29 results about "Mutase" patented technology

The invention provides a uracil production strain and a construction method and application thereof.According to the strain, on an E.coli UR14 genome by means of a CRIPSR / Cas9 gene editing technology, firstly, psuG genes, preTA genes, rutA genes and upp genes are knocked out, so that decomposition of uracil is blocked; then, a uridine phosphorylase gene udp and a pyrimidine-5 '-nucleotide nucleotidase gene ppnN are subjected to overexpression, and synthesis and accumulation of uracil are synergistically enhanced; and finally, overexpression of the ribose phosphate mutase gene pgm further enhances the conversion of a by-product ribose phosphate 1-precursor 5-ribose phosphate 1-pyrophosphate and improves the carbon utilization rate, and the obtained strain has good genetic stability and high fermentation yield, can stably produce uracil, and has wide application prospects.

The invention belongs to the technical field of biochemical engineering, and discloses a production method for improving the content of odd-chain fatty acid in grease by modifying schizochytrium limacinum and optimizing a substrate and application of the method. According to the method, schizochytrium limacinum HX-308 serves as a receptor strain, high-yield odd-chain fatty acid engineering bacteria are obtained through gene overexpression and inhibition, and the high-yield odd-chain fatty acid is obtained. Accumulation of a propionyl CoA precursor pool for synthesizing odd-chain fatty acid is improved, endogenous methylmalonyl-CoA mutase coding gene MUT is inhibited, so that a competitive pathway of the odd-chain fatty acid is inhibited, and then through substrate optimization, the content of fermented grease and the content of the odd-chain fatty acid are obviously improved. The expression of related enzymes in schizochytrium limacinum is adjusted through a genetic engineering strategy to improve the odd-chain fatty acid synthesis, the synthesis and utilization of the key intermediate propionyl coenzyme A in the odd-chain fatty acid synthesis process are increased, the metabolic pathway of the propionyl coenzyme A is inhibited, and substrate optimization is assisted to improve the odd-chain fatty acid yield.

A device for fermenting vegetables, fruits, and herbs that increases the enzyme activity of superoxide dismutases, comprising a fermentation vessel, a raw material processing system, a bacterial culture system, a mycelium and köji culture vessel, and a central control unit, each connected to the fermentation vessel, wherein the central control unit is electrically connected to the raw material processing system, the bacterial culture system, and the mycelium and köji culture vessel, respectively, wherein the raw material processing system first introduces the treated fruits, vegetables, or herbs into the fermentation vessel, and the bacterial culture system is controlled by the central control unit such that different bacterial strains are introduced into the fermentation vessel according to the different fermentation stages.After completion of the fermentation process, the central control unit also controls the mycelium and köji culture containers for different fermentation stages, ensuring that the mycelium and köji are introduced into the fermentation container and, to guarantee submerged fermentation over time, the fermentation container is finally left undisturbed.

The invention relates to the technical field of preparation of L-tyrosine, and discloses a method for enhancing generation of L-tyrosine in nicotiana benthamiana and application. In order to relieve product negative feedback inhibition of a key enzyme in a production path of L-tyrosine in tobacco and to improve the yield of L-tyrosine in tobacco, DAHPS enzyme derived from microorganisms is subjected to L175Q site mutation and then is connected with a signal peptide AtRs1A; shikimic acid mutase / prebenzoate dehydrogenase TyrAfbr and tyrosine aminotransferase TyrB which are derived from microorganisms are introduced into the tobacco chassis after being connected with the signal peptide Rs1A, the expression has orthogonality, and transient expression is performed in the Bensi tobacco plant chassis by utilizing agrobacterium infection, so that the synthesis capability of L-tyrosine in tobacco chassis cells can be improved; a large amount of L-tyrosine can be directly generated in the tobacco chassis plastid.

The invention discloses a method for distinguishing a vibrio alginolyticus wild type virulent strain and a cya gene deletion type low virulent strain based on proteomics. According to the method, a peak area ratio of ATP synthase subunit alpha (atpA) to phosphopentose mutase (deoB) in vibrio alginolyticus, which is measured by proteomics, is utilized to judge an adenylate cyclase coding gene cya-deleted attenuated strain and a wild virulent strain, the vibrio alginolyticus cya gene deleted attenuated strain with a result of atpA / deoB in a sample to be detected being less than 0.2, and the vibrio alginolyticus cya gene deleted attenuated strain with a result of atpA / deoB in the sample to be detected being less than 0.2 is a wild virulent strain with a result of atpA / deoB in the sample to be detected being less than 0.2. The vibrio alginolyticus strain with the result of atpA / deoB being 1.2 or above is a vibrio alginolyticus wild type virulent strain. The distinguishing method is easy to operate, high in detection speed and reliable in result.

The application provides a myo-inositol-3-phosphate synthase mutant, which is obtained by mutating a myo-inositol-3-phosphate synthase with an amino acid sequence as shown in SEQ ID NO: 4 at positions 55, 57 and 273 respectively. The application also provides a preparation method of myo-inositol. The inventors have screened a wild-type myo-inositol-3-phosphate synthase and a myo-inositol monophosphatase with good performance, which can be used in cooperation with a glucan phosphorylase and a glucose phosphate mutase to catalyze starch to generate myo-inositol more efficiently. The myo-inositol-3-phosphate synthase mutant is applied to in-vitro non-fermentation biosynthesis of myo-inositol, combined with myo-inositol monophosphatases E1 and E5, and in a way of feeding whole cells, so that starch can be converted into myo-inositol in a one-pot method, without using NAD + coenzymes, and has a good industrial application prospect.

Provided are methods for producing polysaccharides in bacteria by expressing in a bacterium one or more coding sequences selected from the group consisting of a pglF dehydrogenase coding sequence, a wbpP UDP-N-acetyl-d-glucosamine C4 epimerase coding sequence, a wcfR aminotransferase coding sequence, and a wcfS phospho-glycosyltransferase coding sequence, a wcfQ glycosyltransferase coding sequence, a wcfO pyruvyltransferase coding sequence, a wcfP glycosyltransferase coding sequence, a wcfM UDP-galactopyranose mutase coding sequence, a wcfN glycosyltransferase coding sequence, a wza polysaccharide export protein coding sequence, a wzx fippase coding sequence, a wzy polymerase coding sequence, and a wzz coding sequence, wherein at least one of the coding sequences is heterologous to the bacterium. Also provided are expression cassettes with one or more of the disclosed coding sequences, recombinant bacteria that harbor one or more of the expression cassettes, and methods for producing immunogenic compositions using the polysaccharides produced by the recombinant bacteria.

Methods of treating propionic acidemia (PA), methylmalonic acidemia (MMA) and fatty acid oxidation disorders are described. The methods include administering an anaplerotic agent that can directly enter the tricarboxylic acid cycle, such as a succinate derivative or pro-drug, for example trisuccinylglycerol (TSG). Methods of restoring tricarboxylic acid (TCA) cycle function in a cell deficient for propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT) by contacting the cell with a succinate derivative or pro-drug, such as TSG, are also described.

The invention belongs to the technical field of plant genetic engineering, and relates to an application of a Xanthomonas oryzae hexose phosphate mutase encoding gene in the aspect of adjusting the susceptibility of cruciferous plant arabidopsis thaliana. According to the present invention, the recombinant plasmid is constructed, the Xanthomonas oryzae pv.oryzae (Xoo) PXO99A hexose phosphate mutase coding gene PXO03174 is adopted as the target gene to be introduced into the cruciferous plant arabidopsis thaliana, such that the condition that the plant with the overexpression of the gene is sensitive to the infection of the pathogenic bacterium Pseudomonas syringae pv.tomato (Pst) DC3000 is found, and the significantly enhanced disease infection characteristic is represented; according to the present invention, the discovery can be used for developing the sensitive bacterial disease detection method so as to improve the accuracy of bacterial disease detection; the method can also be used for identifying toxic genes of plant pathogenic bacteria such as pathogenic bacteria sensitive plants and pseudomonas syringae and researching the mechanism of the infected plants, and can also be used for researching the disease resistance of the plants to the pseudomonas syringae and developing antibacterial agents.

The application discloses a kind of recombinant escherichia coli for producing L-tyrosine and application, belong to genetic engineering and bioengineering technical field.The application knocks out aroP and tyrP gene, expresses the yddG gene of escherichia coli endogenous to modify the aromatic amino acid transport system of escherichia coli, then heterologous expression comes from bifidobacterium fpk and expresses the ppsA and tktA gene of escherichia coli endogenous to increase the precursor supply of shikimic acid pathway, again through the anti feedback gene expression aroG and tyrA 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase, branch acid mutase and prephenate dehydrogenase are removed feedback inhibition, increase the carbon flux of shikimic acid pathway.Knock out tyrR gene removes the repression of TyrR protein to shikimic acid pathway key enzyme, again knock out trpE and pheA gene to block partial side reaction of shikimic acid pathway, so that the synthesis flux of L-tyrosine increases.Finally knock out endogenous poxB gene to realize the modification of acetic acid pathway, realizes the stable fermentation performance of high glucose concentration.

This invention discloses a recombinant glucuronide C5 epimerase mutant, expression vector, recombinant expression engineered bacteria, and their culture and induction methods, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (N29–N617) mutation (V173R–V197K–A239V–N269E) with an N-terminal maltose-binding protein (MBP) tag to a human glucuronide C5 epimerase (C5-epi). The recombinant glucuronide C5 epimerase mutant exhibits higher activity, 2.1 times that of the recombinant wild-type, and greater stability, retaining 72.5% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant glucuronide C5 epimerase is increased by 4-fold.

The invention relates to the technical field of biological catalysis, and provides a method for catalytic synthesis of trehalose-6-phosphoric acid, key parameters in a multi-enzyme cascade catalytic reaction system for catalytic synthesis of trehalose-6-phosphoric acid are adjusted, and in the catalytic system, the enzyme activity concentration of maltose phosphorylase is 80-120U / mL, the enzyme activity concentration of maltose phosphorylase is 20-30U / mL, and the enzyme activity concentration of maltose phosphorylase is 20-30U / mL. The enzyme activity concentration of the beta-glucophosphate mutase is 15-35 U / mL, the enzyme activity concentration of the trehalose-6-phosphate phosphorylase is 3-15 U / mL, and the yield of the trehalose-6-phosphate can be remarkably improved by adjusting a corresponding production process, so that an enzyme dosage combination with higher universality and high efficiency is obtained, and the method is suitable for industrial production. The conversion rate from the substrate to the target product trehalose-6-phosphoric acid can be up to 96%, the operation is simple and convenient, the cost is low, and the efficient and low-cost industrial production of the trehalose-6-phosphoric acid is realized.

The invention discloses a preparation method and application of a genetically modified cell drug for expressing MCM, and relates to the field of cell therapy, in particular to a preparation method and application of a genetically modified cell drug for expressing MCM. The methylmalonyl-coenzyme A mutase gene MUT is introduced into cells through a gene delivery vector, then multiplication culture is carried out in vitro, normal MCM can be expressed, and meanwhile, the invention discloses application of the methylmalonyl-coenzyme A mutase gene modified cell medicine in methylmalonic acidmia treatment. According to the invention, normal MCM is expressed through cells, MMA pathogenic factors are radically solved, the cell drug provided by the invention can be used as a drug factory for producing MCM, MCM can be continuously expressed for a long time, the defects of short MCM protein half-life period and frequent lifelong medication in the existing methylmalonic acidmia treatment method are solved, and the method is suitable for clinical application. And a new thought is provided for treatment of genetic diseases caused by other gene mutations.

This invention discloses a glucose phosphate mutase gene Rk PGM Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence encoded by this gene is shown in SEQ ID NO:2; this gene was isolated from *Rhodotorula rubrum* (…). Rhodosporidium kratochvilovae YM25235 was used to ligate the gene to a vector and transfected into Rhodotorula rubrum cells. Experimental results showed that overexpression of Rk... PGM The gene will increase the level of extracellular polysaccharide synthesis in this strain; this invention modifies microorganisms through genetic engineering to improve their ability to secrete extracellular polysaccharides, providing a theoretical basis for increasing the yield of extracellular polysaccharides in Rhodotorula rubrum.

The invention provides an inositol-3-phosphate synthase mutant which is obtained by taking inositol-3-phosphate synthase with an amino acid sequence as shown in SEQ ID NO: 4 as a parent and respectively mutating at a 55th site, a 57th site and a 273th site. The invention also provides a preparation method of inositol. Wild inositol-3-phosphate synthetase and inositol monophosphate which are good in performance are obtained through screening by the inventor, and the wild inositol-3-phosphate synthetase and inositol monophosphate can cooperate with glucan phosphorylase and glucophosphate mutase to catalyze starch to generate inositol more efficiently. The inositol-3-phosphate synthase mutant is applied to in-vitro non-fermentation biosynthesis of inositol, is combined with inositol monophosphates E1 and E5, can efficiently catalyze high-concentration starch to be converted into inositol by a one-pot method in a whole-cell feeding manner, does not need to use coenzymes such as NAD < + >, and has a good industrial application prospect.

The present application discloses mutant enzymes of sucrose phosphorylase, glucose isomerase and kojibiose synthase, fermentation production methods thereof and applications in synthesizing kojibiose. The amino acid sequence of the sucrose phosphorylase mutant enzyme is shown in SEQ.ID.NO.1, the amino acid sequence of the glucose isomerase mutant enzyme is shown in SEQ.ID.NO.3, and the amino acid sequence of the kojibiose synthase mutant enzyme is shown in SEQ.ID.NO.5. The method for synthesizing kojibiose comprises the following steps: adding sucrose phosphorylase mutant enzyme, glucose isomerase mutant enzyme and kojibiose synthase mutant enzyme to sucrose, heating the solution to 40°C and stirring slightly, and completing the reaction after at least 4 hours. Based on the two phosphorylases, the present invention further improves the substrate selectivity and product generation direction through the transformation of specific properties. At the same time, glucose isomerase is used to carry out the mutual conversion between fructose and glucose, so as to realize the effective conversion between sucrose and kojibiose.

The invention relates to the technical field of microorganisms, and particularly discloses application of phosphomannose mutase mutated microorganisms in fermentation production of L-threonine. The research finds that after the phosphomannose mutase of the microorganism is mutated, the yield of threonine can be increased, and then the application of the microorganism with the mutated phosphomannose mutase in fermentation production of L-threonine is provided. The mutation of the phosphomannose mutase means that glutamic acid is mutated into lysine at the 34th site in the protein sequence of the phosphomannose mutase. The invention provides a novel method for fermentation production of threonine.

The invention discloses a genetically engineered bacterium for producing monensin and application of the genetically engineered bacterium, and belongs to the technical field of metabolic engineering. The invention provides a recombinant cinnamon streptomyces capable of increasing the yield of monensin, the recombinant cinnamon streptomyces simultaneously overexpresses phosphofructokinase (pfk), citrate synthase (cs), methylmalonyl coenzyme A isomerase (mutB) and isobutyryl coenzyme A mutase (icm), and the result shows that the recombinant cinnamon streptomyces overexpresses acetyl coenzyme A synthesis and energy supply of the monensin. According to the method, synthesis precursors (methylmalonyl CoA and ethylmalonyl CoA) are increased, the yield of monensin can be increased without increasing the supply of the precursors, the yield can reach 20.4 g / L, the production cost is effectively reduced, and the production benefit is improved.

An aspect provides a vaccine composition for preventing tuberculosis comprising chorismate mutase. The vaccine composition alone may induce immunity specific to Mycobacterium tuberculosis, and when provided together with an immune adjuvant, the vaccine composition may induce immunity specific to tuberculosis more effectively. Furthermore, when an existing vaccine for tuberculosis is used as a prime and the vaccine composition according to an aspect including chorismite mutase is provided as a booster, the immunity specific to tuberculosis may be induced significantly more effectively.

The present invention relates to a mutant enzyme of santalene synthase and its application in synthesizing santalene. Specifically involved mutant enzymes are SanSyn (S533A), SanSyn (S533Q), and SanSyn (Q527A & S533A), which are respectively mutated to alanine, glutamine, glutamine at position 533, and mutated to alanine at position 527 and serine at position 533. A method for synthesizing α-santalene in host Escherichia coli DH5α using glucose as a carbon source mainly includes constructing recombinant plasmids pETDuet-SanSyn (S533A), pETDuet-SanSyn (S533Q), pETDuet-SanSyn (Q527A & S533A), and pMVA; constructing a recombinant strain of Escherichia coli, and further fermenting and culturing. The technical solution involved in the present invention significantly increases the yield of α-santalene, and the protein soluble expression is also improved. It lays a foundation for further metabolic engineering of Escherichia coli to produce α-santalene. Provide a reference for the modification of terpene synthase.

The present invention provides a yeast genetic engineering bacterium for producing resveratrol and a construction method and application thereof. The engineering bacterium overexpresses resveratrol synthase on the chromosome of Saccharomyces cerevisiae BY4742. STS , p-Coumaryl-CoA ligase 4CL , phenylalanine / tyrosine ammonia lyase TAL , cinnamate-4-hydroxylase C4H , cytochrome P450 enzyme reductase CPR1 , DAHP synthase mutant ARO4 , chorismate mutase mutant ARO7 Chorismate synthase ARO2 shikimate kinase AroL , acetyl-CoA synthetase ACS and malonyl-CoA synthetase ACC1 , and knock out bypass genes CIT2, LPP1, DPP1, and PDC6 at the same time, to achieve biosynthesis of resveratrol using glucose as a substrate. The present invention utilizes phenylalanine / tyrosine ammonia lyase to react with phenylalanine and tyrosine as substrates to generate key precursors for resveratrol synthesis, strengthens the precursors required for resveratrol synthesis, and greatly increases the yield of the product resveratrol.

The invention discloses a multienzyme system, a preparation method thereof and application of the multienzyme system in inositol preparation. According to the preparation method of the inositol disclosed by the invention, maltodextrin is converted into the inositol by using a multi-enzyme system; the multi-enzyme system is prepared from alpha-glucan phosphorylase, glucose phosphate mutase, inositol-1-phosphate synthetase and phytase. The multi-enzyme system provided by the invention is used for preparing the inositol, and has the advantages of low cost of raw materials and enzymes, short reaction time, few byproducts, high product purity, high yield and high yield.

The invention discloses an N-acetylglucosamine transferase mutant and application thereof in production of L-tryptophan, and belongs to the technical field of genetic engineering. In order to solve the problems of yield limitation and the like caused by excessive consumption of phosphoenolpyruvic acid in the production of L-tryptophan, the invention weakens a phosphoprotein coding gene crr on escherichia coli; meanwhile, a uridine diphosphate galactose pyranose mutase gene glf and a glucokinase gene glk of the pseudomonas mobilis and an endogenous galactose H (+) transporter enzyme gene galP of escherichia coli are integrated at a pflB site, and a genetically engineered bacterium is obtained; mutagenesis is carried out on the basis to obtain a mutant strain with improved L-tryptophan yield; and the N-acetylglucosamine transferase mutants nagC and nagE are obtained by virtue of sequencing. Fermentation proves that overexpression of the mutant nagC or nagE or simultaneous overexpression of the mutants nagC and nagE in the genetically engineered bacteria can improve the yield of L-tryptophan.

The application discloses a kind of recombinant escherichia coli and its construction method and application, the recombinant escherichia coli can be used for the production of L-phenylalanine.The application utilizes multiple rounds of mutagenesis screening to have high resistance to L-phenylalanine structural analogues, and has tyrosine auxotrophic mutant strain, and is regulated in the form of temperature-sensitive recombinant plasmid to code 3-deoxy-7-phosphoheptanoate synthase and branch acid mutase gene expression in L-phenylalanine synthesis pathway.The recombinant escherichia coli constructed in the application, in 5L fermenter, according to the cell growth stage adopts different temperature control and feeding mode, fermentation time is 56 h, under the condition that L-phenylalanine production is 62 g / L, sugar acid conversion rate is 25.2%, has the prospect and value of industrialization application.

The present disclosure is directed to compositions and methods for the treatment of Metabolic Liver Disorders. The compositions and methods can comprise an adeno-associated virus (AAV) piggyBac polynucleotide comprising a transgene. The transgene may comprise ornithine transcarbamylase (OTC) or methylmalonyl-CoA mutase (MUT1).

The present invention provides, among other things, methods and compositions for treating methylmalonic academia (MMA) based on mRNA therapy. The compositions used in treatment of MMA comprise an mRNA comprising a methymalonyl-CoA mutase (MUT) coding sequence and are administered at an effective dose and an administration interval such that at least one symptom or feature of MMA is reduced in intensity, severity, or frequency or has a delayed onset. mRNAs with optimized MUT coding sequences are provided that can be administered without the need for modifying the nucleotides of the mRNA to achieve sustained in vivo function.

The invention discloses a novel method for synthesizing vidarabine by an enzyme method. The method comprises the following steps: connecting three genes for coding ribokinase, phosphopentose mutase and purine nucleoside phosphorylase in escherichia coli to a pET28a plasmid, and then transferring into host escherichia coli in which adenine is knocked out and converted into hypoxanthine; the obtained recombinant strain can be used for efficiently synthesizing vidarabine by directly utilizing adenine, D-arabinose and ATP (adenosine triphosphate) as substrates. Compared with the existing enzymatic conversion method, the method disclosed by the invention has the advantage that the production cost is obviously reduced.

The present invention belongs to the technical field of plant genetic engineering and relates to a Xanthomonas oryzae ( Xanthomonas oryzae ) Application of hexose phosphate mutase encoding gene in regulating the susceptibility of cruciferous plant Arabidopsis thaliana, the present invention constructs a recombinant plasmid and combines Xanthomonas oryzae Xanthomonas oryzae pv. rice ( Xoo )PXO99 A Hexose phosphate mutase encoding gene PXO_03174 As a target gene, it was introduced into the cruciferous plant Arabidopsis thaliana, and it was found that plants overexpressing this gene were resistant to the pathogenic bacteria Pseudomonas syringae. Pseudomonas syringae pv. tomato ( Psst ) DC3000, showing significantly enhanced susceptibility. The findings of this invention can be used to develop more sensitive bacterial disease detection methods, thereby improving the accuracy of bacterial disease detection. They can also be used to prepare pathogen-sensitive plants, identify toxic genes of plant pathogens such as Pseudomonas syringae, and study the mechanisms of plant infection. They can also be used to study plant resistance to Pseudomonas syringae and develop antibacterial agents.

The invention discloses dsRNA (double-stranded ribonucleic acid) for preventing and treating plant diseases as well as a preparation method and application of the dsRNA. The nucleotide sequences of the eight dsRNAs are as shown in SEQ ID NO. 1 to SEQ ID NO. 8. The dsRNA provided by the invention can accurately target a key gene of phytopathogen, namely a UDP-galactopyranose mutase gene, and the mutase is a synthetase of a main component UDP-galactofuranose of a fungal cell wall. By interfering the expression of the gene, the growth and reproduction of pathogenic bacteria are effectively inhibited, so that the aim of preventing and treating plant diseases is fulfilled. Compared with a traditional prevention and treatment method, the dsRNA provided by the invention can play a role more efficiently, and does not generate the problems of drug resistance and the like. Besides, the dsRNA is environment-friendly, does not cause pollution and damage to soil, water sources and ecological systems, and is beneficial to maintenance of agricultural ecological balance and sustainable development.