121 results about "Sucrose synthetase" patented technology

The invention belongs to the technical field of molecular biology, and discloses a glucosyltransferase mutant, a construction method of the glucosyltransferase mutant, a construction body, a mutant expression strain, application and a preparation method of rebaudioside M. Compared with original glucosyltransferase (comprising an amino acid fragment with the sequence shown as SEQ ID NO: 1), the glucosyltransferase mutant provided by the invention comprises one or more of L79T mutation, S195 V mutation and Y387V mutation. The glucosyltransferase mutant has excellent enzyme activity and can be cooperated with existing sucrose synthase to realize a multi-cascade enzyme catalytic reaction, and sucrose, rebaudioside D and uridine diphosphate are used as raw materials to realize high-efficiency and low-cost industrial production of rebaudioside M.

The invention discloses a light-driven synthetic flora and application thereof. The light-driven synthetic flora comprises phototrophic bacteria and heterotrophic bacteria, the phototrophic bacteria are genetically modified synechococcus slender, and the gene modification comprises construction and expression of heterologous sucrose permease, glyceraldehyde dehydrogenase and sucrose synthase in the synechococcus slender; and the heterotrophic bacteria are the P.adami. According to the light-driven synthetic flora disclosed by the invention, carbon dioxide is converted into cane sugar through photosynthesis of phototrophic bacteria, and the cane sugar is secreted out of cells to provide a carbon source for heterotrophic bacteria; carrying out cell growth and polyhydroxyalkanoate synthesis on the heterotrophic bacteria by using cane sugar; therefore, the light-driven synthetic flora provided by the invention can realize direct conversion of carbon dioxide into polyhydroxyalkanoate without additional carbon sources, has the advantage of environmental friendliness, and provides a new economical and feasible scheme and approach for production of polyhydroxyalkanoate.

The present disclosure provides enzymes and methods of using those enzymes to transfer sugar moieties to the substrate steviol glycosides. The designed beta-1, 3-glycosyltransferase can be used for adding a glucose monomer connected with beta-1, 3 to steviol glycosides. In particular, the designed beta-1, 3-glycosyltransferase is used in a one-pot reaction together with a beta-1, 2-glycosyltransferase and a sucrose synthase to convert stevioside and / or Reb A to Reb M.

The invention relates to the technical field of gene engineering, in particular to a glycosyl transferase mutant, a sucrose synthase mutant and application of the glycosyl transferase mutant and the sucrose synthase mutant in synthesis of rebaudioside M. Wild glycosyl transferase and wild sucrose synthase are modified according to a directed evolution theory to obtain a glycosyl transferase mutant and a sucrose synthase mutant. Rebaudioside M is obtained by catalyzing rebaudioside D through a glycosyl transferase mutant and sucrose synthase mutant co-expression strain, and the catalytic efficiency is superior to that of a wild enzyme. The rebaudioside M is obtained by completely catalyzing 110 g / L of rebaudioside D through the co-expression strain of the glycosyl transferase mutant and the sucrose synthase mutant, only 20 h is needed, the conversion rate reaches 97%, the yield of the rebaudioside M reaches 122 g / L, the raw material conversion rate and the yield of the rebaudioside M are effectively increased, and meanwhile the synthesis time is shortened.

The invention discloses a preparation method of glucosyl stevioside, and relates to the technical field of glucosyl stevioside production, rebaudioside A or stevioside is taken as a raw material, cyclodextrin is taken as an auxiliary material, cyclodextrin glucosyltransferase is added for glycosylation reaction, a glucosyl stevioside solution I is cooled, and a glucosyl stevioside solution II is obtained; the method comprises the following steps: adding sucrose, UGT glycosyl transferase, sucrose synthase and uridine diphosphate into rebaudioside A or stevioside, continuously converting unreacted rebaudioside A or stevioside into rebaudioside D or rebaudioside E, inactivating after the reaction is finished, filtering by a plate frame, removing impurities through resin, analyzing the resin by using ethanol to obtain an analysis solution, and treating the analysis solution at 78-82 DEG C for 2-4 hours to obtain the rebaudioside D or rebaudioside E; and dealcoholizing at the vacuum degree of-0.1 MPa, concentrating and drying to obtain the glucosyl stevioside. The quality of the product is improved, the cost is reduced, and the taste of the product is integrally improved.

The invention provides a method for producing glycosyltransferase or sucrose synthase by fermenting recombinant escherichia coli, and belongs to the technical field of biological fermentation. According to the fermentation method provided by the invention, a three-stage material supplementing control fermentation strategy is adopted, namely after fermentation is carried out for 5 hours, material supplementing operation is started, the material supplementing speed is 6.24-7.20 g / L / h, fermentation is carried out for 8-10 hours, the material supplementing speed is reduced to 4.80 g / L / h, the temperature is reduced to 25 DEG C, and induction is carried out; then maintaining the material supplementing speed until the fermentation OD600 is 65-75, and then reducing the material supplementing speed to 3.84-4.80 g / L / h until the OD600 is not obviously increased any more; the material supplementing mode has remarkable advantages in the aspects of improving fermentation OD600 and fermentation enzyme activity, shortening the fermentation period, reducing the cost and the like.

The invention discloses a recombinant plasmid, a recombinant strain and a method for synthesizing rebaudioside M by a one-step method, and belongs to the technical field of biological catalytic synthesis. The recombinant plasmid contains a coding gene of a glycosyl transferase UGT91C1 mutant, a coding gene of a glycosyl transferase UGT76G1 mutant and a coding gene of sucrose synthase. The mutant of the glycosyl transferase UGT91C1 is 2-12E, and the coding gene sequence of the glycosyl transferase UGT91C1 is as shown in SEQ ID NO. 1; the glycosyl transferase UGT76G1 mutant is 101H10, and the coding gene sequence of the glycosyl transferase UGT76G1 mutant is as shown in SEQ ID NO. 2; the sequence of the coding gene of the sucrose synthase is as shown in SEQ ID NO. 3. The rebaudioside M is synthesized by using a three-enzyme co-expression recombinant strain containing the plasmid through a one-step method, so that the loss of fermentation materials can be reduced, the utilization efficiency of equipment is improved, the energy consumption is reduced, and the large-scale production of the rebaudioside M is facilitated.

The invention relates to the technical field of gene engineering, in particular to a glycosyl transferase mutant and application thereof in synthesis of rebaudioside M. Wild type glycosyl transferase is modified through a directed evolution theory to obtain the glycosyl transferase mutant, so that the efficiency of synthesizing rebaudioside D through reaction of rebaudioside A is effectively improved. Besides, recombinant bacteria capable of simultaneously expressing the glycosyl transferase mutant, a second glycosyl transferase gene and a sucrose synthase gene are constructed, a wet cell obtained by induced culture of the recombinant bacteria or a crude enzyme liquid extracted by crushing the wet cell is used as a catalyst, rebaudioside A is used as a substrate, sucrose is used as an auxiliary substrate, and the rebaudioside M is catalytically synthesized by a one-pot method. According to the present invention, the complete catalysis of 100 g / L of rebaudioside A to produce rebaudioside M only needs 24 h, the conversion rate reaches 97.5%, the yield of rebaudioside M reaches 130 g / L, the raw material conversion rate and the yield of rebaudioside M are effectively improved, and the synthesis time is shortened.

The invention belongs to the technical field of bioengineering, and discloses a glycosyl transferase mutant which is obtained by manufacturing at least one point mutation with an amino acid sequence as shown in SEQ ID NO.1, mutated genes are cloned to corresponding carrier plasmids and transformed into escherichia coli for expression, and the obtained enzyme can be applied to production of stevioside D. The invention further discloses a preparation method of the glycosyl transferase mutant. The obtained glycosyl transferase mutant has the characteristics of high efficiency and stability, and can be coupled with sucrose synthase to efficiently prepare stevioside D.

The invention discloses an escherichia coli recombinant bacterium for whole-cell catalytic synthesis of rebaudioside M as well as a construction method and application of the escherichia coli recombinant bacterium. The Escherichia coli recombinant bacterium for the whole-cell catalytic synthesis of the rebaudioside M, which is constructed by the invention, contains a glycosyl transferase UGT76G1 coding gene mutant, a glycosyl transferase UGT91C1 coding gene mutant, a sucrose synthase atSuS1 gene and a polyphosphoglucokinase cgPPGK gene; a glucose-1-phosphate uridine transferase galU gene, a nucleoside diphosphate kinase ndk gene and a nucleoside diphosphate kinase ndk gene are over-expressed; meanwhile, an encoding gene araA gene of L-arabinose isomerase and an encoding gene ushA gene of 5 '-nucleotidase / UDG hydrolase are knocked out. According to the recombinant escherichia coli constructed by the invention, Reb D is prevented from being directly used as a synthetic substrate, so that the raw materials are easier to obtain, and the conversion rate of the substrate and the synthetic rate and yield of rebaudioside M are remarkably improved.

Disclosed in the present invention are a sucrose synthase mutant and a method for preparing RD using same. The sucrose synthase mutant is based on the amino acid sequence shown in SEQ ID NO: 2 and contains a mutation in at least one of the following sites: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R. By means of site-directed mutagenesis of sucrose synthase, the enzyme activity of the sucrose synthase mutant is improved compared with that of the wild type. Also, some mutants can still maintain a high conversion rate in a wide pH range.

The invention discloses an engineered glycosyl transferase and application thereof in efficient preparation of triterpenoid saponin. The engineered glycosyl transferase is obtained by mutating serine at the 15th site of UGT74AC1 enzyme into alanine, mutating histidine at the 47th site into aspartic acid, mutating leucine at the 48th site into valine, mutating alanine at the 180th site into serine, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid The mutant is obtained by mutating serine at the 332 site into proline, phenylalanine at the 367 site into tryptophan and lysine at the 420 site into arginine. According to the invention, the glycosyl transferase with high catalytic activity is obtained by modifying an enzyme engineering technology, and good application of the glycosyl transferase in synthesis of triterpenoid saponin is realized. The glycosyl transferase disclosed by the invention can also be coupled with sucrose synthase, or coupled with N-acetylhexosamine kinase and N-acetylglucosamine-1-uridine phosphate transferase, and acetylglucosamine is used as a glycosyl donor, so that cascade production of triterpenoid saponin with lower cost and higher efficiency is realized.

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 disclosure provides enzymes and a method to use those enzymes to transfer a sugar moiety to a substrate steviol glycoside. Specifically, designed beta-1,2-glycosyltransferases and sucrose synthases are used in a one-pot reaction to convert stevioside and Reb A into Reb E and Reb D. Additionally, the designed beta-1,2-glycosyltransferases can be used in a one-pot reaction with a sucrose synthase and a beta-1,3-glycosyltransferase to produce Reb M from stevioside and / or Reb A.

The invention discloses a sucrose synthase mutant and a method for preparing RD (Reduced Disorder) by using the sucrose synthase mutant. The sucrose synthase mutant is obtained by mutation of at least one of the following sites on the basis of an amino acid sequence as shown in SEQ ID NO: 2: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R; site-directed mutagenesis is carried out on sucrose synthetase, so that the enzyme activity of the sucrose synthetase mutant is improved compared with that of a wild type, and meanwhile, a part of the mutant still maintains a relatively high conversion rate in a relatively wide pH range.

The invention discloses saccharomyces cerevisiae engineering bacteria for efficiently synthesizing mogroside V on the basis of cytoplasm and lipid droplet double-cell area compartment and a construction method of the saccharomyces cerevisiae engineering bacteria. The construction method comprises the following steps: integrating truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, pentenyl pyrophosphate isomerase IDI1, epoxy squalene cyclization enzyme ERG1, squalene synthetase ERG9, choline kinase ScCK, isopentenyl phosphokinase AtIPK, cucurbitadienol synthetase SgCDS, cyclic epoxy hydrolase SgEPH3, cytochrome P450 enzyme CYP87D18 and cytochrome P450 enzyme reductase AtCPR1 at the site of a saccharomyces cerevisiae genome GAL80; an ABC efflux protein PDR11, a glycosyl transferase UGTMG1, a sucrose synthase Susy and a glycosyl transferase SgUGT94-289-3 are integrated at a site Exg1 of a saccharomyces cerevisiae genome. The yield of mogroside V produced by shake flask fermentation of the saccharomyces cerevisiae engineering strain provided by the invention reaches 96.3 mg / L, so that mogroside V is efficiently synthesized from the beginning in the saccharomyces cerevisiae, and the saccharomyces cerevisiae engineering strain has important practical significance for promoting sustainable development of the mogroside industry.

The invention relates to an enzymatic reaction catalyst and application thereof in synthesis of quercetin 3, 4 '-di-O-beta-d-glucoside, and relates to the technical field of biological catalysis. The enzymatic reaction catalyst comprises glycosyl transferase and sucrose synthase, and the glycosyl transferase comprises glycosyl transferase I and glycosyl transferase II; the amino acid sequence of the glycosyl transferase I is as shown in SEQ ID NO: 4, the amino acid sequence of the glycosyl transferase II is as shown in SEQ ID NO: 5, and the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO: 6. According to the invention, glycosylation reaction of hydroxyls at 3 and 4'sites of quercetin is established, and high-efficiency and low-cost enzymatic synthesis of the natural product quercetin 3, 4 '-di-O-beta-d-glucoside is realized.

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.

Disclosed is a method for efficiently preparing rebaudioside M by using a bio-enzymatic method. The preparation method comprises: adding rebaudioside A, uridine diphosphate glucose, glycosyltransferase, and a salt into a buffer solution, mixing to obtain a mixed solution, performing an enzyme-catalyzed reaction while stirring, and obtaining said rebaudioside M. The amino acid sequence of the glycosyltransferase is one or more of those shown in SEQ ID NO. 1-12. The glycosyltransferase can also be coupled with sucrose synthase for catalysis, so as to use sucrose as a glycosyl donor to, in one step, catalyze rebaudioside A to generate rebaudioside M.

The present application relates to the field of bioengineering, and particularly to a mutant of an enzyme, a mutant of a glycosyltransferase and application thereof in preparing rhodiolin. The present application provides a mutant of an enzyme, which has the following amino acid site mutations on the basis of a wild-type glycosyltransferase: the 308th site is mutated from I to Q, the 333rd site is mutated from V to R, the 363rd site is mutated from W to S and the 386th site is mutated from S to G, and the amino acid sequence of the wild-type glycosyltransferase is shown as SEQ ID NO: 1. The present application obtains a glycosyltransferase and a sucrose synthase with improved thermal stability through structural rational design and deep learning modification, and can glycosylate tyramine to generate rhodiolin in the form of regenerating ADP-glucose, and the price of ADP is only about 20% of UDP, which has a cost advantage.

The present invention provides a glycosyltransferase, and the amino acid sequence of the glycosyltransferase has one or more amino acid mutations at the 129th, 208th, 20th, 69th, 150th, 238th, 273rd, 287th, and 349th amino acids corresponding to the amino acid sequence of the wild-type glycosyltransferase OsUGT91C1. The glycosyltransferase of the present invention has high enzyme activity, so that it can more effectively catalyze the formation of Reb D from Reb A. When the glycosyltransferase of the present invention is used in combination with sucrose synthase SUS1, the addition of expensive UDPG can be avoided, thereby further reducing the production cost of Reb D. The present invention lays a new material foundation for the production of Reb D.

The invention belongs to the technical field of biological catalysis, and particularly relates to a glycosyl transferase mutant and application thereof in catalytic synthesis of rebaudioside M. Engineering bacterium whole cells or broken enzyme liquid for co-expressing a glycosyl transferase G1 mutant, a glycosyl transferase C1 mutant and sucrose synthase SUS is used as a catalyst, Reb A is used as a substrate, sucrose is used as a glycoside donor, Reb M is synthesized under mild conditions, the space-time yield of the generated Reb M is up to 3.33 g / L / h, and the method has the advantages of being high in space-time yield, convenient to purify, green, environmentally friendly and the like, and is suitable for industrial production. The industrial application value is extremely high.

The purpose of the present invention is to provide: a plant having an increased yield of storage tissue; and a method for producing the plant. It was discovered that the yield of storage tissue in a plant can be increased by expressing, in the storage tissue of the plant, a sucrose synthase having a substrate specificity to adenosine diphosphate.

The invention belongs to the technical field of genetic engineering and functional enzyme preparations, particularly relates to sucrose synthase and a coding gene and a mutant thereof, and further discloses application of the sucrose synthase. According to the invention, sucrose synthase is obtained from candidate methylobacterium and is further mutated, so that a sucrose synthase mutant of which the enzyme activity, the conversion rate and the stability are remarkably improved is obtained, and the activity and the efficiency of a stevioside synthesis process are effectively improved.

The invention discloses a glycosyl transferase mutant with improved thermal stability and activity, and belongs to the technical field of biological catalytic synthesis. Through rational design, the 102nd site, the 110th site, the 208th site and the 251th site of glycosyl transferase are mutated to obtain a combined mutant UGT94E13M3 phase starting enzyme, the half-life period of the combined mutant UGT94E13M3 phase starting enzyme at the temperature of 45 DEG C is prolonged from 0.60 h to 8.88 h, the thermal stability is improved by 14.8 times, and the enzyme activity is improved by 1.08 times. According to the invention, the mutant and sucrose synthase are subjected to a coupling reaction, so that the Reb D conversion rate reaches 71.40%, and an efficient and green new way is provided for production of rebaudioside M8.

The present invention discloses a sucrose synthase mutant with improved thermal stability, belonging to the field of enzyme engineering. The present invention provides a sucrose synthase mutant with improved thermal stability, capable of synthesizing UDP-Glc at high temperatures, thereby improving its production efficiency and meeting the needs of industrial production.

The application discloses a sucrose synthetase mutant and a method for preparing RD by using the sucrose synthetase mutant, wherein the sucrose synthetase mutant contains at least one of the following mutations: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R on the basis of the amino acid sequence shown in SEQ ID NO:2; the sucrose synthetase mutant has higher enzyme activity than the wild type, and meanwhile, some mutants still maintain a high conversion rate in a wider pH range.

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M as well as a product and application thereof, and belongs to the technical field of gene engineering. The technical problem to be solved is that the existing glycosyl transferase and sucrose synthase are low in enzyme activity and insufficient in enzyme activity stability, so that the synthesis efficiency of rebaudioside M (RebM) is not high, and the industrial application of preparing RebM by an enzyme method is limited. According to the key points of the technical scheme, the glycosyl transferase mutant is provided, the mutant is obtained by mutating UDP-glycosyl transferase UGT76G1, and the amino acid sequence of the UDP-glycosyl transferase UGT76G is as shown in SEQ ID NO. 1; the mutation comprises mutation on the 83rd site of SEQ ID NO.1, and the amino acid sequence of the glycosyl transferase mutant is shown as SEQ ID NO.5. The invention further discloses a preparation method of the glycosyl transferase mutant.

The invention discloses 2-phenethyl alcohol-beta-D-glucoside catalyzed and synthesized by a cofactor self-circulating multi-enzyme cascade system and application of the 2-phenethyl alcohol-beta-D-glucoside. The system comprises phenylalanine dehydrogenase and a mutant thereof, wherein the phenylalanine dehydrogenase is used for converting L-phenylalanine (L-Phe) into phenylpyruvic acid (PPA); pPA is converted into pyruvate decarboxylase (Aro10) of phenylacetaldehyde; the alcohol dehydrogenase is used for reducing phenylacetaldehyde into 2-phenethyl alcohol (2-PE) or a mutant of the alcohol dehydrogenase; 2-PE is converted into sucrose synthase (SuSy) of 2-phenethyl alcohol-beta-D-glucoside (2PE-Glc) or a mutant of the sucrose synthase, and UDP-glucosyltransferase (UGT) or a mutant of the UDP-glucosyltransferase. A novel multi-enzyme cascade system with cofactor self-circulation capability is realized and is used for efficiently synthesizing 2PE-Glc from L-phenylalanine. The invention not only provides an effective 2PE-Glc biosynthesis strategy, but also shows the potential of a multi-enzyme cascade system in the aspect of synthesizing biologically active complex glycoconjugates.

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.