668 results about "Synthetic enzyme" patented technology

The invention discloses a novel santalene synthase gene as well as an encoding protein and application thereof. The amino acid sequence of the santalene synthetase is as shown in SEQ ID NO. 2. The invention relates to a method for carrying out functional verification on a new sandalene synthase gene encoding enzyme from sandalwood and carrying out synthesis of sandalene and bergamoene in escherichia coli and tobacco leaves. According to the invention, the enzyme can be massively prepared by utilizing escherichia coli, is applied to synthesis of santalene and bergamoene, can be further applied to synthesis of bergamoene or santalene, and has important significance; the heterologous synthesis of the high-value santalene and the bergamoene in plants can also be realized, and the method has a wide industrial practical prospect and a large-scale development potential.

The invention relates to the technical field of microbial fermentation, and discloses a genetically engineered bacterium of high-yield D-pantothenic acid as well as a preparation method and application of the genetically engineered bacterium. The D-PA yield of the engineering bacteria is greatly improved through the following effects that acetolactate synthase IlvB and IlvN shunted by pyruvic acid are subjected to overexpression, meanwhile, a transcription attenuation region is deleted, and valine feedback inhibition sites are mutated; the method comprises the following steps: overexpressing keto-alcohol acid reductoisomerase IlvC and dihydroxy acid dehydratase IlvD, and converting the IlvC coenzyme preference from NADPH into NADH, so as to break through the upstream of D-PA synthesis; overexpression of PanB is carried out, overexpression of a serine-glycine transport system is carried out, and synthesis of 5, 10-methylenetetrahydrofolic acid is promoted; meanwhile, the expression of ketopantoic acid reductase PanE is enhanced; and finally, over-expressing pantothenic acid synthetase PanC, and increasing the content of a cofactor ATP required by the PanC. According to the engineering bacteria provided by the invention, through overall reinforcement of the multiple modules, cell growth is improved due to balance of cofactors and increase of D-PA, and the yield of D-PA reaches an unprecedented height.

The invention belongs to the technical field of chondroitin sulfate biosynthesis, and discloses a recombinant strain for high-yield chondroitin sulfate, application of the recombinant strain and a preparation method of the chondroitin sulfate. The recombinant strain provided by the invention can be used for simultaneously expressing UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase and glucose phosphate mutase; according to the present invention, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase are adopted, and the enzyme molecules are matched with the original carbon metabolism pathway in the recombinant strain cell to jointly form the enzyme-linked pathway having the efficient CSA synthesis ability, such that the high-yield and high-purity preparation of the CSA can be achieved; the method has the advantages of simple operation, high biological safety of the obtained product, easy realization of large-scale production and the like, and has a good application prospect.

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.

Provided are S-adenosylmethionine synthetase mutants and the use thereof, belonging to the technical field of biological enzyme engineering. Site-directed combinatorial mutations are performed on positions 189, 200, 234, 266 and 371 of S-adenosylmethionine synthetase MAT to obtain a series of mutants with improved enzyme activity, wherein the specific enzyme activity of I189V / V266H and V266H / N371R is increased by more than 10 times relative to the parent. The yields of S-adenosylmethionine synthesized by whole cells of recombinant bacteria expressing mutants I189V / V266H / N371R, V266H / Q234N / N371R and I189V / V266H / Q234N / N371R all reach 1550 mg / L or more. The provided enzyme mutants have broad application prospects in the production of S-adenosylmethionine and the construction of genetically engineered microorganisms for S-adenosylmethionine.

The invention relates to the technical field of synthetic biology and microbial fermentation, in particular to a 5-aminolevulinic acid synthetase mutant and application thereof. According to the invention, 5-aminolevulinic acid synthetase derived from rhodobacter capsulatus is expressed in recombinant Escherichia coli, point mutation is carried out on non-conservative amino acid of wild-type ALAS to obtain I86C mutants, the enzymatic activity of the 5-aminolevulinic acid synthetase is improved, and the ability of the 5-aminolevulinic acid synthetase in synthesis of 5-aminolevulinic acid is improved. The method can be used for solving the problems that the enzyme activity of the ALAS is low and the activity of the ALAS is easily inhibited by high-concentration substrate glycine and the like at present, and the yield of the 5-ALA is increased.

The invention discloses a genetically engineered bacterium for producing uridine diphosphate glucose as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The following genes are overexpressed in the genetically engineered bacterium: a pyrimidine nucleoside operon gene, a carbamyl phosphate synthetase gene, an aspartate carbamyl transaminase gene, an orotic acid phosphoribose transferase gene, a phosphoribose pyrophosphate synthase gene, a uridylate kinase gene and a glucophosphate mutase gene. A uridine triphosphate glucose-1-phosphate uridine group transferase gene; the genetically engineered bacterium does not express the following genes: a purine nucleotide repressor protein gene, a deoxyribonucleoside triphosphate reductase gene, a nucleoside triphosphate pyrophosphohydrolase gene, a cytidine triphosphate synthase gene, a pyrimidine nucleotidase gene, a nucleotidase gene, an ornithine carbamoyltransferase gene, a UDPG dehydrogenase gene and a lacI repressor protein gene. The genetically engineered bacterium can be used for efficiently synthesizing UDPG by taking glucose as a substrate.

The invention provides a 9-hydroxycamptothecin synthetase and a method for synthesizing the 9-hydroxycamptothecin. The invention discloses the 9-hydroxycamptothecin synthetase for the first time, and establishes a method for producing the 9-hydroxycamptothecin. According to the technical scheme, biological synthesis of the 9-hydroxycamptothecin is achieved, and a new way is provided for producing camptothecin derivatives based on strategies such as synthetic biology.

The invention discloses a double-function flavin adenine dinucleotide synthetase mutant and application, the double-function flavin adenine dinucleotide synthetase is called RFK / FADS for short, and the amino acid sequence of the double-function flavin adenine dinucleotide synthetase is shown as SEQ ID NO.1; and the RFK / FADS mutant is RFK / FADSF55A, RFK / FADSG43Q, RFK / FADSG43Q-F55H, RFK / FADSG43Q-F55N or RFK / FADSG43Q-F55N-F129Y, and the RFK / FADSG43Q-F55N and the FADSG43Q-F55N-F129Y can be used for preparing the According to the genetically engineered bacterium disclosed by the invention, flavine adenine dinucleotide is produced through a simple fermentation process by taking glucose as a substrate under a shake flask aerobic condition, and the yields of the strains ZF02, ZF03, ZF04, ZF05 and ZF06 are respectively increased by 120%, 130%, 166%, 246% and 286% in sequence compared with those of ZF01.

The invention belongs to the technical field of immunotherapy, and particularly relates to a method for improving NK cell functions by targeted inhibition of lactylation. The invention provides a method for improving functions of NK cells by targeted inhibition of lactylation, NK cell lactic acid transporters or modification enzymes in inhibition of the lactylation process are targeted, the modification enzymes are NK cell lactylation modification enzymes Writer or lactic acid coenzyme A synthetase, NK92MI lactylation modification is blocked, so that function depletion of the NK cells is reversed, and the NK cell function is improved. The function of the NK cell can be partially reversed by inhibiting the lactic acid modification, and the expression level of effector molecules of the NK92MI cell can be recovered.

The invention discloses a pyranopyridine compound as well as a preparation method, a pharmaceutical composition and application thereof. The invention provides a compound as shown in a formula (I), a pharmaceutically acceptable salt or a stereoisomer thereof, and the compound has a relatively strong inhibition effect on aldosterone synthetase, almost has no influence on cortisol synthetase, has high selectivity and relatively high safety, and can be used for preventing and / or treating various diseases related to aldosterone. # imgabs0 #

The invention relates to a method for increasing the yield of L-malic acid produced by fermentation by adding ethylene glycol, which comprises the following steps: constructing genetic engineering escherichia coli, and introducing ethylene glycol metabolism genes (overexpression) including glycerol dehydrogenase, lactaldehyde reductase, lactaldehyde dehydrogenase and the like; and overexpression of other genes involving malic acid production, such as malic acid synthase, phosphoenolpyruvate carboxylase and malic acid export protein. The gene engineering strain is an E.coli BW25113 strain of which ldhA, pta, poxB, adhE, pflB, mgsA, frdA, maeA, maeB, yqhD, ycdW and iclR genes are substantially inactivated, and the deleted genes are beneficial to reducing the generation of metabolic byproducts and increasing the accumulation of malic acid. The genetic engineering strain is subjected to fermentation culture by taking glucose and ethylene glycol as carbon sources, and L-malic acid is separated from a culture after culture is finished.

The invention discloses a mutant with improved catalytic activity of S-adenosylmethionine synthetase and application of the mutant, and belongs to the technical field of biological enzyme engineering. According to the present invention, the site 189, the site 234, the site 266 and the site 371 of the S-adenosylmethionine synthetase MAT are subjected to site-specific combinatorial mutation to obtain a series of mutants with improved enzyme activity, the I189V / V266H improvement is the most obvious, and the specific enzyme activity is 3.5833 + / -0.1373 U.mg <-1 >, which is improved by 1353.08% compared to the parent WT; and for a whole-cell catalysis system, the yield of S-ademetionine synthesized by the mutant I189V / V266H / Q234N / N371R reaches 1751.01 mg / L, and the increase is most obvious. The enzyme mutant provided by the invention has a wide application prospect in the production of S-adenosylmethionine and the construction of genetic engineering microorganisms of S-adenosylmethionine.

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.

Provided are a (-)-germacene A synthetase and a preparation method therefor, which effectively achieve the synthesis of (+)-β-elemene.

The invention relates to a series of GDP-fucose synthase polypeptides which can be applied to synthesis of GDP-fucose which is a precursor of human milk oligosaccharides (HMOs), and belongs to the technical field of enzyme engineering. In certain embodiments, the polypeptide capable of catalyzing the conversion of GDP-4-keto-6-deoxymannose into GDP-fucose comprises a polypeptide of which the amino acid sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% identical to the amino acid sequence of SEQ ID NO: 1 and / or the amino acid sequence of the polypeptide M1-14. The invention provides a series of polypeptides for synthesizing GDP-fucose.

The invention provides a method for producing borneol, the method comprising culturing the transgenic microorganism in a culture medium, and the microorganism comprises or expresses borneol diphosphate synthetase and phosphatase.

The invention relates to the field of bioengineering, in particular to an enzyme composition, a product and application of the enzyme composition in preparation of Ectoine. The invention provides an enzyme composition. The enzyme composition comprises a mutant of 2-hydroxy-4-aminobutyric acid oxidase, a mutant of keto transaminase, a mutant of hydroperoxide hydrolase, a mutant of Ectoine synthetase, a mutant of pyruvic acid-CoA oxidase and lactobacillus helveticus lactic dehydrogenase. According to the method disclosed by the invention, a plurality of mutant enzymes are utilized for combined transformation for the first time, and cheap 2-hydroxy-4-aminobutyric acid is taken as a starting raw material to quickly and efficiently produce Ectoine; the enzyme method has the characteristics of easiness in control, short conversion time, few generated impurities and the like, so that the method has unique advantages in the aspects of low-cost and large-scale mass production of Ectoin.

The present invention relates to a biofermentation method of tagatose, and in particular to a tagatase enzyme for catalytic synthesis at high temperature and its application. The enzyme comprises tagatose six phosphate synthase and tagatose six phosphate phosphatase, wherein the amino acid sequence of the tagatose six phosphate synthase is shown in SEQ ID NO.1, and the amino acid sequence of the tagatose six phosphate phosphatase is shown in SEQ ID NO.5. The tagatase enzyme provided by the present invention has higher temperature stability and is more conducive to improving the efficiency of converting starch into tagatose.

The invention discloses a yarrowia lipolytica engineering strain with high yield of resveratrol as well as construction and application of the yarrowia lipolytica engineering strain, and belongs to the technical field of synthetic biology and metabolic engineering. The invention provides a yarrowia lipolytica engineering strain. A 4-coumaric acid-CoA ligase gene, a multi-copy resveratrol synthase gene, a transketolase 1 gene, a fusion gene of a histidine phosphate aminotransferase gene and a tyrosine ammonia lyase gene, a glycolytic pathway hexokinase gene, a 6-phosphofructokinase-1 gene and a phosphoglycerate kinase gene are integrated, so that the recombinant protein is obtained. According to the present invention, by using glucose as the substrate, the high yield of resveratrol can be achieved by using glucose as the substrate, the resveratrol yield during shake-flask culture fermentation can achieve 3.157 g / L, the resveratrol yield can be increased to 30.7 g / L during 5L-scale fed-batch fermentation, and the resveratrol yield can be increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L when the resveratrol yield is increased to 30.7 g / L; the maximum value is reported by the yarrowia lipolytica system at present.

The invention provides application of acidophilous glycosyltransferase in salidroside production, and belongs to the technical field of biological engineering. The problem of producing salidroside under the acidic condition is solved. Comprising an application of acidophilous glycosyl transferase with an amino acid sequence as shown in SEQ ID NO.1 in salidroside production under an acidic condition and an acidophilous escherichia coli engineering strain for producing salidroside. The escherichia coli engineering strain overexpresses a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthetase gene aroGfbr, a cyclohexadiene dehydrogenase gene tyrC, a glucose phosphate mutant enzyme gene pgm and a UDP-glucose pyrophosphorylase galU, overexpresses a phenylpyruvate decarboxylase gene ARO10 derived from saccharomyces cerevisiae, and can be used for producing a mutant 3-deoxy-D-arabinoheptulose-7-phosphate mutant enzyme. The kit comprises an ethanol dehydrogenase gene ADH6 and a glycosyl transferase gene LrUGT85AF8. The method is mainly used for producing salidroside under an acidic condition.

The application discloses a Weizmannia coagulans with fat-reducing and weight-reducing functions, and belongs to the technical field of microorganisms. The Weizmannia coagulans screened by the application can inhibit the content and activity of fat synthesis enzyme on the basis of reducing blood fat and weight, can relieve liver damage caused by high-fat diet, has excellent acid tolerance, bile salt tolerance and high fat hydrolysis capacity, and is a multifunctional Weizmannia coagulans, and has a wide application prospect in the preparation of fat-reducing, weight-reducing and weight-losing products.

The invention discloses construction and application of a saccharomyces cerevisiae engineering strain for synthesizing vanillin by taking ferulic acid as a substrate, and belongs to the technical field of genetic engineering. The invention provides a recombinant saccharomyces cerevisiae, which is characterized in that a series of genes for producing vanillic acid and vanillyl alcohol from vanillin through oxidation and reduction are knocked out, and feruloyl coenzyme A synthetase FCS and p-hydroxycinnamyl coenzyme A hydratase ECH which are obtained by screening from a metagene library in a West Sichuan hot spring extreme environment are overexpressed. According to the engineering strain VAN002 with high yield of vanillin, the accumulation of vanillin reaches 16.8 g / L, and the molar conversion rate of catalyzing ferulic acid to synthesize vanillin reaches 93%, which is the highest level reported by synthesizing vanillin by saccharomyces cerevisiae at present.

To identify lipid metabolites that control Th17 cell differentiation and lipid biosynthetic enzymes involved therein, and based on that, to provide a screening method for a therapeutic agent for diseases caused by excessive Th17 cell responses.SOLUTION: Provided is 1-oleoyl-lysophosphatidylethanolamine (LPE(1-18:1)) as a lipid metabolic product that controls Th17 cell differentiation. Since LPE(1-18:1) binds to RORγt with high specificity and promotes differentiation into Th17 cells, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained by selecting a compound that inhibits the binding of LPE(1-18:1) to RORγt. Also provided are five lipid metabolic enzymes (Gpam, Gpat3, Lplat1, Pla2g12a, and Scd2) that are responsible for the biosynthesis of LPE(1-18:1) and control Th17 cell differentiation. By selecting a compound that inhibits the activity or expression of these lipid metabolic enzymes, a candidate compound for a therapeutic agent for diseases caused by excessive Th17 cell responses can be obtained.SELECTED DRAWING: None

The present invention provides modified aminoacyl-tRNA synthetases (ARSs) having increased reactivity with N-methyl amino acids compared to natural aminoacyl-tRNA synthetases. The modified aminoacyl-tRNA synthetases according to the present invention can aminoacylate tRNAs with their corresponding N-methyl-substituted amino acids such as N-methyl-phenylalanine, N-methyl-valine, N-methyl-serine, N-methyl-threonine, N-methyl-tryptophan and N-methyl-leucine more efficiently than natural aminoacyl-tRNA synthetases. The present invention enables a more efficient production of polypeptides containing N-methyl amino acids.

The present invention belongs to the field of cellulose synthesis, and specifically relates to a heterologous expression Escherichia coli biosynthesis system of cellulose synthase subunits PtCesA1 and PtCesA8 and its application, including a heterologous expression vector of cellulose synthase subunit PtCesA1 or a heterologous expression vector of cellulose synthase subunit PtCesA8; and a heterologous cell containing the above vector. PtCesA1 and PtCesA8 are expressed in heterologous cells respectively to form cellulose synthase, which is used to catalyze the synthesis of cellulose microfibrils. The present invention realizes the biosynthesis of cellulose in vivo, and directly synthesizes a cellulose product with high purity similar to plants and free of lignin and hemicellulose from the bacteria by heterologously expressing plant cellulose synthase subunits in Escherichia coli.

The invention discloses cloning and functional identification of a liverwort flavone synthetase gene, and belongs to the technical field of gene engineering. According to the invention, one flavone synthase I (flavones synthase I, FNS I) is obtained through screening, and is named as MpFNS I; a prokaryotic expression system is utilized, an MpFNS I recombinant protein is obtained through purification, and the result of in-vitro enzyme activity identification shows that an MpFNS I enzyme substrate is relatively wide in selectivity, has a flavone synthetase function and also has a part of flavonol synthetase function. The liverwort flavonoid has a significant difference on affinity and catalytic efficiency of different substrates, the in-vivo function of the liverwort flavonoid is verified by utilizing heterologous expression of arabidopsis thaliana, experimental evidence is provided for comprehensively analyzing a biosynthetic pathway of the liverwort flavonoid, and meanwhile, a research target is provided for deeply researching a mechanism for synthesizing and responding to UV-B radiation of the liverwort flavonoid.

The present invention provides modified arninoacyl-tRNA synthetases (ARSs) having increased reactivity with N-methyl amino acids compared to natural aminoacyl-tRNA synthetases. The modified aminoacyI-tRNA synthetases according to the present invention can aminoacylate tRNAs with their corresponding N-methyl-substituted amino acids such as N-methyl-phenylalanine, N-methyl-valine, N-methyl-serine, N-methyl-threonine, N-methyl-tryptophan, and N-methyl-leucine more efficiently than natural aminoacyl-tRNA synthetases. The present invention enables a more efficient production of polypeptides containing N-methyl amino acids.

The invention discloses a method for gene modification of aureobasidium pullulans, which belongs to the technical field of gene engineering, and comprises the following steps: determining a malic acid synthase gene, obtaining a malic acid synthase gene segment, searching a gene sequence of the malic acid synthase in the disclosed aureobasidium pullulans as a template on NCBI (National Center of Biotechnology Information), and carrying out gene modification on the malic acid synthase gene segment to obtain a malic acid synthase gene segment; the method comprises the following steps: respectively designing an upstream primer and a downstream primer, taking extracted genome DNA of aureobasidium pullulans M1 as a template, respectively carrying out PCR amplification and recombinant plasmid pUC57-PMA-ms construction and linearization treatment on the upstream primer and the downstream primer, and introducing the plasmid into the aureobasidium pullulans M1 to obtain an aureobasidium pullulans recombinant strain; according to the method, the polymalic acid synthetase gene (PMA-ms) on an aureobasidium pullulans genome is knocked out from aureobasidium pullulans, the yield of pullulan in the obtained recombinant plasmid M1-2 is about 39.11 g / L, and compared with an original strain M1, the yield of M1-2 is increased by 9.79 g / L, and the practical application prospect of the method is improved.

The invention relates to the technical field of enzyme engineering, and discloses a pantothenic acid synthetase mutant and application thereof. According to the invention, site-specific mutagenesis is carried out on 62nd, 123rd and 189th amino acids of pantothenic acid synthase from escherichia coli, and site-specific and directed mutagenesis is carried out through an alanine scanning strategy near an active pocket to change protein amino acid residues; the 62nd phenylalanine is mutated into isoleucine and / or the 123rd amino acid arginine is mutated into glutamine and / or the 189th arginine is changed into isoleucine, so that the efficiency of catalyzing the D-panthenol by the pantothenic acid synthetase is improved, and the yield of the D-panthenol is increased. Finally, when the sites are mutated at the same time, recombinant escherichia coli of the pantothenic acid synthetase can improve the efficiency of catalyzing synthesis of D-panthenol by the pantothenic acid synthetase by 1.32 times, and the yield of a genetic engineering strain carrying the constructed pantothenic acid synthetase mutant in shake flask fermentation of panthenol is improved by 3.65 times.