66 results about "Fucosyltransferase" patented technology

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 relates to a genetically modified cell, and belongs to the technical field of biological genetic engineering. The invention provides a genetically modified cell, which contains a heterologous nucleic acid sequence of an alpha-1, 3-fucosyltransferase polypeptide. The alpha-1, 3-fucosyltransferase polypeptide further comprises but is not limited to derived polypeptides M1-M21 of the polypeptide with the amino acid sequence as shown in SEQ ID NO: 1, and compared with the polypeptide as shown in SEQ ID NO: 1, the 3-FL catalytic synthesis capability of the derived polypeptides and the specificity of a catalytic substrate are both improved.

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 invention discloses a production method of fucosylated oligosaccharide. The core of the method is as follows: the GDP-D-rhamnose is converted into the GDP-L-fucose by using the GDP-D-rhamnose-3, 5-epimerase; the GDP-D-rhamnose is obtained by converting the GDP-D-mannose-4, 6-dehydratase and the GDP-4-keto-6-deoxy D-mannose reductase by taking the GDP-D-mannose as a substrate, and the GDP-D-rhamnose is obtained by taking the GDP-D-mannose as a substrate. The invention relates to a method for producing a fucosylated oligosaccharide, which is characterized in that a GDP-D-mannose-4, 6-dehydratase, a GDP-4-keto-6-deoxyD-mannose reductase, a GDP-D-rhamnose-3, 5-epimerase and a fucosyltransferase are expressed in a genetically modified host cell, and the fucosylated oligosaccharide can be produced by the way mentioned above.

The invention discloses recombinant escherichia coli capable of increasing the yield of fucosyllactose and a construction method and application of a fucosyltransferase mutant of the recombinant escherichia coli, and belongs to the field of synthetic biology and metabolic engineering. According to the invention, recombinant escherichia coli B20 and B21 are constructed based on key enzyme engineering transformation. Under the shake flask condition, the 3-FL production capacity of the recombinant strain is obviously improved to 6.20 g / L and 6.34 g / L from the initial 3.45 g / L, and the amplification reaches 79.7% and 83.8% respectively. The yields of 3-FL of the strain B20 and the strain B21 respectively reach 53.88 g / L and 54.64 g / L under the culture condition of a 3L fermentation tank. The recombinant escherichia coli for expressing the 2 '-FL is constructed, and the yield of the 2'-FL reaches 64.86 g / L in a 3L fermentation tank. The invention provides a new technical scheme for efficient microbial synthesis of the 3-FL.

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.

The invention relates to a method for producing 6 '-fucosylated chitosan disaccharide GlcNacbeta1-4 (Fucalpha1-6) GlcNAc through double-bacterium coupling fermentation, and belongs to the technical field of fermentation engineering. The method comprises the following steps: taking fucose and N-acetyl-D-glucosamine as substrates in a fermentation system; the 6 '-fucosylated chitosan disaccharide is produced through coupled fermentation of an engineering escherichia coli strain and yeast, and a GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc synthesis pathway including N-acetylhexosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucose kinase Fkp and N, N-diacetylchitosan phosphorylase Chbp is constructed in the engineering escherichia coli, so that the 6'-fucosylated chitosan disaccharide is obtained. According to the invention, the yeast is added to realize cyclic regeneration from GMP to GTP, so that not only is the problem of high oligosaccharide production cost caused by pure exogenous supplement of GTP effectively solved, but also efficient production of GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc is realized, and the yield of GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc is increased to 25.50 g / L by an optimized coupling fermentation production technology. The invention provides a novel synthesis technology of complex N-glycans, and has a wide application prospect.

The invention discloses recombinant yarrowia lipolytica with high yield of 3-fucosyllactose as well as a construction method and application of the recombinant yarrowia lipolytica. According to the invention, a reinforced lactose transporter lac12 gene, a reinforced GDP-mannose-4, 6-dehydratase gmd gene, a reinforced GDP-L-fucose synthase gmer gene and a 3-fucosyltransferase SUMO-fut3Bc gene with a reinforced SUMO tag are subjected to integrated expression on a recombinant yarrowia lipolytica chromosome, and a 6-phosphogluconolactonase pgl gene is subjected to overexpression, so that a recombinant yarrowia lipolytica strain is obtained. The constructed recombinant yarrowia lipolytica can maintain the genetic stability of a target gene, the yield and production efficiency of 3-fucosyllactose are remarkably improved, and the requirements of industrial production can be met.

The invention relates to Escherichia coli for producing 2 '-fucosyllactose as well as a construction method and application of the Escherichia coli, and belongs to the technical field of genetic engineering. A CRISPR / Cas9 system is used for transforming a genome of escherichia coli MG1655, a wcaJ gene and a lacZ gene are knocked out, the utilization rate of lactose and GDP-L-fucose in a metabolic pathway is increased, a promoter of a lacY gene is replaced with PJ23119, an alpha-1, 2-fucosyltransferase mutant with a dissolution promoting tag GST added to the N end is heterologously introduced, the expression quantity of the alpha-1, 2-fucosyltransferase mutant is increased through multi-copy integration, and the expression quantity of the alpha-1, 2-fucosyltransferase mutant is increased. Meanwhile, a gmd gene, an fcl gene, a manB gene and a manC gene are co-expressed at proper sites, so that the synthesis route of the GDP-L-fucose is optimized. The strain obtained through transformation can efficiently convert lactose into 2 '-FL and has the advantages of being low in metabolic burden, sufficient in precursor supply, high in enzyme stability and the like, the construction method is clear in step and suitable for industrial production, the yield of 2'-FL can be remarkably increased through microbial agents and fermentation application, and the production cost is reduced.

The invention relates to a gene modified cell for producing HMOs, which can be applied to synthesis of difucosyllactose, and belongs to the technical field of gene engineering. The invention provides a genetically modified cell, which contains an alpha-1, 2-fucosyltransferase polypeptide and a heterologous nucleic acid sequence of the alpha-1, 3-fucosyltransferase polypeptide. The genetically modified cell has higher activity of synthesizing difucosyllactose, and does not contain synthetic 3-fucosyllactose or lower side activity of synthetic 2 '-fucosyllactose. The technical scheme of the invention has positive significance for industrial production of human milk oligosaccharide, and the method is green, efficient and sustainable, is beneficial to industrial large-scale production, and has important practical value.

Provided herein are host cells capable of producing a human milk oligosaccharide (HMO), such as yeast cells that include one or more heterologous nucleic acids encoding one or more enzymes of the HMO biosynthetic pathway, such as a fucosyltransferase, GDP-mannose dehydratase, lactose permease, and / or fucose synthase. Also provided are fermentation compositions including the disclosed host cells, as well as related methods of producing and recovering HMOs generated by the host cells.

The invention relates to a high-throughput screening method of alpha-1, 2-fucosyltransferase and a mutant obtained by screening, and belongs to the technical field of enzyme engineering. The invention provides a high-throughput screening method of alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase). The method comprises the following steps: constructing an indicator strain of 2 '-fucosyllactose, enabling the content of the 2'-fucosyllactose to be positively correlated with the fluorescence intensity, then expressing an L-fucokinase and alpha-1, 2-fucosyltransferase mutant library in a chassis strain, carrying out water-in-oil droplet embedding on the chassis strain, and carrying out fusion co-culture on the chassis strain and the indicator strain to obtain the 2 '-fucosyllactose. A chassis strain containing the alpha-1, 2-fucosyltransferase forward mutant is obtained through droplet microfluidic sorting according to a fluorescence detection result, the enzyme activity of the mutant FutCV93I is 2.31 times that of an original enzyme, and the enzyme activity is greatly improved. By using the method provided by the invention, the alpha-1, 2-fucosyltransferase can be subjected to high-throughput screening, and industrial production of the 2 '-fucosyllactose and large-scale application and popularization of the 2'-fucosyllactose in the fields of foods, medicines and the like are facilitated.

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 present application relates to a kind of 2'-fucosyllactose producing recombinant escherichia coli and its construction method and application, belong to the field of bioengineering technology.The present application expresses 2'-fucosyllactose remediation synthesis pathway related gene in recombinant escherichia coli, including the gene fkp of coding GDP-fucose pyrophosphorylase FKP and the gene futC of coding fucosyltransferase FutC, respectively in the upstream of fkp gene and futC gene expression macromolecular coagulation material protein FUSLCD after the modification of coagulation tag GCN4, and replaces replicon, constructs a strain of high-yield 2'-fucosyllactose recombinant escherichia coli, yield and production intensity reach 38.68g / L and 0.35g / L / h respectively.It has good application prospect, has the potential of industrial application.

The invention discloses plasmid-free engineered Escherichia coli for synthesizing lactoyl-N-fucosyl pentasaccharide I as well as a construction method and application of the plasmid-free engineered Escherichia coli, and belongs to the field of genetic engineering. Starting from a genome integration strain for efficiently synthesizing lactoyl-N-tetraose, alpha-1, 2-fucosyltransferase TeSA is introduced, the integration copy number of the alpha-1, 2-fucosyltransferase TeSA is optimized, a plasmid-free strain for efficiently synthesizing the LNFPI is constructed through multi-step reinforcement of a GDP-fucose synthesis route and metabolic flux optimization, finally, the extracellular titer of the LNFPI is 3.98 g / L, and the extracellular titer of the LNFPI is 3.98 g / L; and the method has a good industrialization prospect.

The invention belongs to the technical field of biochemistry, and particularly relates to an efficient enzymatic synthesis method of holothurian glycosaminoglycan. According to the invention, a high-efficiency enzymatic synthesis approach of the sea cucumber glycosaminoglycan is established, sulfated fucose and chondroitin sulfate are used as raw materials, and the sea cucumber glycosaminoglycan with anticoagulant activity is efficiently prepared by using the alpha 1, 3-fucosyltransferase mutant and L-fucose kinase / GDP-fucose pyrophosphorylase and adopting a one-pot multi-enzyme synthesis strategy. And important technical support is provided for development of novel anticoagulant drugs.

The present invention relates to a method for producing triterpenoids using one or more of the following polypeptides: (i) Saponaria officinalis synthase (SobAS), (ii) S. officinalis C28 oxidase (SoC28), (iii) S. officinalis C28C16 oxidase (SoC28C16), (iv) S. officinalis C23 oxidase (SoC23), (v) S. officinalis QA 3-O-glucuronosyltransferase (“SoCSL”), (vi) S. officinalis QA-GlcA galactosyltransferase (“SoC3Gal”), (vii) S. officinalis QA-GlcA-Gal xylosyltransferase (“SoC3Xyl”), (viii) S. officinalis QA-Tri fucosyltransferase (“SoC28Fu”), (ix) S. officinalis QA-TriF rhamnosyltransferase (“SoC28Rha”), (x) S. officinalis QA-TriFR xylosyltransferase (“SoC28Xyl1”), (xi) S. officinalis QA-TriFRX xylosyltransferase (“SoC28Xyl2”), (xii) S. officinalis QA-TriFRXX quinovosyltransferase (“SoGH1”), and (xiii) S. officinalis QA-TriF(Q)RXX acetyltransferase (“SoBAHD1”). Methods, host cells, isolated polypeptides, nucleic acids, and plants are provided.

The invention discloses a double-fucosyl transferase complex and application thereof in synthesis of difucosyl lactose, and belongs to the technical field of biology. In order to solve the problem of high residual quantity of a by-product 2 '-FL in the existing difucosyl lactose (DFL) cell factory, the invention constructs a novel difucosyl transferase complex, random mutation and screening are performed on a key element connecting peptide in the complex, and the catalytic effect of an enzyme is optimized. Through the strategy, the constructed recombinant strain can control the residual quantity of a byproduct 2 '-FL while efficiently synthesizing the DFL, so that the subsequent separation and purification cost is remarkably reduced, the production efficiency is improved, and important data support is provided for industrial large-scale production of the DFL.

The invention discloses application of 2-fluorofucose in preparation of a medicine for relieving acute kidney injury, and belongs to the technical field of biology. In-vitro cell experiments and animal experiments prove that 2-fluorofucose can inhibit core fucosylation, competitively inhibit the activity of fucosyltransferase, down-regulate the expression of FUT8 and reduce the core fucosylation level in kidney tissues and renal tubular epithelial cells; by inhibiting core fucosylation, effectively reversing EMT phenotypic transformation induced by hypoxia reoxygenation or ischemia reperfusion, up-regulating E-cadherin expression, down-regulating alpha-SMA and Snail expression and reducing cell migration ability, phosphorylation activation of an ERK pathway is remarkably inhibited, and downstream inflammatory factor release and cell injury signal transduction are blocked, so that the ERK pathway can be used for inhibiting ERK pathway phosphorylation activation. The invention provides application of 2-fluorofucose to preparation of a medicine for relieving acute kidney injury. The invention provides a new choice for treating acute kidney injury, has the outstanding advantages of remarkable curative effect, high safety and the like, and has a wide application prospect.

The invention discloses an FUT2 high-expression cell strain as well as application and a method of the FUT2 high-expression cell strain in vaccine quality control, and belongs to the technical field of vaccines. The technical problem to be solved is that a simple and easy-to-operate cell model is lacked in the prior art, and the cell model is used for evaluating the effectiveness of NoV vaccines on the cellular level. According to the key points of the technical scheme, the FUT2 high-expression cell strain is provided, the cell strain is an HEK 293T cell strain (HEK 293T-FUT2-EGFP) with EGFP fluorescence for overexpressing human alpha 1, 2-fucosyltransferase 2 (FUT2), and the cell strain stably expresses H, Lewis b and Lewis y type HBGA antigens; meanwhile, an alternative neutralization experiment of the cell level of the norovirus vaccine based on the cell strain is developed, a basis is provided for interaction research of the norovirus and host cells, and the cell strain has high practical application value.

Provided are compositions and methods for specific fucosyltransferase inhibition for treatment of a variety of diseases. The compositions of the invention comprise a glycomimetic of L-Fucose that selectively inhibits the generation of sialyl Lewis X by FTVI and FTVII but has no effect on the generation of Lewis X by FTIX.

Provided is a polypeptide M1-M21 having an activity of synthesizing 3-FL, which polypeptide is obtained by means of modifying a polypeptide as set forth in SEQ ID NO: 1. Further provided is a genetically modified cell containing the gene of the polypeptide. The provided polypeptide has improved activity in catalyzing the synthesis of 3-FL and also has improved specificity in catalyzing a substrate compared to the polypeptide as set forth in SEQ ID NO: 1.

A method for producing fucosylated oligosaccharides. The core of the method lies in converting GDP-D-rhamnose into GDP-L-fucose by using GDP-D-rhamnose-3,5-epimerase, wherein the GDP-D-rhamnose is obtained by means of converting GDP-D-mannose as a substrate via GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose reductase. The expression of GDP-D-mannose-4,6-dehydratase, GDP-4-keto-6-deoxy-D-mannose reductase, GDP-D-rhamnose-3,5-epimerase, and fucosyltransferase in genetically modified host cells enables the production of fucosylated oligosaccharides via the described pathway.

A genetically modified Corynebacterium for production of fucosyllactose, wherein the Corynebacterium has been modified to express a permease for lactose import, GDP-D-mannose-4,6-dehydratase (GMD), GDP-L-fucose synthase (WcaG) and fucosyltransferase (FucT) from exogenous nucleic acid sequences. The exogenous nucleic acid sequences encode a permease for lactose import and the GMD, WcaG and FucT are chromosomally integrated. The Corynebacterium additionally may comprise chromosomally integrated exogenous nucleic acid sequences for expression of phosphomannomutase (ManB) and GTP-mannose-1-phosphate guanylyltransferase (ManC). In certain embodiments, the Corynebacterium is Corynebacterium glutamicum. The Corynebacterium of the invention may be defective for functional expression of one or more glycosyltransferases involved in corynebacterial cell wall biosynthesis.

The invention discloses an engineering bacterium and a method for synthesizing 3 '-sialic acid lactose based on an RIAD-RIDD self-assembled multienzyme compound. The present invention provides a recombinant microorganism for the production of 3 '-sialic acid lactose wherein the recombinant microorganism has (a) enhanced enzymatic activity of CMP-N-acetylneuraminic acid synthetase and / or expression level of coding gene thereof compared to a wild-type microorganism or an original strain; (b) enhancing the enzymatic activity of the alpha-1, 3-fucosyltransferase and / or the expression level of the encoding gene of the alpha-1, 3-fucosyltransferase; and (c) the CMP-N-acetylneuraminic acid synthetase and the [alpha]-1, 3-fucosyltransferase enzyme form a complex. The recombinant microorganism provided by the invention can improve the intermediate channelization efficiency and yield, has improved substrate utilization rate and is strong in universality.

The application provides a preparation method and application of a kind of α-1, 6-fucosyltransferase targeted inhibitor. Specifically, the application provides a compound as shown in formula I or its pharmaceutically acceptable salt, isomer or mixture of isomers, solvate or crystal form and its application as α-1, 6-fucosyltransferase targeted inhibitor.R 1 -(CH2) n -(C(R 3 )2)‑O‑R 2 (I)

The invention relates to application of gallic acid in killing pine wood nematodes, gallic acid can effectively kill the pine wood nematodes, the median lethal concentration of gallic acid to the pine wood nematodes within 24 hours is 100 micrograms / mL, gallic acid kills the pine wood nematodes by inhibiting the activity of fucosyltransferase of the pine wood nematodes, the movement and oviposition ability of the pine wood nematodes can be effectively inhibited, and the effect of killing the pine wood nematodes is achieved. A good inhibition effect is achieved on the pathogenicity of the pine wood nematodes. The gallic acid is used as an inhibitor of the fucosyltransferase, pine wood nematodes are killed by inhibiting the activity of the fucosyltransferase of the pine wood nematodes, and a potential drug action target is provided; the gallic acid is high in biological safety, low in cost and efficient in nematicidal activity, has a good application prospect in the aspect of controlling pine wood nematode diseases, and can be applied to pesticide preparations for killing pine wood nematode.

The present application relates to the technical field of fermentation engineering, and particularly relates to a mutant of alpha-1,2-fucosyltransferase and application thereof. The mutant is based on the amino acid sequence of alpha-1,2-fucosyltransferase and comprises any one or more of the following mutations: K125D, A242C, Y269A, P284L or V292L. The present application provides a plurality of mutants of alpha-1,2-fucosyltransferase, which can effectively improve the ability of Escherichia coli to produce 2'-fucosyllactose and inhibit the generation of impurities. The mutant of alpha-1,2-fucosyltransferase provided by the present application can solve the problem of by-product generation in the fermentation process while improving the yield of 2'-fucosyllactose, which has important significance in the field of 2'-fucosyllactose production.

The invention relates to an alpha-1, 3-fucosyltransferase mutant and an application thereof in preparation of 3-fucosyllactose, the alpha-1, 3-fucosyltransferase mutant is based on SEQ ID NO.1, and any one or combination of more sites in 221, 225, 250 and 254 of the SEQ ID NO.1 is mutated. Compared with the prior art, the multiple alpha-1, 3-FucT mutants provided by the invention can effectively improve the 3-FL secretion capability of alpha-1, 3-FucT, and the 3-FL production level can reach 3.68 g / L after shake-flask culture, which is 11.15 times that of the original alpha-1, 3-FucT strain; the level of 3-FL produced by amplified fermentation culture can reach 25.28 g / L, and the strain has good industrial application potential.

The present invention relates to a genetically modified cell and a method for producing fucosyllactose using the same, and in particular to a genetically modified cell of Saccharomyces cerevisiae and its application, and belongs to the field of biological genetic engineering technology. The present invention provides a genetically modified cell, which comprises a recombinant nucleic acid sequence encoding a polypeptide having α-1,2-fucosyltransferase polypeptide (α-1,2-fucosyltransferase) activity. The α-1,2-fucosyltransferase polypeptide, including but not limited to a polypeptide having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% identical amino acid sequence to SEQ ID NO: 1-25. The genetically modified cell improves the ability of the starting strain to synthesize fucosyllactose.