Novel steviol glycoside derivative rebaudioside m9

Abstract

Disclosed in the present invention is a method for efficient biosynthesis of a novel steviol glycoside derivative rebaudioside M9, belonging to the technical field of biocatalytic synthesis. The present invention provides a glycosyltransferase UGT94E13 and a mutant thereof capable of catalyzing the monoglycosylation activity of rebaudioside M2, and the mutant is used for preparing a brand-new steviol glycoside-rebaudioside M2 monoglycosylated derivative, named rebaudioside M9. The present invention constructs an enzyme cascade reaction between a glycosyltransferase mutant and a sucrose synthase, and realizes efficient catalytic synthesis of rebaudioside M9 with rebaudioside M2 as a substrate. 43.56 g / L (30 mmol / L) rebaudioside M2 is used as a substrate and is subjected to a reaction for 8 h, and 42.81 g / L (29.14 mmol / L) rebaudioside M9 can be obtained with a yield of 97.14%, which provides a new route and a new method for the production of new rebaudioside M9 and other rebaudioside derivatives.

Description

A novel steviol glycoside derivative, rebaudioside M9 Technical Field

[0001] This invention relates to a novel steviol glycoside derivative, rebaudioside M9, and a method for its biosynthesis, belonging to the field of biocatalytic synthesis technology. Background Technology

[0002] Steviol glycosides (SGs) are natural sweeteners extracted from stevia, existing in various similar structures, and more than 60 different SG derivatives have been purified. Due to their high sweetness, ranging from 50 to 450 times that of sucrose, and the absence of calories and side effects, these compounds are considered the most attractive calorie-free sweeteners. However, the bitter aftertaste of steviol glycosides and rebaudioside A limits their application, a key obstacle to their widespread commercial use as sweeteners. The number and type of glycosyl units at C-13 and C-19 of SGs significantly affect their sensory properties, and structural modification strategies to reduce the bitter aftertaste of SGs have been widely adopted. In 2022, Yang et al. used the glycosyltransferase YjiC derived from Bacillus subtilis to catalyze rebaudioside A, producing a novel β-1,6-glycosylated derivative, rebaudioside L2, which showed improved sweetness compared to rebaudioside A. Therefore, it is of great significance to develop novel SG derivatives to overcome the existing shortcomings of steviol glycosides as sweeteners and to enrich their functions. Summary of the Invention

[0003] To address the aforementioned issues, this invention explores the glycosyltransferase UGT94E13 from Gardenia jasminoides to catalyze the synthesis of a novel monosaccharide derivative, rebaudioside M9, from rebaudioside M2. This enzyme can be expressed efficiently and solublely in Escherichia coli and exhibits catalytic activity for the synthesis of rebaudioside M9 from rebaudioside M2 ​​in the presence of uridine diphosphate glucose (UDPG). Based on the rational design and modification of UGT94E13, its efficiency in catalyzing the synthesis of rebaudioside M9 from rebaudioside M2 ​​is significantly improved. Furthermore, by constructing a uridine diphosphate glucose (UDPG) recycling system, efficient biosynthesis of rebaudioside M9 is achieved using E. coli lysates containing UGT94E13 or its mutants, providing an effective method for the industrial application of rebaudioside M9.

[0004] This invention provides a novel compound, 13-[(2-oxo-β-D-glucopyranosyl-3-oxo-(6-oxo-β-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaurenoic acid-[(2-oxo-β-D-glucopyranosyl-6-oxo-β-D-glucopyranosyl-β-D-glucopyranosyl)ester], the chemical structure of which is 13-[(2-O-β-D-glucopyranosyl-3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester], the chemical structure of which is shown below:

[0005] The compound of this invention is rebaudioside M9.

[0006] The present invention also provides a sweetener composition containing the said compound.

[0007] In one embodiment, the sweetener composition further comprises one or more of the following: steviol glycoside, rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside M, inulin, β-glucan, phloroglucin, mogroside, xylitol, erythritol, maltitol, sucralose, aspartame, sorbitol, isomaltitol, acesulfame K, rebaudioside I, rebaudioside O, rebaudioside C, rebaudioside M2, rebaudioside F, rebaudioside H, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside N, rebaudioside V, tagatose, allulose, or neotame.

[0008] In one embodiment, the sweetener composition further comprises a sweetener.

[0009] In one embodiment, the flavoring agent is one or more of ribose, xylose and xylitol, glucose, sorbitol, lactose, sucrose, palaginose, trehalose, maltodextrin, or starch, lactic acid, malic acid, and citric acid.

[0010] The present invention also provides the use of the compound or the sweetener composition in the preparation of food, beverage or pharmaceutical.

[0011] The present invention also provides food products containing the said compound or the said sweetener composition.

[0012] This invention also provides an oral consumer product comprising rebaudioside M9, wherein the oral consumer product is selected from beverage products or consumer products. Beverage products are selected from soft drinks, spring water beverages, frozen beverages, ready-to-drink beverages, coffee, tea, dairy beverages, liquid concentrates, flavored water, fortified water, fruit juice-flavored beverages, sports drinks and energy drinks, and fruit juices; the consumer product is selected from food products, nutritional preparations, pharmaceuticals, dietary supplements, oral hygiene compositions, edible gel compositions, cosmetic products, and flavorings.

[0013] The present invention also provides a glycosyltransferase mutant, which is based on the glycosyltransferase UGT94E13 shown in SEQ ID NO.1, wherein phenylalanine at position 169 is mutated to alanine, and / or isoleucine at position 185 is mutated to alanine.

[0014] In one embodiment, phenylalanine at position 169 is mutated to alanine, and isoleucine at position 185 is mutated to alanine. The amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO.3, the sequence of phenylalanine at position 169 mutated to alanine is shown in SEQ ID NO.6, and the sequence of isoleucine at position 185 mutated to alanine is shown in SEQ ID NO.7.

[0015] The present invention also provides a gene encoding the glycosyltransferase UGT94E13, the nucleotide sequences of which are shown in SEQ ID NO.2, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0016] The present invention also provides an expression vector carrying the gene.

[0017] This also provides a recombinant bacterium expressing the glycosyltransferase mutant. This recombinant bacterium is a recombinant *Escherichia coli*.

[0018] In one embodiment, the recombinant bacteria may also express sucrose synthase.

[0019] In one embodiment, the amino acid sequence of the sucrose synthase can be any amino acid sequence with sucrose synthase activity from any source.

[0020] In one embodiment, the amino acid sequence of the sucrose synthase is shown in SEQ ID NO.5.

[0021] In one embodiment, the nucleotide sequence encoding the sucrose synthase is shown in SEQ ID NO.4.

[0022] In one embodiment, the recombinant bacteria uses Escherichia coli as the host, pET-21b(+) as the vector to express the glycosyltransferase mutant, and pACYCDuet-1 as the vector to express the sucrose synthase.

[0023] The present invention also provides a method for catalytic synthesis of rebaudioside M9, wherein the method uses rebaudioside M2 ​​as a substrate and a biocatalyst is used for catalytic reaction; wherein the biocatalyst is the glycosyltransferase mutant, or the recombinant cell, or the cell lysate of the recombinant bacteria.

[0024] In one embodiment, the cell lysis buffer is the supernatant obtained by culturing the recombinant bacteria in a culture medium for a period of time, inducing the expression of the glycosyltransferase mutant with an inducer, and then further lysing the cells.

[0025] In one embodiment, the method involves culturing the recombinant bacteria in LB medium to obtain a seed culture, then transferring it to 2×YT medium and culturing at 35–40°C until the OD reaches 0.6–0.8. The bacteria are then induced with IPTG, and the bacterial cells in the fermentation broth are collected. After cell disruption, the supernatant is collected as a cell lysis buffer.

[0026] In one embodiment, the induction is performed by incubation at 16–20°C for 14–18 hours.

[0027] In one embodiment, the method specifically involves: picking a single colony and inoculating it into 5 mL of LB liquid medium containing the corresponding antibiotic, incubating overnight at 37°C and 200 rpm; subsequently, inoculating the resulting seed culture at a ratio of 1:100 into 500 mL of 2×YT medium containing the corresponding antibiotic, incubating at 37°C and 115 rpm until OD (dose retardation) occurs. 600 The concentration was increased to 0.6-0.8. The culture temperature was lowered to 18℃, and 100 μL of 1M isopropyl-β-thiogalactoside (IPTG) was added. The culture was then incubated at 18℃ for 16 h. The fermentation broth was collected and centrifuged at 7000 rpm and 4℃ for 7 min to collect the cells. The cells were resuspended in cell lysis buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol) at a ratio of 1 g of cells per 10 mL of cell lysis buffer. The cells were homogenized using a high-pressure homogenizer and then centrifuged at 40000×g and 4℃ for 30 min to obtain the supernatant, which is the coupling reaction cell lysis buffer.

[0028] In one embodiment, the catalytic reaction is carried out under the following conditions: using 1-100 mmol / L rebaudioside M2, 1-1000 mmol / L sucrose, 10-1000 mmol / L KPi buffer, and 10-1000 mmol / L NaCl as the reaction system, and reacting at 20-60°C for 1-50 h.

[0029] In one embodiment, the concentration of rebaudioside M2 ​​is 1-30 mmol / L.

[0030] In one embodiment, the sucrose concentration is 100–800 mmol / L, particularly 400–800 mmol / L.

[0031] In one embodiment, the catalytic reaction is carried out at 32–38°C or 34–36°C; particularly at 35 ± 0.5°C.

[0032] In one embodiment, the buffer solution has a pH of 6.0-8.0.

[0033] This invention also protects the use of the above-mentioned glycosyltransferase mutant, the above-mentioned gene, the above-mentioned expression vector, the above-mentioned microbial cell, the above-mentioned recombinant bacteria, or the above-mentioned method in the preparation of products containing rebaudioside M9. Beneficial effects:

[0034] (1) This invention uses UDPG as a glycosyl donor and rebaudioside M2 ​​as a substrate to synthesize a novel steviol glycoside derivative—rebaudioside M9, a monosaccharide derivative of rebaudioside M2—using glycosyltransferase UGT94E13 as a catalyst, providing a new sweetener for the food industry.

[0035] (2) In this invention, the amino acid sequence of glycosyltransferase UGT94E13 was subjected to site-directed mutagenesis to obtain the optimal mutant UGT94E13-F169A / I185A. This mutant, using UDPG as a glycosyl donor, significantly improved the efficiency of catalyzing the synthesis of rebaudioside M2 ​​into rebaudioside M9, and its k cat / K m Significantly increased to 22.88mM -1 min -1 The relative enzyme activity reached 12.04 times that of the wild-type enzyme.

[0036] (3) The recombinant strain constructed in this invention co-expresses the gardenia-derived glycosyltransferase mutant UGT94E13-F169A / I185A and the Arabidopsis-derived sucrose synthase AtSuSy. The cell lysate prepared after induction of expression by the recombinant strain catalyzes the synthesis of rebaudin M2 into rebaudin M9. Through optimization of the cascade reaction system, a rebaudin M9 of 42.81 g / L (29.14 mmol / L) was obtained after 8 h of reaction with 43.56 g / L (30 mmol / L) rebaudin M2 as the substrate, achieving a yield of 97.14%. This provides a highly efficient new route and method for the production of novel rebaudin M9 and other rebaudin derivatives. Attached Figure Description

[0037] Figure 1 shows the biosynthetic pathway of rebaudioside M2 ​​to rebaudioside M9 catalyzed by glycosyltransferase UGT94E13.

[0038] Figure 2 shows the protein expression and purification analysis of the mutant UGT94E13-F169A / I185A of glycosyltransferase UGT94E13 in Example 2; Lane M: Marker; Lane 1: Sample without IPTG induction; Lane 2: Crude enzyme solution; Lane 3: Crude enzyme solution supernatant; Lane 4: Crude enzyme solution precipitate; Lane 5: Purification permeate; Lane 6: Washed sample containing other proteins; Lane 7: Target protein sample.

[0039] Figure 3 is a UPLC analysis of the synthesis of rebaudioside M9 from rebaudioside M2 ​​catalyzed by glycosyltransferase UGT94E13 in Example 3.

[0040] Figure 4 shows the mass spectrometry analysis of rebaudioside M9, the product of the glycosylation reaction of rebaudioside M2 ​​in Example 4.

[0041] Figure 5 shows the proton NMR spectrum of the product rebaudioside M9 from Example 4.

[0042] Figure 6 shows the carbon NMR spectrum of the product rebaudioside M9 from Example 4.

[0043] Figure 7 shows the COSY spectrum of the nuclear magnetic resonance spectroscopy of the product rebaudioside M9 in Example 4.

[0044] Figure 8 shows the TOCSY spectrum of the nuclear magnetic resonance spectroscopy analysis of the product rebaudioside M9 in Example 4.

[0045] Figure 9 shows the HSQC spectrum of the product rebaudioside M9 from Example 4.

[0046] Figure 10 shows the HMBC spectrum of the product rebaudioside M9 in Example 4, analyzed by nuclear magnetic resonance spectroscopy.

[0047] Figure 11 shows the ROESY spectrum of the nuclear magnetic resonance spectrum of the product rebaudioside M9 in Example 4.

[0048] Figure 12 shows the effect of pH on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A in Example 5.

[0049] Figure 13 shows the effect of temperature on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A in Example 6.

[0050] Figure 14 shows the protein expression analysis of the lysate from the UGT94E13-F169A / I185A-AtSusy glycosylation coupling reaction in Example 9. Lane M: Marker; Lane 1: Sample without IPTG induction; Lane 2: Crude enzyme solution; Lane 3: Crude enzyme solution supernatant; Lane 4: Crude enzyme solution precipitate.

[0051] Figure 15 shows the effect of pH on the preparation of Reb M9 by the cascade reaction in Example 10.

[0052] Figure 16 shows the effect of temperature on the preparation of Reb M9 by the cascade reaction in Example 11.

[0053] Figure 17 shows the effect of sucrose concentration on the preparation of Reb M9 by the cascade reaction in Example 12.

[0054] Figure 18 shows the effect of reaction time on the preparation of Reb M9 by the cascade reaction in Example 13. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0056] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0057] The culture media involved in the following examples:

[0058] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder.

[0059] 2×YT liquid culture medium: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl.

[0060] The methods involved in the following embodiments:

[0061] Rebaudioside M9 Yield Calculation: Rebaudioside M9 was dissolved in water to prepare a 5 mM mother liquor. The mother liquor was then diluted with methanol to prepare solutions of various concentrations: 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, and 1.75 mM. These solutions were filtered through a 0.22 μm filter and analyzed by UPLC to obtain peak areas. A standard concentration curve of rebaudioside M9 was fitted using Origin 8.0 software, with rebaudioside M9 concentration as the x-axis and peak area as the y-axis. The yield of rebaudioside M9 was calculated based on the standard curve. Yield = Actual yield of rebaudioside M9 / Theoretical yield of rebaudioside M9.

[0062] In the biosynthesis of rebaudioside M9 (Reb M9), glycosyltransferases (GTs) are key catalysts, regulating the physicochemical properties of the product through glycosylation reactions. This invention screened UGT94E13 from a variety of candidate glycosyltransferases through phylogenetic analysis and substrate structure comparison. In vitro experiments showed that UGT94E13 can specifically catalyze the β-1,6-O-glycosylation of the precursor compound Reb M2 at the C-13 site to generate rebaudioside M9.

[0063] Wild-type glycosyltransferase UGT94E13 can achieve the directed synthesis of rebaudioside M9, but its catalytic efficiency is low. This invention also discovered that the F169 and I185 residues near the active site may restrict substrate binding through steric hindrance. Mutating these residues to alanine (F169A / I185A) increases the volume of the enzyme's active site and shortens the distance between the C-13 hydroxyl group of the substrate Reb M2 and the C1 atom of UDP-glucose. Molecular dynamics simulations further show that the key transition state energy barrier of the mutant reaction is significantly reduced, and the catalytic efficiency is 12-fold higher than that of the wild type after modification. Furthermore, the mutant maintains >90% activity in continuous reactions, exhibiting excellent stability.

[0064] Example 1: Acquisition of the glycosyltransferase UGT94E13 gene and construction of the strain

[0065] The nucleotide sequence (Genbank accession number: MN944055.1) and protein sequence (Genbank accession number: ARU08119.1) of the gardenia-derived glycosyltransferase were downloaded from Genbank. E. coli codon preference optimization and gene synthesis were performed by Yixin Biotechnology Co., Ltd. The nucleotide sequence of the synthesized gene is shown in SEQ ID NO.2 and was ligated to the NdeI / BamHI site of the vector pET-21b(+) to obtain the recombinant plasmid pET-21b(+)-UGT94E13.

[0066] Using the recombinant plasmid pET-21b(+)-UGT94E13 as a template, full plasmid PCR was performed sequentially using primers F169A-1 / F169A-2 and I185A-1 / I185A-2 (primers are shown in Table 1). The phenylalanine at position 169 of UGT94E13 was mutated to alanine, and the isoleucine at position 185 was mutated to alanine, thus constructing the mutant recombinant plasmid pET-21b(+)UGT94E13-F169A / I185A.

[0067] The obtained plasmid pET-21b(+)-UGT94E13-F169A / I185A was sequenced and identified, and then transformed into E. coli BL21(DE3) competent cells. The recombinant strain E. coli BL21(DE3)pET-21b(+)-UGT94E13-F169A / I185A was obtained by screening on LB solid medium containing 100 μg / mL ampicillin.

[0068] Table 1 Primer names and primer sequences

[0069] Example 2: Induction of recombinant strain expression and purification of target protein

[0070] Positive colonies of the recombinant mutant strains *E. coli* BL21(DE3)pET-21b(+)-UGT94E13 and *E. coli* BL21(DE3)pET-21b(+)-UGT94E13-F169A / I185A constructed in Example 1 were picked and cultured in 5 mL LB medium containing 100 μg / mL ampicillin for 12 hours. Subsequently, the resulting seed culture was inoculated at a ratio of 1:100 into 500 mL 2×YT medium containing 100 μg / mL ampicillin and cultured at 37°C and 115 rpm until OD (Organic Dysplasia) was reached. 600 The culture temperature was lowered to 18°C, and 50 μL of 1M isopropyl-β-thiogalactoside (IPTG) was added. The cells were then incubated at 18°C ​​for 8-12 hours. Subsequently, the cells were collected by centrifugation at 7000 rpm for 7 minutes at 4°C for protein purification. The cells were resuspended in cell lysis buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol) at a ratio of 1 g of cells per 10 mL of cell lysis buffer. The cells were homogenized using a high-pressure homogenizer, and then centrifuged at 40000 × g at 4°C for 30 minutes to obtain the supernatant, which yielded the crude enzyme solution.

[0071] The crude enzyme solution was utilized with Ni + The column was used for affinity chromatography purification. After sample loading, 20 times Ni was used.+ The column volume was washed with a wash buffer (50 mmol / L Tris-HCl pH 8.0, 250 mmol / L NaCl, 20 mmol / L imidazole, 10% glycerol) to remove contaminating proteins, followed by elution with 4 mL of elution buffer (50 mmol / L Tris-HCl pH 8.0, 250 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerol). The target protein was then desalted using a Hisrip™ 5 mL desalting column and desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl). After desalting, the concentration was increased to 10 mg / mL for subsequent reactions. The purified protein was detected by 12% SDS-PAGE gel electrophoresis, and the results are shown in Figure 2. This method successfully obtained a pure enzyme with a clear target band and accurate protein size. Figure 2 shows the protein expression and purification analysis of the mutant UGT94E13-F169A / I185A of the glycosyltransferase UGT94E13 obtained in this embodiment; Lane M: Marker; Lane 1: Sample without IPTG induction; Lane 2: Crude enzyme solution; Lane 3: Crude enzyme solution supernatant; Lane 4: Crude enzyme solution precipitate; Lane 5: Purification permeate; Lane 6: Washed sample containing other proteins; Lane 7: Target protein sample.

[0072] Example 3: Glycosylation reaction of rebaudioside M2 ​​to rebaudioside M9 catalyzed by UGT94E13

[0073] The purified glycosyltransferase UGT94E13 obtained in Example 2 was used for glycosylation reaction (Figure 1). Figure 1 shows the biosynthetic pathway of rebaudioside M2 ​​to rebaudioside M9 catalyzed by glycosyltransferase UGT94E13.

[0074] The glycosylation reaction was carried out in a 200 μL reaction system as follows: 50 mmol / L Tris pH 8.0, 5 mmol / L UDPG, 1.5 mmol / L rebaudioside M2, and 0.5 mg / mL of the purified enzyme obtained in Example 2. The reaction was carried out at 35 °C and 800 rpm for 3 h. Immediately after the reaction was completed, the reaction was terminated by heating at 95 °C for 5 min, and 400 μL of methanol was added to the mixture. The above reaction system was centrifuged at 20000 × g for 5 min to remove precipitates. The supernatant was filtered through a 0.22 μm filter membrane and used for UPLC and LC-MS analysis. The UPLC system used a CORTECS C18 1.6μm column (2.1×50mm). The liquid phase conditions were: organic phase - acetonitrile, aqueous phase - ultrapure water; flow rate 0.3mL / min; column temperature 35℃; UV detection wavelength 210nm; detection program: 0-1min 15% organic phase, 6min 40% organic phase, 7-8min 15% organic phase.

[0075] Figure 3 shows the UPLC analysis of the synthesis of rebaudioside M9 from rebaudioside M2 ​​catalyzed by glycosyltransferase UGT94E13. Liquid phase analysis reveals the formation of a significant new product in the glycosyltransferase-catalyzed reaction system (Figure 3), indicating that glycosyltransferase UGT94E13 can undergo glycosylation with UDPG as the glycosyl donor and rebaudioside M2 ​​as the substrate to generate the rebaudioside M2 ​​derivative. As shown in Figure 4, mass spectrometry analysis of the rebaudioside M9 glycosylation product reveals a peak for [MH] ions at m / z 1451.5798 in negative ion mode. This molecular weight is similar to [Reb M2-H]. - The molecular weight increased by 162 compared to the theoretical molecular weight of 1289.5476, which is exactly the molecular weight of a single glucose unit. Therefore, this indicates that the glycosylation modification of rebaudioside M2 ​​by UGT94E13 introduces a glucose unit into its structure.

[0076] Example 4: Structural Identification of Novel Rebaudioside M2 ​​Monosaccharide Derivatives

[0077] Rebaudioside M2 ​​monosaccharide derivatives were prepared in large quantities using glycosyltransferase UGT94E13. The 100 mL reaction system was as follows: 5 mM rebaudioside M2, 10 μM glycosyltransferase, 5 mM UDPG, 50 mM Tris, pH 8.0. The reaction system was reacted at 35℃ for 24 h. After the reaction, the reaction was quenched by heating at 95℃ for 5 min, and the resulting mixture was diluted with 5 times its volume of methanol. The reaction system was centrifuged at 20000×g for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and purified using a semi-preparative high-performance liquid chromatography (HPLC) system. The conditions were: Shim-pack GIST C18 column (10×250 mm, 0.5 mg / mL, SHIMADZU, Japan); organic phase: acetonitrile, aqueous phase: ultrapure water; flow rate: 5 mL / min; time program: 0-5 min 15% organic phase, 5-30 min 40% organic phase, 31-35 min 100% organic phase, 35-40 min 15% organic phase; column temperature: 40℃; UV detection wavelength: 210 nm. As shown in Figure 5-11, the obtained sample was dissolved in heavy water and purified by 1D (…). 1 H and 13 The complete structure of the product was analyzed using C) and 2D NMR (COSY, TCOSY, HSQC, HMBC, and ROESY) spectra. Data were collected using a Bruker Avance III 600MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany). 1 The H-spectrum detection frequency is 600MHz. 13 The C spectrum is 151 MHz. Figure 5 shows the proton NMR spectrum of rebaudioside M9. Figure 6 shows the carbon NMR spectrum of rebaudioside M9. Figure 7 shows the COSY NMR spectrum of rebaudioside M9. Figure 8 shows the TOCSY NMR spectrum of rebaudioside M9. Figure 9 shows the HSQC NMR spectrum of rebaudioside M9. Figure 10 shows the HMBC NMR spectrum of rebaudioside M9. Figure 11 shows the ROESY NMR spectrum of rebaudioside M9.

[0078] Analysis of the 1H-NMR and HSQC spectra revealed the presence of seven anomeric protons with chemical shifts of δH 5.50 (δC 92.71), δH 4.69 (δC 95.57), δH 4.78 (δC 102.14), δH 4.66 (δC 102.43), δH 4.70 (δC 102.49), δH 4.36 (δC 102.83), and δH 4.39 (δC 102.56). This indicates the presence of seven glycosidic units in the product, consistent with the LC-MS results. These units were named residues I, II, III, IV, V, VI, and VII.

[0079] Next, the linkage of the seven glucose residues was confirmed using HMBC spectroscopy. Based on the correlation between H-I1 (δH 5.50) and C-19 (δC 177.98), residue I was linked to C-19 of the terpene backbone. Based on the correlation between H-II1 (δH 4.69) and C-13 (δC 87.64), residue II was linked to C-13 of the terpene backbone. Based on the correlation between H-III1 (δH 4.78) and C-II2 (δC 78.61), residue III was linked to C-2 of residue II, forming a 1→2 glycosidic bond. Based on the correlation between H-IV1 (δH 4.66) and C-II3 (δC 86.40), residue IV was linked to C-3 of residue II, forming a 1→3 glycosidic bond. Based on the correlation between H-V1 (δH 4.70) and C-I2 (δC 77.50), residue V is linked to residue I at C-2, forming a 1→2 glycosidic bond. Based on the correlation between H-VI1 (δH 4.36) and C-I6 (δC 68.12), residue VI is linked to residue I at C-6, forming a 1→6 glycosidic bond. Based on the correlation between H-VII1 (δH 4.39) and C-IV6 (δC 68.65), residue VII is linked to residue IV at C-6, forming a 1→6 glycosidic bond. The chemical shifts of H and C in the new derivatives were assigned in detail by 1D and 2D NMR (Figures 5-11), as shown in Table 2.

[0080] Table 2 1 H and 13 C Chemical Shift Assignment Table (D2O)

[0081] In summary, the glycosyltransferase UGT94E13 catalyzes the formation of a novel monoglucosylated product from rebaudioside M2, with the newly added glucose residue VII linked to residue IV of rebaudioside M2 ​​via a 1→6 glycosidic bond at C-6. This structure represents a previously unreported steviol glycoside derivative, with the structural formula shown as Reb M9 in Figure 1, and is named rebaudioside M9. The structural formula is 13-[(2-oxo-β-D-glucopyranosyl-3-oxo-(6-oxo-β-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]kaurenoic acid-[(2-oxo-β-D-glucopyranosyl-6-oxo-β-D-glucopyranosyl-β-D-glucopyranosyl)ester], 13-[(2-O-β-D-glucopyranosyl-3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester].

[0082] Example 5: Effect of pH on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A

[0083] In this embodiment, the glycosylation reaction system was placed in buffer solutions with different pH values ​​to carry out the reaction, and the effect of pH on the reactivity of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A was measured.

[0084] The glycosylation reaction system consisted of 200 μL, including (by final concentration) 50 mmol / L Tris-HCl, 10 mmol / L UDPG, 5 mmol / L rebaudioside M2, and the purified enzyme UGT94E13 or UGT94E13-F169A / I185A obtained in Example 2 at a concentration of 0.5 mg / mL. The reaction was carried out at 35 °C for 20 min. Immediately after the reaction was completed, the reaction was terminated by heating at 95 °C for 5 min, and 400 μL of methanol was added to the mixture. The above reaction system was centrifuged at 20000 × g for 5 min to remove precipitates, and the supernatant was filtered through a 0.22 μm filter membrane for UPLC analysis. The liquid chromatography detection method was the same as in Example 3. Figure 12 shows the effect of pH on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A. The results showed that the optimal reaction pH of the mutant UGT94E13-F169A / I185A did not change compared with the wild type (Figure 12). It could maintain good reactivity in the pH range of 7.5-8.5, and the optimal pH was 8.0.

[0085] Example 6: Effect of temperature on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A

[0086] In this embodiment, the glycosylation coupling reaction system was placed at different temperatures to determine the effect of temperature on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A. The selected temperature range was 20-50℃.

[0087] The glycosylation reaction system consisted of 200 μL, including 50 mmol / L Tris-HCl pH 8.0, 10 mmol / L UDPG, 5 mmol / L rebaudioside M2, and the purified enzyme UGT94E13 or UGT94E13-F169A / I185A obtained in Example 2 at a concentration of 0.5 mg / mL. The reaction time was 20 min. Immediately after the reaction, the mixture was heated at 95 °C for 5 min to terminate the reaction, and 400 μL of methanol was added to the mixture. The reaction system was centrifuged at 20000 × g for 5 min to remove precipitates. The supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography detection method was the same as in Example 3. Figure 13 shows the effect of temperature on the reaction of glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A. The results showed that the optimal reaction temperature of the mutant UGT94E13-F169A / I185A did not change compared with the wild type (Figure 13). It could maintain good reactivity in the range of 30℃-40℃, and the optimal reaction temperature was 35℃.

[0088] Example 7: Determination of the enzymatic kinetics of glycosyltransferase UGT94E13 and its mutant catalyzing rebaudioside M2.

[0089] In this embodiment, the purified wild-type enzyme UGT94E13 and the mutant enzyme UGT94E13-F169A / I185A obtained in Example 2 were subjected to glycosylation. The glycosylation reaction was carried out in a 200 μL reaction system as follows: 50 mmol / L Tris-HCl pH 8.0, 10 mmol / L UDPG, 5 mmol / L rebaudioside M2, and the concentration of the purified enzyme UGT94E13 or UGT94E13-F169A / I185A obtained in Example 2 was 0.5 mg / mL. The reaction time was 20 min. After the reaction was completed, the reaction was immediately terminated by heating at 95 °C for 5 min, and 400 μL of methanol was added to the mixture. The above reaction system was centrifuged at 20000×g for 5 min to remove precipitate, and the supernatant was filtered through a 0.22 μm filter membrane for UPLC analysis. The liquid phase detection method was performed as described in Example 3, and the detection results are shown in Table 3. Compared with the wild type, UGT94E13-F169A / I185A has a greater affinity for the substrate and a faster rate of substrate catalytic formation. cat / K m The value increased significantly, from 1.88 mM -1 min -1 Increased to 22.88mM -1 min -1 The enzyme activity increased by 12.04 times.

[0090] Table 3 Kinetic constants of UGT94E13 and its mutants with respect to the substrate rebaudioside M2

[0091] Example 8: Construction of recombinant plasmids expressing glycosyltransferase UGT94E13 and sucrose synthase AtSuSy, and construction of recombinant strains.

[0092] The amino acid sequence of sucrose synthase AtSuSy from Arabidopsis thaliana (accession number: NP_001031915.1) was downloaded from GenBank. Codon preference optimization and gene synthesis for *E. coli* were performed by Yixin Biotechnology Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5. This sequence was then ligated to the NcoI / NflII sites of the vector pACYCDuet-1 to obtain the recombinant plasmid pACYCDuet-1-AtSuSy. The resulting plasmid was sequenced and identified. It was then co-transformed with the recombinant plasmid pET-21b(+)-UGT94E13-F169A / I185A constructed in Example 1 into *E. coli* E. coli BL21(DE3) competent cells. Correct colonies were screened on LB solid medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol to obtain the recombinant strain *E. coli*. BL21(DE3)pET-21b(+)-UGT94E13-F169A / I185ApACYCDuet-1-AtSuSy.

[0093] Example 9: Preparation of cell lysates from recombinant strains of glycosyltransferase UGT94E13 and sucrose synthase AtSuSy

[0094] Using the recombinant strain *E. coli* BL21(DE3)pET-21b(+)-UGT94E13-F169A / I185ApACYCDuet-1-AtSuSy obtained in Example 8 as the fermentation strain, a single colony was picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, and cultured overnight at 37°C and 200 rpm. Subsequently, the resulting seed culture was inoculated at a ratio of 1:100 into 500 mL of 2×YT medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, and cultured at 37°C and 115 rpm until OD (dose expiratory time) was reached. 600The concentration was reduced to 0.6-0.8. The culture temperature was lowered to 18℃, and 100 μL of 1M isopropyl-β-thiogalactoside (IPTG) was added. The cells were cultured at 18℃ for 16 h. Subsequently, the cells were collected by centrifugation at 7000 rpm and 4℃ for 7 min for protein purification. The cells were resuspended in cell lysis buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol) at a rate of 1 g of cells per 10 mL of cell lysis buffer. The cells were homogenized using a high-pressure homogenizer and then centrifuged at 40000×g and 4℃ for 30 min to obtain the supernatant, which was the coupling reaction cell lysis buffer. The lysis buffer was analyzed by 12% SDS-PAGE gel electrophoresis. Figure 14 shows the protein expression analysis of the UGT94E13-F169A / I185A-AtSusy glycosylation coupling reaction lysis buffer. Lane M: Marker; Lane 1: Sample without IPTG induction; Lane 2: Crude enzyme solution; Lane 3: Crude enzyme solution supernatant; Lane 4: Crude enzyme solution precipitate. Results showed that both glycosyltransferase and sucrase synthase were well expressed. Protein concentrations in cell lysates were determined using a Nano-Drop 2000 UV-Vis spectrophotometer. The prepared cell lysates were aliquoted and stored at -80°C or used directly for coupling reactions.

[0095] Example 10 Effect of pH on the cascade reaction for the preparation of Reb M9

[0096] Cell lysis buffer was prepared as described in Example 9. To investigate the effects of various factors in the cascade reaction, a standard reaction system was used. 1 mL of the standard reaction system (based on final concentration) contained: cell lysis buffer to a protein concentration of 12 mg / mL, 25 mmol / L rebaudioside M2, 200 mmol / L sucrose, and 100 mmol / L phosphate buffer (KPi, pH 8.0, containing 100 mmol / L NaCl). The reaction mixture was reacted at 35°C for 3 h; then the reaction was quenched by heating at 95°C for 5 min, followed by the addition of 4 volumes of methanol; centrifugation at 20000×g for 5 min was performed to remove the precipitate; the supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography detection method was the same as in Example 3, and the yield of rebaudioside M9 was calculated. Each reaction was performed in triplicate.

[0097] The effect of pH on the cascade reaction for Reb M9 preparation was determined: Different pH buffers were selected: Bis-Tris (pH 6.0-7.5) (containing 100 mM NaCl), KPi (pH 5.5-8.0), and Tris-HCl (pH 7.5-9.0) (containing 100 mM NaCl). Under the protein concentration conditions determined in the above experiments, the effect of different pH values ​​on the cascade reaction for Reb M9 preparation was tested according to the standard reaction system method. The results are shown in Figure 15. When the buffer was 100 mmol / L KPi at pH 6.5 (containing 100 mmol / L NaCl), the yield of Rebaudioside M9 was 23.23 g / L (16 mmol / L), with the highest yield reaching over 64% (Figure 15).

[0098] Example 11 Effect of temperature on the preparation of Reb M9 by cascade reaction

[0099] The effect of reaction temperature on the cascade reaction preparation of Reb M9 was determined: At the optimal pH (pH 6.5) determined in the above experiments, different temperature gradients (20-50℃, with temperature intervals of 5℃) were set, and other conditions remained unchanged. The effect of temperature on the cascade reaction preparation of Reb M9 was tested according to the standard reaction system of Example 10. The results are shown in Figure 16. When the temperature is 35℃, the yield of Rebaudioside M9 is the highest, reaching more than 70% (Figure 16).

[0100] Example 12 Effect of sucrose concentration on the preparation of Reb M9 by cascade reaction

[0101] The effect of sucrose concentration on the preparation of Reb M9 in the cascade reaction system was determined: different sucrose concentration gradients of 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, and 800 mM were set. At the optimal pH (pH 6.5) and temperature (35 °C) determined in Examples 10 and 11, the effect of sucrose concentration on the preparation of Reb M9 in the cascade reaction was tested according to the standard reaction system of Example 10. The results, as shown in Figure 17, indicate that the yield of Reb M9 was highest, reaching 80%, when the sucrose concentration was 500 mmol / L (Figure 17).

[0102] Example 13 Effect of reaction time on the preparation of Reb M9 by cascade reaction

[0103] Following the preparation of wild-type glycosyltransferase UGT94E13 and its mutant UGT94E13-F169A / I185A, and cell lysates of sucrose synthase AtSuSy as described in Example 9, the effect of reaction time on the preparation of Reb M9 in the cascade reaction system was determined.

[0104] Reb M9 was prepared in a 20 mL reaction system containing 100 mM KPi at pH 6.5 (containing 100 mM NaCl) to achieve a protein concentration of 12 mg / mL. -1 Cell lysis buffer, 30 mM Reb M2, and 500 mM sucrose were added, and the reaction was carried out at 35°C for 8 h. Samples were taken every 1 h to detect the yield of Reb M9. Wild-type UGT94E13 was used as a control. The results, shown in Figure 18, indicate that UGT94E13-F169A / I185A, reacting with 43.56 g / L (30 mmol / L) rebaudioside M2 ​​as a substrate for 8 h yielded 42.81 g / L (29.14 mmol / L) of rebaudioside M9, achieving a yield of 97.14% (Figure 18), while the yield of wild-type UGT94E13 was only 23.46% (7.04 mmol / L).

[0105] Example 14 Sweetness test of rebaudioside M9

[0106] Rebaudioside M2 ​​was used as a control in the sweetness test of rebaudioside M9. The rebaudioside M2 ​​sample was purchased from Rhine Biosciences. The purity of rebaudioside M9 was 98%.

[0107] Sweetness analysis of glycosylated products was performed using the SA402B electronic tongue system. First, the sample solution for sweetness analysis was prepared by dissolving 20 mg of rebaudioside M2 ​​or 20 mg of the glycosylated product in 50 mL of ultrapure water (400 ppm concentration). The SA402B electronic tongue system uses a lipid membrane sensor (GL1) and a reference electrode (Ag / AgCl). Before use, the sensor was soaked in the reference solution (30 mM KCl and 0.3 mM tartaric acid) for at least 24 hours. Then, after equilibration in the reference solution, the activated sensor was sequentially immersed in the reference solution and the sample solution, and the membrane potential V was measured. S and V R The taste signal value (R) was calculated using the following formula. Each sample solution was measured four times, and the average of the last three measurements was taken as the test result: R = V S -V R (1);

[0108] As shown in Table 4, the analysis results indicate that the R (taste signal value) value of rebaudine M9 is higher than that of rebaudine M2, meaning that the sweetness of rebaudine M9 is higher than that of rebaudine M2. According to publicly available information, the sweetness of rebaudine M2 is approximately 30% higher than that of rebaudine D (Reb D), and the sweetness of Reb D can reach 300 times that of sucrose. This demonstrates that mono-β-1,6-O-glycosylation at the C-13 position can effectively improve the sweetness properties of rebaudine M2, and its sweetness can significantly surpass that of existing commonly used sweeteners, providing important information for the development of novel steviol glycoside natural sweeteners. The steviol glycoside M9 (Reb M9) of this invention has a sweetness 300-450 times that of sucrose and possesses zero calorific value. Reb M9 has a purer sweetness and almost eliminates bitterness, showing significant application potential in the food, beverage, and pharmaceutical industries.

[0109] Table 4. Electronic tongue analysis of lebodiin M9 and lebodiin M2

[0110] Comparative Example

[0111] The specific implementation method is the same as in Example 1, except that the mutants shown in Table 5 were constructed in this invention. With the enzyme activity of WT as 100%, the enzyme activity was detected according to the method in Example 3, and the relative enzyme activity of the mutants was calculated. The results are shown in Table 5: the relative activity of F169A was 384.12%, the relative activity of I185A was 407.44%, and the relative activity of F169A / I185A was 1204.73%.

[0112] Table 5 Comparison of relative activities of different mutants

[0113] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A compound, characterized in that, The chemical structure is:

2. The compound of claim 1 is rebaudioside M9.

3. A sweetener containing the compound of claim 1 or 2.

4. The sweetener according to claim 3, characterized in that, It also contains one or more of the following: steviol glycosides, rebaudioside A, rebaudioside D, rebaudioside E, rebaudioside M, inulin, β-glucan, phloroglucin, mogroside, xylitol, erythritol, maltitol, sucralose, aspartame, sorbitol, isomaltitol, acesulfame K, rebaudioside B, rebaudioside I, rebaudioside O, rebaudioside C, rebaudioside M2, rebaudioside F, rebaudioside H, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside N, rebaudioside V, tagatose, allulose, or neotame.

5. The use of the compound of claim 1 or 2 or the sweetener of any one of claims 3 to 4 in the preparation of food, beverage or pharmaceutical.

6. An oral consumer product, characterized in that, It comprises the compound of claim 1 or 2, wherein the oral consumer product is selected from beverage products or consumer products.

7. The oral consumer product according to claim 6, characterized in that, The beverage products are selected from soft drinks, spring water beverages, frozen beverages, ready-to-drink beverages, coffee, tea, dairy beverages, liquid concentrates, flavored water, fortified water, fruit juice flavored beverages, sports drinks and energy drinks, and fruit juices; the consumer products are selected from food products, nutritional preparations, pharmaceuticals, dietary supplements, oral hygiene compositions, edible gel compositions, cosmetic products, and flavorings.

8. A glycosyltransferase mutant, characterized in that, Based on the glycosyltransferase UGT94E13 with the amino acid sequence shown in SEQ ID NO.1, phenylalanine at position 169 is mutated to alanine, and / or isoleucine at position 185 is mutated to alanine.

9. The glycosyltransferase mutant according to claim 8, characterized in that, Their amino acid sequences are SEQ ID NO.3, SEQ ID NO.6, or SEQ ID NO.7, respectively.

10. The glycosyltransferase mutant according to claim 8 or 9, characterized in that, The glycosyltransferase mutant is encoded by nucleotides, the nucleotide sequences of which are SEQ ID NO.2, SEQ ID NO.8, and SEQ ID NO.9, respectively.

11. A biomaterial, characterized in that, For (a), (b), (c), or (d): (a) The gene encoding the glycosyltransferase UGT94E13 of claim 8; (b) An expression vector carrying the gene described in (a); (c) A recombinant bacterium expressing the glycosyltransferase mutant of claim 8; (d) Recombinant bacteria expressing the glycosyltransferase mutant and sucrose synthase of claim 8.

12. Recombinant Escherichia coli, characterized in that, Express the glycosyltransferase mutant as described in claim 8 or 9.

13. Recombinant Escherichia coli, characterized in that, Express the glycosyltransferase mutant of claim 8 and express sucrose synthase with the amino acid sequence shown in NCBI accession number: NP_001031915.

14. A method for the catalytic synthesis of rebaudioside M9, characterized in that, Using rebaudioside M2 ​​as a substrate, a catalytic reaction is carried out using a biocatalyst; the biocatalyst is the glycosyltransferase mutant of claim 8 or 9, or recombinant cells expressing the mutant of claim 8 or 9, or cell lysate of the recombinant cells.

15. The method according to claim 14, characterized in that, The catalytic reaction system contains, by final concentration: 1-100 mmol / L rebaudioside M2 ​​and 1-1000 mmol / L sucrose, and the catalytic reaction is carried out at 20-60℃ for 1-50 h.

16. The use of the glycosyltransferase mutant of claim 7, or the biological material of claim 11, or the recombinant Escherichia coli of claim 12 or 13, or the method of any of claim 14 or 15 in the preparation of a product containing rebaudioside M9.

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