36 results about "Rebaudioside D" patented technology

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

The invention relates to an engineered glycosyltransferase and a steviol glycoside glucosylation method. The present invention provides engineered glycosyltransferases (GTs), polypeptides having GT activity and polynucleotides encoding the enzymes, as well as vectors and host cells comprising the polynucleotides and polypeptides. The present invention provides engineered sucrose synthases (SuS), polypeptides having SuS activity and polynucleotides encoding the enzymes, as well as vectors and host cells comprising the polynucleotides and polypeptides. The invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to prepare products having beta-glucose ligation. The present invention also provides compositions and methods for producing rebaudioside (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The invention also provides compositions comprising the SuS enzymes and methods of using the same. Methods for producing a GT enzyme and a SuS enzyme are also provided.

The present application relates to methods for preparing rebaudioside D and rebaudioside M by reaction of uridine diphosphate (UDP)-glycosyltransferases; and compositions for preparing rebaudioside D and rebaudioside M, comprising uridine diphosphate (UDP)-glycosyltransferases.

The present invention discloses a method for producing a target stevioside. According to the method for producing the target stevioside, the distribution change amount of the target stevioside reaches 90% or above within 2 hours, so that a precipitate containing the target stevioside is formed. The target stevioside is rebaudioside D and / or rebaudioside M. The target stevioside is rebaudioside M.

The invention provides a method for efficient biosynthesis of rebaudioside M2 using glycosyltransferase, belonging to the technical field of biocatalytic synthesis. A glycosyltransferase UGT94D1 catalyzing rebaudioside D to synthesize rebaudioside M2 is obtained by mining, providing a new, efficient and environmentally friendly way of producing rebaudioside M2. During the whole catalytic reaction process, no by-products are produced, facilitating downstream purification and further reducing production costs.

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a glycosyl transferase mutant and application thereof in synthesis of rebaudioside M. According to the invention, site-directed mutagenesis is carried out on an amino acid sequence SEQ ID NO.1 of wild type glycosyl transferase, and mutants: I29M, L149C and F374C are obtained through screening; the invention also provides a nucleic acid sequence for coding the mutant, an expression vector and a recombinant strain which are used for catalytically converting rebaudioside D to prepare rebaudioside M, and the conversion rate is obviously improved. Experimental results show that under optimized reaction conditions, the mutant can increase the conversion rate of converting rebaudioside D into rebaudioside M to 100%, which is increased by more than 30% compared with a wild type, and the mutant has significant industrial application value.

The present invention relates to novel rebaudioside D and rebaudioside M-like steviol glycosides and a method for producing same. The novel rebaudioside D and rebaudioside M-like steviol glycosides obtained by the production method according to the present invention exhibit improved water solubility, and thus can be easily applied to beverages and foods with a high water content, and can significantly improve the industrial utilization of steviol glycosides.

The present disclosure provides enzymes and methods of using those enzymes to transfer sugar moieties to substrate steviol glycosides. Specifically, designed beta-1,2-glycosyltransferases and sucrose synthases are used in one-pot reactions to convert stevioside and Reb A to Reb E and Reb D. Additionally, designed beta-1,2-glycosyltransferases can be used in one-pot reactions with sucrose synthases and beta-1,3-glycosyltransferases to produce Reb M from stevioside and / or Reb A.

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.

The application discloses a preparation method of rebaudioside D and rebaudioside M and application of a beta-1,2-glucosyltransferase in the method. The beta-1,2-glucosyltransferase has an amino acid sequence as shown in SEQ ID NO: 4, or has a mutant amino acid sequence with at least 99% sequence identity compared with the amino acid sequence as shown in SEQ ID NO: 4. The application improves the yield of rebaudioside D, an intermediate product in the catalysis of rebaudioside A to generate rebaudioside M by the beta-1,2-glucosyltransferase, and accordingly, the yield of the final product rebaudioside M is improved.

Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleic acid expression cassette that expresses an ABC-transporter capable of transporting steviol glycosides to the extracellular space or to the luminal space of an intracellular organelle. In some embodiments, the host cell further comprises one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing one or more steviol glycosides in the host cell. The host cells, compositions, and methods described herein provide an efficient route for the heterologous production of steviol glycosides, including but not limited to, rebaudioside D and rebaudioside M.

The application discloses a novel steviol glycoside composition and application thereof, and belongs to the technical field of sweeteners. The composition comprises rebaudioside M2, rebaudioside D and rebaudioside M, wherein the weight percentage of each component is as follows: rebaudioside M2 60%-85%, rebaudioside D 5%-15%, and rebaudioside M 10%-25%. The composition is highly simulated to sucrose in terms of sensory properties such as sweetness intensity, sweetness peak speed, after-sweetness residue, bitterness and astringency through ternary synergistic compounding, and is suitable for edible products such as dairy products, beverages, candies, baked products, frozen drinks, condiments and health foods, and can realize partial or complete replacement of sucrose, and has the advantages of pure naturalness, zero calorie, pure taste, wide application and the like.

The invention belongs to the field of enzyme engineering, and discloses a glycosyl transferase pgUGT-M8 mutant which is subjected to at least one of the following mutations on the basis of an amino acid sequence shown in SEQ ID NO.1: M87H, I146 F / Y / H / W / P / M / C, Y164 W / H / F / M / C, N178 H / W / Y, L182V and K207R. The activity of the mutant is greatly improved, compared with the initial UDP-glucosyltransferase mutant M8, the relative activity is maximally improved by 30 times, kcat / Km is improved by more than 30 times, and the mutant can be applied to synthesis of rebaudioside D.

The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to make products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudiosides (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising the SuS enzymes and methods of using them. Methods for producing GT and SuS enzymes are also provided.

This invention proposes a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. Site-directed mutagenesis of UDP-glucosyltransferase is performed, and the mutant plasmid is transferred to *E. coli* to obtain recombinant engineered bacteria. The recombinant engineered bacteria express the protein through fermentation to obtain a crude enzyme solution of the UDP-glucosyltransferase mutant. ADP-dependent enzyme activity and temperature-dependent enzyme activity of the UDP-glucosyltransferase mutant were measured. The mutant ADP-dependent enzyme activity was significantly increased, and it exhibited thermostability. Rebaudioside D was prepared using rebaudioside A, sucrose, ADP, the UDP-glucosyltransferase mutant, and AtSUS sucrose synthase hypercatalytic synthase.

A glucosyltransferase mutant catalyzing RD to produce RM and a use thereof are provided. The glucosyltransferase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO: 1 to at least one of the following mutations: mutating proline at position 84 to tryptophan; and / or mutating methionine at position 88 to valine; and / or mutating leucine at position 126 to phenylalanine; and / or mutating asparagine at position 196 to histidine; and / or mutating leucine at position 379 to isoleucine. The use of the mutant in preparation of the RM is also provided. The enzyme activity of the mutant is significantly higher than that of the wild type, and the conversion rate for the preparation of the RM is high.

The application discloses a glycosyltransferase UGT91C1 mutant and a method for catalyzing synthesis of rebaudioside D, and belongs to biological catalytic synthesis. The glycosyltransferase UGT91C1 mutant is any one of the following (A)-(C): A) a protein obtained by making any one or more mutations in the amino acid sequence shown in SEQ ID NO. 1: the 89th amino acid is mutated from N to Y; the 155th amino acid is mutated from M to L; the 274th amino acid is mutated from S to T; and the 361st amino acid is mutated from N to S; (B) a protein having 95% or 98% or above identity with the amino acid sequence defined in (A) and having the same function; and (C) a fusion protein obtained by connecting a tag to the end of the protein defined in (A) or (B). The glycosyltransferase UGT91C1 mutant is obtained by mutation screening from a wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can efficiently synthesize rebaudioside D by taking rebaudioside A as a substrate.

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and / or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.

This invention discloses a glycosyltransferase UGT76G1 mutant, which is (a) a protein with the amino acid sequence shown in SEQ ID NO.3; or (b) a protein derived from (a) having one or more amino acid sequences substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO.3, and possessing enzymatic activity catalyzing the conversion of rebaudioside D to rebaudioside M. This invention also discloses the gene sequence encoding the glycosyltransferase UGT76G1 mutant of claim 1. This invention significantly improves the enzyme activity of glycosyltransferase UGT76G1 through site-directed mutagenesis. Compared with wild-type glycosyltransferase UGT76G1, the optimal mutant enzyme activity is increased by 4.72 times, thus significantly reducing the amount of enzyme used. This invention heterologously expresses the glycosyltransferase UGT76G1 mutant and sucrose synthase SUS in Pichia pastoris, without purification, and constructs a "UDP-UDPG" cyclic regeneration system using crude enzyme solution, avoiding the need to add additional UDPG as a glycosyl donor, thus significantly reducing costs.

The invention relates to a method for synthesizing rebaudioside D by utilizing immobilized glycosyl transferase, which comprises the following steps: S1, pretreating and activating carrier resin: pretreating and activating the carrier resin to obtain an activated carrier; s2, immobilization of glycosyl transferase: mixing the activated carrier and glycosyl transferase YojK in a buffer solution, and incubating to obtain immobilized glycosyl transferase; s3, immobilized enzyme catalytic reaction: adding the immobilized glycosyl transferase obtained in S2 into a buffer solution containing 3 to 10 mM of rebaudioside A, 10 to 15 mM of UDPG and 100 to 200 mM of NaCl, and carrying out mixed reaction, so as to obtain rebaudioside D. According to the method provided by the invention, the glycosyl transferase YojK is fixed on the carrier resin, so that efficient catalysis and enzyme reutilization are realized, and the method has a good application prospect.

To provide a method for producing rebaudioside D and rebaudioside M by reaction of glycosyltransferase, and to provide a composition for producing rebaudioside D and rebaudioside M containing glycosyltransferase.SOLUTION: Provided is a method for preparing rebaudioside D, the method including preparing rebaudioside D by reacting glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B), wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins consisting of an amino acid sequence represented by a specific sequence.SELECTED DRAWING: Figure 1

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a stevia rebaudiana low-phosphorus response gene PHR family in improving the content of glycoside in stevia rebaudiana. The invention provides application of a stevia rebaudiana low-phosphorus response gene PHR family in improving the content of glycoside in stevia rebaudiana, the stevia rebaudiana low-phosphorus response gene PHR family comprises a SrPHR1 gene and a SrPHR13 gene, and the relative content of rebaudioside C, steviobioside, rebaudioside D and total glycoside is improved by overexpressing the SrPHR1 gene; the relative content of stevioside and rebaudioside A is reduced through overexpression of the SrPHR1 gene; the relative contents of rebaudioside A, rebaudioside C and total glycosides are increased by overexpressing the SrPHR13 gene; the relative content of stevioside and rebaudioside D is reduced through overexpression of the SrPHR13 gene. The invention clarifies the influence of low phosphorus on the yield and quality of stevia rebaudiana, and preliminarily explores the application value of the phosphorus regulatory gene in improving the quality of stevia rebaudiana.

The invention discloses a Ningxia wolfberry-derived glycosyltransferase mutant and application thereof, and belongs to the field of gene engineering. The technical problem to be solved by the invention is how to produce rebaudioside D. The glycosyl transferase mutant disclosed by the invention contains a protein of which the amino acid sequence is SEQ ID No.4. Experiments prove that the glycosyl transferase mutant and the recombinant bacterium containing the glycosyl transferase mutant coding gene can be used for producing rebaudioside D by taking rebaudioside A as a substrate, the rebaudioside D can be obtained by carrying out a whole-cell catalytic reaction by utilizing the obtained recombinant bacterium, and the conversion rate can reach 91.3% under the optimal reaction condition.

The invention discloses a composition containing rebaudioside A, rebaudioside D and rebaudioside M. The composition comprises rebaudioside A, rebaudioside D and rebaudioside M. The content of the rebaudioside A accounts for less than 1% of the total content of the rebaudioside A, the rebaudioside D and the rebaudioside M, the content of the rebaudioside D accounts for less than 9.5% of the total content of the rebaudioside A, the rebaudioside D and the rebaudioside M, and the content of the rebaudioside M accounts for more than 84% of the total content of the rebaudioside A, the rebaudioside D and the rebaudioside M.

The application discloses a preparation method of a novel steviol glycoside derivative rebaudioside M8 and belongs to the field of biological catalysis synthesis. The application obtains a glycosyltransferase UGT94E13 with catalytic rebaudioside D monosaccharide glycosylation activity through excavation, analyzes the complete structure of a product through LC-MS, 1D and 2D NMR spectrum, and obtains a novel steviol glycoside, rebaudioside M8, which is a rebaudioside D monosaccharide glycosylation derivative. The application constructs a coupling reaction of the glycosyltransferase UGT94E13 and a sucrose synthase AtSuSy, realizes efficient catalytic synthesis of rebaudioside M8 with rebaudioside D as a substrate, and efficiently synthesizes 5.71 g / L of rebaudioside M8, with a rebaudioside M8 yield of 88.52%, thereby providing a new efficient and green way for rebaudioside M8 production.

Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. The host cells are genetically modified to contain a heterologous nucleic acid that expresses novel and optimized variants of Ro.KAH. The host cell further contains one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing one or more steviol glycosides in the host cell. The host cells, compositions, and methods described herein provide an efficient route for the heterologous production of steviol glycosides including rebaudioside A, rebaudioside D, and rebaudioside M.

Provided are a preparation method for rebaudioside D and rebaudioside M, and a use of β-1,2-glucosyltransferase in same. The β-1,2-glucosyltransferase has an amino acid sequence as shown in SEQ ID NO: 4, or has a mutant amino acid sequence having at least 99% sequence identity to the amino acid sequence as shown in SEQ ID NO: 4. The provided β-1,2-glucosyltransferase increases the yield of the intermediate product rebaudioside D when catalyzing rebaudioside A to generate rebaudioside M, thereby correspondingly improving the yield of the final product rebaudioside M.

The application discloses a glycosyltransferase UGT76G1 mutant and a method for catalyzing synthesis of rebaudioside M, and relates to the technical field of biological catalytic synthesis. The glycosyltransferase UGT76G1 mutant is any one of the following (A)-(C): (A) a protein obtained by making any one or more mutations in the amino acid sequence shown in SEQ ID NO. 1: the 89th amino acid is mutated from M to H; the 380th amino acid is mutated from L to M; and the 411th amino acid is mutated from A to Y; (B) a protein having 95% or 98% or more identity with the amino acid sequence defined in (A) and having the same function; and (C) a fusion protein obtained by connecting a tag to the terminal of the protein defined in (A) or (B). The glycosyltransferase UGT76G1 mutant in the application is screened from the glycosyltransferase UGT76G1 after mutation, has higher catalytic activity, can take rebaudioside D and UDPG as substrates, and can efficiently synthesize rebaudioside M.

The invention discloses a glycosyl transferase UGT94B1 mutant with improved thermal stability and activity, and belongs to the technical field of biological catalytic synthesis. Through rational design, the 11th site, the 96th site, the 219th site, the 281th site and the 327th site of glycosyl transferase are mutated to obtain the combined mutant UGT94B1M3, compared with a starting enzyme, the half-life period at 40 DEG C is prolonged from 0.43 h to 8.88 h, the thermal stability is improved by 20.47 times, and the enzyme activity is improved by 1.45 times. According to the invention, the mutant is expressed in escherichia coli, and a cascade reaction is constructed with sucrose synthase, so that the Reb D conversion rate reaches 84.67%, and an efficient new way is provided for production of rebaudioside D.