32 results about "Phosphotransferase" patented technology

The invention discloses an attenuated salmonella typhimurium EN-VNP (Enhanced Natriuretic Peptide) strain as well as a preparation method and application of the attenuated salmonella typhimurium EN-VNP strain. Comprising the following steps: on the basis of a salmonella VNP20009 strain, flagellin genes fliC and fljB, a phosphoethanolamine transferase gene eptA, a 4-amino-4-deoxy-L-arabinose transferase gene arnT and phosphotransferase yeiU are sequentially knocked out or silenced, and a strain is formed after a phosphotransferase gene lpxE is knocked in or overexpressed, so that the attenuated salmonella typhimurium EN-VNP strain is prepared. According to the attenuated salmonella typhimurium EN-VNP with low toxicity and the T cell adjuvant effect and an EN-OMV antigen delivery system prepared from the attenuated salmonella typhimurium EN-VNP, an improved strain does not contain flagellin, an LPS molecular structure is changed, the attenuated salmonella typhimurium EN-VNP has the low toxicity and the T cell adjuvant effect, and normal multiplication culture can be achieved.

The invention discloses recombinant escherichia coli for producing N-acetylneuraminic acid as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of genetic engineering. According to the invention, 6-phosphofructokinase is knocked out, glycerol kinase is introduced into a glycerol kinase mutant, a diffusion transporter is promoted by glucose to replace PTS phosphotransferase I, and a promoter is optimized, so that recombinant escherichia coli can grow by double carbon sources of glucose and glycerol, N-acetylneuraminic acid is synthesized, and meanwhile, the generation amount of acetic acid is extremely low. On the basis, a 6-phosphofructokinase coding gene pfkB is knocked out, a heterologous ED pathway from pseudomonas mobilis is introduced, and the yield under the pore plate level is further increased to 9.44 g / L through copy number optimization and RBS strength optimization.

The invention discloses a production method of N-acetylneuraminic acid based on artificial double carbon sources, and belongs to the technical field of genetic engineering. Escherichia coli with high yield of N-acetylneuraminic acid as a carbon source is taken as an original strain, 6-phosphofructokinase is knocked out, glycerol kinase is knocked out, a glycerol kinase mutant is introduced, PTS phosphotransferase I is replaced by glucose-promoted diffusion transporter, and a promoter is optimized, so that the N-acetylneuraminic acid with high yield is obtained. The recombinant escherichia coli can grow with glucose and glycerol as double carbon sources, N-acetylneuraminic acid is synthesized, the yield under the pore plate level reaches 9.2 g / L, meanwhile, the generation amount of acetic acid is extremely low, and a new direction is provided for large-scale production of N-acetylneuraminic acid.

Provided herein are compositions and methods of using a bicistronic vector for treating or preventing a lysosomal storage disorder (LSD) in a subject. The disclosed compositions comprise a bicistronic vector comprising a promoter, an Internal Ribosome Entry Site (IRES), a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). The present methods comprise administering to the subject a pharmaceutical composition comprising the bicistronic vector as disclosed herein.

The invention discloses a construction method of an escherichia coli mutant for producing succinic acid by fermentation of a synthetic medium. The method comprises the following steps: firstly, knocking out a lactic dehydrogenase gene ldhA, a pyruvate formate lyase gene pflB, a ptsG gene responsible for encoding a phosphotransferase system EIIBC protein, an ethanol dehydrogenase gene adhE, an acetokinase-phosphate transacetylase gene ackA-pta, and a ptsG gene responsible for encoding a phosphotransferase system EIIBC protein in escherichia coli; a phosphoenolpyruvate carboxykinase gene pck from bacillus subtilis is integrated at an SS9 safety site of a strain to obtain escherichia coli ESC6 with high succinic acid yield; and mutating one or more loci in one or more genes of a glucose-transcriptional inhibition factor gene mlc, a nitrate response regulatory factor gene narL and a cyclic adenylate receptor protein gene crp to obtain the escherichia coli mutant capable of producing succinic acid by fermentation of a synthetic culture medium, wherein the one or more loci in one or more genes of the glucose-transcriptional inhibition factor gene mlc, the nitrate response regulatory factor gene narL and the cyclic adenylate receptor protein gene crp are mutated. The strain can be fermented in a synthetic medium to produce succinic acid, so that the fermentation cost is greatly reduced, and the strain has a very wide application prospect.

The invention discloses a salmonella typhimurium EN-VNP strain based on flagellin and LPS attenuation as well as a preparation method and application of the salmonella typhimurium EN-VNP strain, and particularly relates to the technical field of biology. The bacterial strain is formed by sequentially knocking out or silencing flagellin genes fliC and fljB, a phosphoethanolamine transferase gene eptA, a 4-amino-4-deoxy-L-arabinose transferase gene arnT and phosphotransferase yeiU on the basis of a salmonella VNP 20009 bacterial strain, and knocking in or overexpressing a phosphotransferase gene lpxE. The salmonella typhimurium EN-VNP strain disclosed by the invention has relatively high safety, the attenuated strain does not contain flagellin, and a lipid A molecule of LPS only retains one phosphate group, so that the safety of the strain is improved, and the strain is more suitable for serving as a vaccine and a drug delivery carrier.

The invention provides a multi-enzyme composition and a method for carbon sequestration synthesis of 3-hydracrylic acid, the multi-enzyme composition is composed of acetyl-CoA ligase, acetyl-CoA carboxylase, malonyl-CoA reductase and polyphosphoric acid-AMP phosphotransferase, and can be used for producing 3-hydracrylic acid, and the specific method is that acetic acid which is cheap and easy to obtain is used as a substrate; the 3-hydracrylic acid is prepared through carbon sequestration by means of in-vitro multienzyme driven by the light engine, the yield of the 3-hydracrylic acid is increased by adjusting the adaptability of the system, and the method is low in production cost, simple in step and environmentally friendly.

The present invention relates to a novel and efficient method for the sialylation of a glycoside, the method comprising mixing said glycoside with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: at least one polypeptide having cytidine monophosphate kinase activity, at least one polypeptide having N / -acylneuraminate citydyltransferase activity, and at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside.

The invention discloses a method for efficiently detecting various microbial limits of tobramycin, belongs to the technical field of microbial detection, and initiates an enzyme-chemical synergistic neutralization system constructed by aminoglycoside-3-acetyltransferase, aminoglycoside-3 '-phosphotransferase, magnesium chloride hexahydrate and a sodium chloride-peptone buffer solution. According to the system, the bacteriostatic activity of tobramycin can be thoroughly eliminated through a direct enzymatic inactivation way, and the limitation that neutralization is not thorough in a traditional method is broken through. Based on the system, the efficient detection method is successfully developed by combining process optimization of adding bacteria and adding a passivating enzyme and a chemical neutralizer into a culture medium. The method has four core advantages of high efficiency, specificity, environmental protection and synergy, can thoroughly eliminate antibacterial interference of tobramycin, and remarkably improves the accuracy and reliability of a microbial limit test result.

The invention provides a reagent and a kit for detecting ADP (adenosine diphosphate), a detection method of ADP and application of the detection method, and belongs to the technical field of ADP detection. The reagent for detecting the ADP comprises ATP sulfating enzyme, pyrophosphatase, an enzyme inhibitor, invertase for catalyzing the ADP into ATP, a substrate of the invertase, bioluminescent enzyme and a substrate of the bioluminescent enzyme, wherein the enzyme inhibitor inhibits the activity of the ATP sulfating enzyme and the pyrophosphatase. According to the present invention, the ADP detection accuracy and the ADP detection sensitivity are high, the fluorescence signal is stable, and the reagent can be used for detecting the enzyme activity of phosphotransferase and / or ATP hydrolase.

The present invention relates to a novel and efficient method for sialylation of a glycoside comprising mixing said glycoside with sialic acid, cytidine monophosphate, nucleoside triphosphate and one or more cell-free extracts of a microorganism, the present invention relates to a microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein the one or more cell-free extracts comprise: at least one polypeptide having cytidine monophosphate kinase activity, at least one polypeptide having N-acyl neuraminic acid cytidyltransferase activity, and at least one polypeptide having sialyltransferase activity, thereby sialylating the glycoside.

The invention discloses a construction method and application of escherichia coli for producing a heparin precursor by using cane sugar. The invention provides a method for constructing a recombinant strain, which comprises the following steps: overexpressing a heterologous sucrose-6-phosphohydrolase gene sacA and a sucrose phosphorylase gene spI in escherichia coli to obtain an engineering strain which can grow by using sucrose as a carbon source and can effectively synthesize a heparin precursor; a sucrose specific enzyme IIBC component gene sacP of a phosphotransferase system and a sucrose-6-phosphohydrolase gene sacA are co-expressed, and recombinant bacteria can more efficiently synthesize a heparin precursor in shake-flask culture, so that the yield of the heparin precursor is increased. The recombinant strain constructed by the invention has a good application prospect.

The invention discloses a phosphotransferase mutant with improved catalytic activity, and belongs to the field of biology and food. On the basis of a phosphotransferase female parent sequence, the 72nd site, the 104th site and the 136th site are substituted. The enzyme activity of the constructed mutant is improved from 46.2 U / mg to 213.2 U / mg in a whole-cell catalytic enzyme activity test of inosine compared with that of a female parent mature enzyme. The phosphotransferase variant disclosed by the invention benefits from the improved catalytic activity, is more beneficial to stable application in a harsh industrial environment, can be used for producing food flavoring agents such as disodium inosinate and the like, and is widely applied to the fields of food and biology.

This invention relates to the field of biotechnology, and discloses an engineered *Yarrowia lipolytica* strain that produces p-coumaric acid using glucose as a substrate, its construction method, and its applications. Construction method: A tyrosine ammonia-lyase gene is integrated using a CRISPR / Cas9 localization and integration method. TAL In *Yarrowia lipophila* strains, the DAHP synthase gene was enhanced. ARO4 , DHS1 and AROG Overexpression of tyrosine synthase TYR Genes and histidine phosphotransferases HIS5 The protease gene was further integrated to synthesize exogenous phenylalanine deamination and hydroxylation pathway genes for p-coumaric acid, including phenylalanine ammonia-lyase gene, cinnamate hydroxylase gene, and P450 reductase gene. The p-coumaric acid produced by the engineered *Yarrowia lipolytica* strain of this invention can reach a maximum yield of 1.7 g / L in shake flasks, and a yield of 30 g / L in a 5 L fed-batch fermentation tank, demonstrating significant industrial application value.

The invention discloses an anti-streptomycin gene as well as an encoding protein and application thereof, and belongs to the technical field of phytopathology. The invention discloses a novel streptomycin resistance gene aphh (3) derived from tomato canker pathogen. The gene is located on a newly found plasmid pCM3 of tomato canker pathogen, and is used for coding a protein APH (3) belonging to an aminoglycoside antibiotic phosphotransferase family. The functions of the gene are verified through tests of gene knockout, function complementation, heterologous expression and the like, meanwhile, mass spectrometric analysis proves that APH (3) can catalyze streptomycin molecules to generate phosphorylation reaction to generate streptomycin monophosphate, so that the inhibitory activity on bacteria is lost, and a strain carrying the gene has remarkable resistance to streptomycin. The streptomycin resistance gene aphh (3) can be widely applied to the fields of pathogenic bacterium resistance mechanism research, resistance strain screening, molecular marker selection and the like, and has important scientific research and application values.

The invention discloses an OMV vaccine capable of preventing group B streptococcus III serotype infection and a preparation method thereof. The invention belongs to the field of synthetic biology, and particularly relates to an OMV vaccine capable of preventing group B streptococcus III serotype infection and a preparation method of the OMV vaccine. The recombinant escherichia coli contains a lipoid A deacylase coding gene, a lipoid A dephosphatase coding gene and a group B streptococcus capsular polysaccharide length control gene coding gene; the gene does not contain an acetyl glucosamine phosphotransferase coding gene, an escherichia coli O antigen synthesis gene cluster coding gene, a lipoid A myristoyl transferase coding gene, an escherichia coli O antigen chain length control gene coding gene, a lipoid A palmitoyl transferase coding gene and an escherichia coli sialic acid degradation gene cluster coding gene. The composition also contains a group B streptococcus III capsular polysaccharide synthesis cluster coding gene. The recombinant escherichia coli can be used for producing an OMV vaccine for preventing group B streptococcus III serotype infection.

The present invention provides a polypeptide that blocks the PTS pathway and its use. In vitro binding experiments demonstrate that phage-displayed peptide 1C6 binds to HPr in a dose-dependent manner. 1C6 blocks the PTS pathway, specifically the EI-HPr interaction and phosphotransferase. This suggests that peptide 1C6 has potential antibacterial properties.

Provided are compositions comprising vectors for the co-expression of a modified GlcNAc-1-Phosphotransferase gene and a lysosomal enzyme. The gene encoding the lysosomal enzyme is operably linked to a first promoter and the gene encoding the GlcNAc-1-Phosphotransferase is operably linked to a second promoter. Also provided herein are methods of treating a lysosomal storage disorder comprising administering to a subject the compositions of the disclosure.

The application discloses an L-homoserine phosphotransferase mutant, a coding gene, a plasmid, a genetically engineered bacterium and application, and belongs to the technical field of bioengineering, wherein a cysteine at the 223th position of L-homoserine phosphotransferase is mutated into a glycine single mutation or a double mutation of mutating isoleucine at the 180th position into serine on the basis of the single mutation, namely the L-homoserine phosphotransferase mutant, a corresponding coding gene is obtained, and then a corresponding plasmid and genetically engineered bacterium are obtained; the enzyme mutant not only has high enzyme activity, but also is resistant to feedback inhibition of alpha-ketoglutarate; compared with simply improving enzyme activity, the application can further promote the synthesis of L-homoserine by affecting the reversible reaction direction. The application is suitable for applying the enzyme mutant to industrial fermentation production of L-homoserine, can significantly improve production efficiency, and can improve the yield of L-homoserine by about 60% by adding L-glutamine.

This invention discloses a streptomycin resistance gene, its encoded protein, and its applications, belonging to the field of plant pathology technology. This invention discovers a novel streptomycin resistance gene derived from *Tomato Canker*. aph(3) This gene is located on a newly discovered plasmid pCM3 of *Tomato Canker*, encoding a protein APH(3) belonging to the aminoglycoside antibiotic phosphotransferase family. This invention verified the function of this gene through gene knockout, functional complementation, and heterologous expression experiments. Simultaneously, mass spectrometry analysis demonstrated that APH(3) catalyzes the phosphorylation of streptomycin molecules, generating streptomycin monophosphate, thereby eliminating its inhibitory activity against bacteria. Strains carrying this gene exhibit significant resistance to streptomycin. This invention relates to a streptomycin resistance gene. aph(3) It can be widely used in the study of pathogen resistance mechanisms, screening of resistant strains, and selection of molecular markers, and has important scientific research and application value.

The invention provides a phosphoketolase mutant, a related product of the phosphoketolase mutant and application of the phosphoketolase mutant in production of acetyl phosphate, acetyl coenzyme A and / or 3-hydracrylic acid. The phosphoketolase mutant provided by the invention has relatively high catalytic capability on hexanediose and pentose, and can catalyze hexanediose and pentose to be converted into acetyl phosphoric acid at relatively high efficiency, so that subsequent products acetyl coenzyme A and 3-hydracrylic acid are efficiently generated; therefore, the method has great application potential in production of acetyl coenzyme A and products (such as 3-hydracrylic acid) taking acetyl coenzyme A as a precursor substance.

The present invention discloses an aminoglycoside 2'-phosphotransferase mutant and its application, relating to the field of biotransformation technology, including the following method for preparing the aminoglycoside 2'-phosphotransferase mutant: S1, cloning and expressing the aminoglycoside 2'-phosphotransferase; S2, purification of the aminoglycoside 2'-phosphotransferase; S3, determination of the enzyme activity and substrate specificity of the aminoglycoside 2'-phosphotransferase; S4, construction of a single-point mutant; S5, construction, expression and screening of an AphIf mutant library. The present invention obtains an aminoglycoside 2'-phosphotransferase mutant capable of specifically degrading GTP without degrading UTP through cloning, expression, purification and screening, which can solve the problems of difficult separation and purification of GTP in the UTP production process, high GTP impurity content in the UTP production process, and low UTP yield and purity.

The invention belongs to the technical field of biology, and particularly relates to a recombinant bacterium for producing a heparin precursor as well as a construction method and application of the recombinant bacterium. According to the invention, by knocking out or reducing a phosphoenolpyruvate sugar phosphotransferase PTS system of a heparin precursor producing strain, glucose transport, glycolysis and TCA circulation rate are balanced, and generation of by-products acetic acid and lactic acid is reduced, so that more carbon metabolism enters a heparin precursor synthesis pathway, and the yield of heparin precursor is improved. 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 / or conversion rate of the heparin precursor can be further improved.

The present invention provides non-natural microorganisms containing enzymatic pathway and / or metabolic modifications for increasing carbon flux by acetyl-CoA or by oxaloacetic acid and acetyl-CoA. Embodiments of the invention include a microorganism having a pathway for obtaining acetyl-CoA and oxaloacetic acid (PK pathway) comprising a phosphoketolase. The microorganism also has (i) a genetic modification that enhances the sugar uptake activity of a non-phosphotransferase (non-PTS) system, and / or (ii) a genetic modification in the electron transport chain (ETC) of the microorganism that increases the efficiency of ATP production that increases the availability of reducing equivalents, or both. The microorganism may optionally include (iii) a genetic modification that maintains, attenuates, or eliminates the sugar uptake activity of a phosphotransferase system (PTS). The increased carbon flux by acetyl-CoA and oxaloacetic acid may be used for the production of biologically derived compounds, and the microorganisms may further include pathways capable of producing biologically derived compounds.

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a lactobacillus engineering bacterium with improved stress resistance as well as a preparation method and application thereof. In order to improve the stress resistance of lactic acid bacteria, a genetic engineering technology is utilized, and an Hpr protein for regulating and controlling an intracellular phosphotransferase system of the lactic acid bacteria is over-expressed in the lactic acid bacteria, so that the lactic acid bacteria engineering bacteria with improved stress resistance are prepared. Compared with wild type lactococcus lactis, the survival rate of the lactobacillus engineering bacterium L.lactis (ptsH) prepared by the invention under the acid stress condition that the pH value is 5.0 is increased to 43.95% from 4.49%, and the survival rate is increased by 9.79 times; under the stress of 20% NaCl (v / v), the survival rate is increased to 7.62% from 0.19%, and the survival rate is increased by 40.10 times. Therefore, when applied to industrial production and facing various stress environments, the engineering lactobacillus prepared by the invention has a huge application prospect.

The present application relates to the field of agricultural biotechnology, and in particular to a method for constructing an engineered strain of Rhodopseudomonas palustris for producing inositol using glucose, glycerol and CO2 as carbon sources, and the strain. The present application mutates the gene nagE encoding N-acetylglucosamine-specific phosphotransferase system in the genome of Rhodopseudomonas palustris (G265R), and knocks out the gene nagR encoding a transcriptional regulator of the GntR family, so that Rhodopseudomonas palustris can efficiently utilize glucose. On this basis, an engineered strain of Rhodopseudomonas palustris H16 is constructed for synthesizing inositol using renewable substrates (glucose and glycerol) and CO2 as carbon source. Based on this technology, not only the synthesis of bioactive substances can be achieved, but also a method for efficiently eliminating greenhouse gases in the atmosphere.

Provided are amino acid sequences for modified, truncated forms of human GlcNA-1-Phosphotransferase (PTase) that retain phosphotransferase activity and the ability to phosphorylate proteins, lysosomal or non-lysosomal. Truncated forms of PTase lacking or with modified linkers and / or lacking the C-terminal transmembrane and cytosolic domain are demonstrated to retain phosphotransferase activity and the ability to phosphorylate target proteins.

The invention provides a method for synthesizing p-hydroxybenzaldehyde from p-hydroxybenzoic acid through light-driven enzyme catalysis. A used reaction system comprises carboxylic acid reductase (CAR), a photosynthesis unit, ferredoxin (Fdx) and phosphotransferase (PAP). According to the method, light energy is utilized to drive spinach capsule membranes to co-regenerate NADPH and ATP, and the problem that carboxylic acid reductase needs coenzyme supply of NADPH and ATP at the same time is solved. According to the method, a light energy driven enzyme catalysis method is adopted, a biological photosynthetic reaction is used for replacing a traditional high-energy-consumption and high-pollution chemical process, and green and efficient synthesis of p-hydroxybenzaldehyde is achieved through cooperation of double-coenzyme light regeneration and enzyme specific catalysis.

The application discloses a recombinant escherichia coli for producing N-acetylneuraminic acid as well as a construction method and application thereof, and belongs to the technical field of genetic engineering. The recombinant escherichia coli can grow in glucose and glycerol double carbon sources, synthesize N-acetylneuraminic acid and has extremely low acetic acid generation by knocking out 6-phosphofructokinase, introducing a glycerol kinase mutant, replacing PTS phosphotransferase I with a glucose-promoted diffusion transporter and optimizing a promoter. On this basis, the application further knocks out the 6-phosphofructokinase coding gene pfkB, introduces a heterologous ED pathway from Pseudomonas aeruginosa, and further improves the yield under the hole plate level to 9.44 g / L through copy number optimization and RBS intensity optimization.

The invention discloses recombinant corynebacterium glutamicum for high yield of N-acetylglucosamine, which is characterized in that corynebacterium glutamicum ATCC13032 is used as an original strain; an N-acetylglucosamine-6-phosphate deacetylase encoding gene nagA, a glucosamine-6-phosphate deaminase encoding gene nagB, a glucose-6-phosphate 1-dehydrogenase encoding gene zwf, a lactic dehydrogenase encoding gene ldhA, an N-acetylmannosamine-6-phosphate 2-epimerase encoding gene nano E, a glucosamine-6-phosphate deacetylase encoding gene nagA, a glucosamine-6-phosphate deacetylase encoding gene nagB, a glucose-6-phosphate 1-dehydrogenase encoding gene zwf, a lactic dehydrogenase encoding gene ldhA, a lactic dehydrogenase encoding gene ldhA, a an N-acetylglucosamine specific phosphotransferase encoding gene cgl2642 and a glucose transporter encoding gene ptsG are used as the specific gene of the N-acetylglucosamine specific phosphotransferase; the invention also discloses a method for preparing the recombinant glucosamine-6-phosphate acetyltransferase gene, and expresses an exogenous glucosamine-6-phosphate acetyltransferase coding gene GNA1, an exogenous glutamine-fructose-6-phosphate transaminase coding gene glmS, an exogenous glucose transporter coding gene galP and an exogenous glucose transporter coding gene glf.