497 results about "Glycosyltransferase" patented technology

The invention discloses a glycosyl transferase UGT76G1 mutant and a method for catalytically synthesizing rebaudioside M by using the glycosyl transferase UGT76G1 mutant, and relates to the technical field of biological catalytic synthesis. The glycosyl transferase UGT76G1 mutant is any one of the following components (A)-(C): (A) a protein obtained by performing any one or more of the following mutations on the basis of an amino acid sequence as shown in SEQ ID NO.1: the 89th amino acid is mutated into H from M; the 380th amino acid is mutated from L to M; the 411th amino acid is mutated from A to Y; (B) a protein which has 95% or 98% or more of identity with the amino acid sequence defined by (A) and has the same function as the amino acid sequence defined by (A); and (C) a fusion protein obtained by connecting a tag to the terminal of the protein defined in (A) or (B). The glycosyl transferase UGT76G1 mutant disclosed by the invention is obtained by mutating and screening glycosyl transferase UGT76G1, has higher catalytic activity, and can be used for efficiently synthesizing rebaudioside M by taking rebaudioside D and UDPG as substrates.

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.

Glycosyltransferase UGT76G1 and UGT91C1 mutants and a method for catalytic synthesis of rebaudioside A, D, and M by using same, pertaining to the technical field of biocatalytic synthesis. The glycosyltransferase UGT76G1 mutant can effectively synthesize rebaudioside A using stevioside STV as a substrate. The glycosyltransferase UGT91C1 mutant is obtained by mutation screening of wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can effectively synthesize rebaudioside D using rebaudioside A as a substrate. The glycosyltransferase UGT76G1 mutant can also be obtained by mutation and subsequent screening of glycosyltransferase UGT76G1, has higher catalytic activity, and can effectively synthesize rebaudioside M using rebaudioside D and UDPG as substrates.

Provided is a glycosyltransferase UGTSL2 mutant, which is produced through any one or more selected from the group consisting of mutations based on an amino acid sequence shown in SEQ ID NO: 1. Also provided is a glycosyltransferase mutant, which is the following A1) or A2): A1) a protein produced through an amino acid residue substitution in an amino acid sequence shown in SEQ ID NO: 66, where the protein has an identity of 90% or more with and the same function as the amino acid sequence shown in SEQ ID NO: 66; and A2) a fusion protein produced by linking a tag to an N-terminus and / or a C-terminus of the protein in the A1).

The present invention provides a fucosyltransferase polypeptide, which can be applied to the synthesis of human milk oligosaccharides (HMOs) and belongs to the field of enzyme engineering technology. The fucosyltransferase polypeptide described in the present application comprises a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100% identical amino acid sequence to the polypeptide shown in SEQ ID NO:1-25. The transferase polypeptide described in the present application improves the ability of the polypeptide shown in SEQ ID NO:1 to catalyze the synthesis of 2'-FL while also improving the specificity of the polypeptide catalyzing the substrate.

The invention discloses a flavonoid glycoside glycosyltransferase LbUGT71BX1 in laportea bulbifera as well as the coding gene and the use thereof. The amino acid sequence of the flavonoid glycoside glycosyltransferase LbUGT71BX1 is shown as SEQ ID NO: 2. The nucleotide sequence of the coding gene of the flavonoid glycoside glycosyltransferase LbUGT71BX1 is shown as SEQ ID NO: 1. According to the invention, on the basis of relevant results of the second-generation transcriptome and the third-generation full-length transcriptome sequencing of the laportea bulbifera, the last-step key enzyme LbUGT71BX1 for the synthesis of the flavonoid glycoside in the laportea bulbifera is screened and identified by using a reverse genetics method, filling the terminal blank of the biosynthesis pathway of the flavonoid glycoside in the laportea bulbifera.

The invention discloses a haemonchus contortus subunit vaccine, and belongs to the field of veterinary vaccines. The subunit vaccine comprises an antigen protein MEP3, an antigen protein AP1, an antigen protein AP8, an antigen protein H11-2, an antigen protein H11-4 and an antigen protein AP5 of galactosylated and modified haemonchus contortus, and the antigen protein MEP3, the antigen protein AP1, the antigen protein AP8, the antigen protein H11-2, the antigen protein H11-4 and the antigen protein AP5 of galactosylated and modified haemonchus contortus. The galactosylation modification of the antigen protein is realized by jointly expressing glycosyl transferase HcGALT of haemonchus contortus and one or more of the proteins in eukaryotic cells. The antigen protein modified by galactosylation in the subunit vaccine is closer to the glycosylation modification of natural protein, and can provide effective immune protection after the goat is infected with haemonchus contortus, and the egg laying rate is reduced by 81.43%. The invention lays a foundation for development of haemonchus contortus subunit vaccines, provides an effective technical means for prevention and treatment of the haemonchus contortus subunit vaccines, and has important application value and popularization prospect.

The invention discloses a production method for biosynthesis of rebaudioside M. Reb A / Reb D has poor solubility at a low temperature, and the reaction efficiency can be obviously accelerated when the Reb A / Reb D is catalytically produced in a high-temperature environment, so that the whole reaction system is carried out in the high-temperature environment, and the production efficiency can be improved. Under the reaction temperature environment of 70 DEG C, the wild type first glycosyltransferase or / and the wild type second glycosyltransferase are / is basically inactivated, and the catalytic enzyme combination cannot meet the requirement of industrial biosynthesis of rebaudioside M. The wild-type catalytic enzyme is mutated, so that the wild-type catalytic enzyme can keep high catalytic activity at 70 DEG C. Therefore, the production cost of the rebaudioside M produced by the production method disclosed by the invention is obviously lower than that of the prior art, and the method is suitable for industrial popularization.

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.

According to the invention, a glycosyl transferase mutant derived from Bacillus licheniformis ZSP01 is used as a research object, AlphaFold 2 is used for constructing a three-dimensional structure of a protein, then technologies such as molecular docking and molecular dynamics simulation are used for analyzing amino acids far away from a catalytic center in glycosyl transferase, site-directed mutagenesis is carried out on the amino acids, and the glycosyl transferase is obtained. The activity of the mutant for catalytically synthesizing the icariside D2 glycosyltransferase is 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 discloses an application of burkholderia in root system microorganisms in regulation and control of glycosylation of ginsenoside. The invention provides application of burkholderia or a composition containing the burkholderia in any one of the following applications: 1) regulating and controlling proportions of different glycosylated saponins in ginseng; 2) increasing the proportion of monoglycosylated saponin and / or the proportion of disglycosylated saponin in ginseng; 3) preparing ginseng with a high monoglycosylated saponin ratio and / or a high disglycosylated saponin ratio; and 4) improving ginseng cultivation. In a greenhouse environment, the roots of ginseng seedlings are inoculated with burkholderia, metabolome data shows that the burkholderia regulates glycosylation formation of ginsenoside, and the proportion of monoglycosylated saponin and / or the proportion of disglycosylated saponin can be increased. Further, transcriptome sequencing data analysis shows that the burkholderia regulates and controls the expression of different glycosyl transferases, so that the diversity of glycosylation of ginsenoside is influenced. The burkholderia provides a new thought for refined improvement of a ginseng cultivation technology.

The invention relates to a series of fucosyl transferase polypeptides which can be applied to synthesis of human milk oligosaccharides (HMOs), and belongs to the technical field of enzyme engineering and genetic engineering. The invention provides an alpha-1, 3-fucosyltransferase polypeptide, which comprises a polypeptide with an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to that of a polypeptide shown in SEQ ID NO: 1. Or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% of the same amino acid sequence as the polypeptide M1-21. The invention provides a polypeptide as shown in SEQ ID NO: 1 and M1-M21 polypeptides. The 3-FL catalytic synthesis capability of the polypeptide as shown in SEQ ID NO: 1 is improved; and the specificity of a catalytic substrate is improved.

The application belongs to the technical field of genetic engineering, and discloses a kind of saccharomyces cerevisiae for producing rare ginsenoside by seaweed biomass and a construction method and application thereof.The saccharomyces cerevisiae has the following characteristics: overexpression of agarase, neojuncanohydrolase, hydroxymethylglutaryl coenzyme A reductase, isopentenyl diphosphate delta isomerase, dammaradienol synthase, protopanaxadiol synthase, cytochrome P450 reductase, protopanaxatriol synthase and glycosyltransferase.The application combines enzymolysis of seaweed biomass with fermentation of rare ginsenoside, which not only endows wild-type yeast with the ability to degrade seaweed biomass that it originally does not have, but also effectively improves the yield of squalene and downstream terpenes (rare ginsenoside Rh1) by overexpression of tHMG1 and IDI1.The saccharomyces cerevisiae can effectively utilize seaweed biomass to obtain squalene and rare ginsenoside Rh1, and has the characteristics of simplicity, economy and ecological friendliness, can convert cheap biomass into high-value products, and provides a way for the development of marine bioeconomy.

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 relates to an alpha-1, 3-fucosyltransferase mutant as well as a preparation method and application thereof. Compared with wild type alpha-1, 3-fucosyltransferase, the mutant has the advantages that mutation sites of the mutant comprise H129W and / or A188H, and any one of the mutation sites or accumulative mutation can obtain higher yield than that of the wild type in 3-FL synthesis. The obtained mutant obviously improves the yield of 3-FL, has important significance on biosynthesis of 3-FL, is suitable for the requirements of industrial production, and has wide application prospects.

Methods of producing bioconjugates of O-antigen polysaccharides covalently linked to a carrier protein using recombinant host cells are provided. The recombinant host cells used in the methods described herein encode a particular oligosaccharyl transferase enzyme depending on the O-antigen polysaccharide bioconjugate to be produced. The oligosaccharyl transferase enzymes can be PglB oligosaccharyl transferase or variants thereof. Also provided are compositions containing the bioconjugates, and methods of using the bioconjugates and compositions described herein to vaccinate a subject against extra-intestinal pathogenic E. coli. (ExPEC).

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.

The invention discloses a multi-site mutant of glucosyltransferase and an application of the multi-site mutant. A GT 1 family O-glycosyl transferase UGT73C33 derived from liquorice is mutated, an excellent multi-site mutant of the O-glycosyl transferase is obtained through screening, the multi-site mutant contains three mutation sites, F148 is mutated into W, F388 is mutated into W, and A389 is mutated into T. The novel O-glycosyl transferase mutant C33-F148W-F388W-A389T is high in specificity, the selectivity of the novel O-glycosyl transferase mutant C33-F148W-F388W-A389T for generating luteolin-4 '-O-glucoside through specific conversion of luteolin reaches 80%, the novel O-glycosyl transferase mutant is high in catalytic activity, and the yield of luteolin-4'-O-glucoside reaches 156.8 mg / L under suitable conditions. The invention provides a unique biocatalyst and a conversion process thereof for the preparation of luteolin-4 '-O-glucoside with high activity and high added value.

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 discloses arabinose glycosyl transferase PnUGT73B4 and application thereof to preparation of panax japonicus saponin IV, the amino acid sequence of the arabinose glycosyl transferase is shown as SEQ ID NO: 1, under the catalysis of the arabinose glycosyl transferase PnUGT73B4, UDP-arabinose is added to a glucuronic acid group on the C3 site of panax japonicus saponin IVa, and the panax japonicus saponin IV is generated; according to the method, the requirement for raw material plants can be reduced, agricultural land is saved, the product is single, preparation of the panax japonicus saponin IV is facilitated, the biosynthesis route of oleanane type triterpenoid saponin is analyzed, and a breakthrough solution is provided for biosynthesis of the panax japonicus saponin IV.

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 invention belongs to the technical field of biology, and particularly relates to a construction method and application of a tobacco plant for expressing rare ginsenoside. According to the method, dammarendiol synthase DDS, cytochrome P450 CYP716A47 and glycosyl transferase UGTPg1 / UGTPg45 are added into tobacco, heterologous synthesis of rare ginsenosides CK and Rh2 is achieved, dependence on traditional ginseng cultivation extraction is avoided, and cost is remarkably reduced and efficiency is remarkably improved. The characteristics of short growth cycle and easy culture of tobacco are utilized to overcome the problems of low efficiency and low yield of traditional synthesis; positive transgenic tobacco leaves are dedifferentiated to form callus cells, so that materials are provided for large-scale fermentation, and an industrial production foundation is laid.

The invention belongs to the technical field of biology, and particularly relates to a genetically engineered bacterium capable of reducing by-products and improving yield of heparin precursors as well as a construction method and application of the genetically engineered bacterium. According to the technical scheme, acetic acid synthesis pathway genes ackA and poxB and lactic acid synthesis pathway genes ldhA are knocked out, and synthesis of acetic acid and lactic acid is blocked, so that more carbon metabolic flows enter heparin precursor synthesis pathways, the yield and conversion rate of heparin precursors are increased, and / or by-products are reduced. According to the invention, heparin precursor synthetases KfiA (UDP-GlcNAc glycosyl transferase), KfiC (UDP-GlcA glycosyl transferase) and KfiD (UDP-glucose dehydrogenase) are further enhanced, the expression of UDP-GlcA generated by UDP-Glc is catalyzed, and the yield and the conversion rate of the heparin precursor can be further improved and / or byproducts can be further reduced.

The invention discloses glycosyl transferase for catalytic production of rebaudioside M and a recombinant gene of the glycosyl transferase. Compared with wild type glycosyl transferase, the glycosyl transferase variant disclosed by the invention has the advantages that the thermal stability and the enzyme activity are obviously improved. In a high-temperature environment of 70 DEG C, the wild type glycosyl transferase is completely inactivated, and the enzyme activity of the glycosyl transferase variant disclosed by the invention is more than 50U / mg. Meanwhile, in a high-temperature environment of 70 DEG C, the solubility of a reaction substrate rebaudioside D is improved, the thermal motion of enzyme molecules and substrate molecules is intensified, and the collision frequency and energy between the enzyme molecules and the substrate molecules are increased, so that the catalytic reaction speed can be further increased. Therefore, the glycosyl transferase variant disclosed by the invention is suitable for being used as a biocatalyst and is used for large-scale production of a sweetening agent rebaudioside M with a better taste.

The present invention relates to a glycosyltransferase mutant, and a composition and a method for producing steviol glycosides using the same, and more specifically, to a glycosyltransferase of rebaudioside that transfers glucose to steviol glycosides and its mutant, a recombinant strain expressing the enzymes, and a method for producing rebaudioside using the same.

The invention relates to the technical field of oligosaccharide preparation through enzyme catalysis, in particular to a method for preparing a worm oligosaccharide product through cascade catalysis of glycosyltransferase. The method comprises the following steps: secreting and expressing truncated beta 1, 4-N-acetamino galactosyl transferase and alpha 1, 3-fucosyl transferase E by using an insect cell baculovirus expression system to obtain GalNAcT delta TM and FucT-E delta TM; gn [beta] GlyFmoc and Gn2Man3Gn2 [beta] AsnFmoc are used as substrates, and continuous catalysis is carried out, so as to prepare an oligosaccharide intermediate containing LDN epitopes and an oligosaccharide product containing LDN-F epitopes. The method solves the problem of high production cost of an LDN-F epitope-containing oligosaccharide product.

The invention discloses saccharomyces cerevisiae with high yield of salidroside as well as a construction method and application of the saccharomyces cerevisiae. According to the recombinant saccharomyces cerevisiae, ribulose-5-phosphate isomerase RKI1 and transketolase TKL1 are expressed in host bacteria, so that precursor supply is increased; reverse methyltransferase ARO2 and phenylalanine decarboxylase ARO10 are expressed at the same time, and a tyrosine branch pathway is adjusted; then, UDP-glycosyl transferase of different sources is expressed, and iterative site-specific mutagenesis is carried out on the UDP-glycosyl transferase to enhance conversion of hydroxytyrosol to salidroside; the salidroside production performance of the recombinant strain is verified on the basis of a precursor supply pathway, a tyrosine regulation pathway and key enzyme screening, and the salidroside production capacity of the saccharomyces cerevisiae is further improved.