conversion rates of Reb A to Reb D are shown in Tables 3 and 4.

[0052] Table 3. Effects of different substrate concentrations on glycosyltransferase mutants

[0053]

[0054]

[0055] Table 4. Effects of different UDPG concentrations on glycosyltransferase mutants

[0056] 1mM UDPG 2mM UDPG 3mM UDPG wild type 13.99％ 24.06％ 13.61％ M77K 0 0 0 M77R 6.41％ 10.12％ 9.9％ M77Y 53.6％ 70.94％ 71.7％ M77N 0 14.71％ 11.87％ M142W 28.59％ 31.89％ 44.27％ M142W / M77R 0 0 0 M142W / M77Y 21.92％ 47.61％ 29.68％ M142W / M77N 0 8.36％ 4.5％ .

[0057] As shown in Tables 3 and 4, under optimized reaction conditions (1 g / L Reb A - 3 mM UDPG), the efficiency of glycosyltransferase mutants in catalyzing the synthesis of Reb D from Reb A was significantly improved compared to the wild type (13.61%). Among them, mutant M77Y showed the highest catalytic efficiency, with a Reb D conversion rate of 71.7%, which was 5.27 times that of the wild type. The conversion rates of M142W and M142W / M77Y also reached 44.27% and 29.68%, respectively, which were 3.25 times and 2.18 times that of the wild type.

Claims

1. A glycosyltransferase mutant, characterized in that, The glycosyltransferase mutant is M77Y and / or M142W relative to the wild-type glycosyltransferase; wherein: M77Y is that the 77th methionine is mutated to tyrosine, and M142W is that the 142th methionine is mutated to tryptophan; The nucleic acid sequence encoding the glycosyltransferase is shown as SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, The nucleic acid sequence comprising the glycosyltransferase mutant of claim 1.

3. The recombinant expression vector of claim 2, wherein, The vector is any vector or derivative vector carrying the MBP solubility tag.

4. The recombinant expression vector of claim 3, wherein, The vector is a pMAL series vector or a pET28a-MBP derivative vector.

5. A recombinant bacterium, characterized in that, The chassis cell comprises the nucleic acid sequence of the glycosyltransferase mutant of claim 1 or the recombinant expression vector of claim 2.

6. The recombinant bacteria of claim 5, wherein, The chassis cell is Escherichia coli.

7. The recombinant bacteria of claim 6, wherein The chassis cell is E.coli BL21 or E.coli JM109.

8. Use of the glycosyltransferase mutant of claim 1, or the recombinant expression vector of any one of claims 2-4, or the recombinant bacteria of any one of claims 5-7 in the preparation of steviol glycosides.

9. Use according to claim 8, characterized in that, The gene encoding the glycosyltransferase mutant is constructed in a recombinant expression vector using rebaudioside A as the substrate, the host bacteria is transformed to obtain recombinant engineering bacteria, and the crude enzyme solution thereof is used to catalyze the conversion to prepare rebaudioside D; the reaction system is: rebaudioside A, sucrose, UDPG, buffer, and crude enzyme solution.

10. Use according to claim 9, characterized in that, In the reaction system, the amount of substrate rebaudioside A is 0.5 g / L-3 g / L, the amount of sucrose is 200 g / L-400 g / L, the amount of UDPG is 1 mM-3 mM, the amount of buffer is 10-50 mM PBS, and the amount of crude enzyme solution is 25-50 g / L.

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