30 results about "Glutamate kinase" patented technology

The invention discloses recombinant corynebacterium glutamicum and application thereof in production of L-glutamine, and belongs to the technical field of bioengineering. The activity of gamma-glutamate kinase, glutamic acid secretion mechanical channel protein and adenosine acylase of glutamine synthetase in cells of the recombinant corynebacterium glutamicum is lost, and plasmid pXMJ19 is used for co-expressing the glutamine synthetase from saccharomyces cerevisiae and polyphosphate kinase from escherichia coli. The invention provides the recombinant corynebacterium glutamicum CGQ03 / pXMJ19-glnASc-ppkEc capable of producing the L-glutamine at high yield; the recombinant corynebacterium glutamicum is inoculated into a 5L fermentation tank and is fermented for 66h, so that the yield of the L-glutamine in fermentation liquid reaches 73.5 + / -3.1g / L and the conversion rate of glucosamine reaches 0.368 + / -0.034.

The invention discloses a method for fermentation production of L-proline by utilizing recombinant Escherichia coli. The recombinant Escherichia coli carries recombinant plasmid with gamma-glutamyl kinase genes with site mutagenesis and tryptophan series promoters, wherein the recombinant plasmid is obtained by connecting the cloned gamma-glutamyl kinase genes and the tryptophan series promoter in the lab to the same plasmid and performing whole plasmid PCR site-specific mutagenesis. The invention also discloses an application of the Escherichia coli in production of L-proline.

The invention discloses an N-acetylgutamate kinase mutant with improved catalytic efficiency and thermal stability and belongs to the field of biological engineering. Gene of N-acetylgutamate kinase from Corynebacterium crenatum SYPA5-5 is subjected to site-specific mutagenesis so that coding amino acid sequence of this gene is mutated at 91-th site into histidine H by phenylalanine F. The N-acetylgutamate kinase mutant described herein is 2.12 times as high as a wild one in catalytic efficiency, and has half-life period that is 2.78 times as high as pre-mutation period. The N-acetylgutamate kinase mutant obtained herein is more satisfactory to the need for production of arginine and lays basis for efficient synthesis of L-arginine.

The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, and / or alleviating a limitation such that yield is increased, or a combination of these and that have been transformed using a novel vector construct including a synthetic N-acetyl glutamate kinase (NAGK) gene that modulates nitrogen use in plants. The invention also includes the overexpression and enzymatic characterization of an arginine-insensitive NAGK isolated from a bacterial strain that improves stress tolerance and nitrogen uptake, metabolism or both. In various embodiments, the vector construct includes one or more nucleic acid sequences including SEQ ID NO: 1. The invention also relates to isolated vectors for transforming plants and to antibodies used for detecting transformed plants. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants or are modulated by nitrogen conditions.

The invention provides a Corynebacterium glutamicum for producing L-proline, and a method for producing L-proline by using the strain. The L-proline-producing Corynebacterium glutamicum constructed by the present invention, wherein proline dehydrogenase / pyrrole-5-carboxylate dehydrogenase PutA is inactivated, glutamate kinase ProB, glutamate-5- Enhanced activity of semialdehyde dehydrogenase ProA, pyrrole-5-carboxylate dehydrogenase ProC, pyruvate carboxylase Pyc, glyceraldehyde-3-phosphate dehydrogenase GapN, L-proline efflux protein ThrE or SerE, The L-glutamate efflux protein MscCG is inactivated, and the L-proline yield, transformation rate and production intensity of the obtained strain are significantly improved compared with the starting strain, which can reduce the production cost of L-proline.

The invention discloses an N-acetylgutamate kinase mutant with improved catalysis efficiency and belongs to the field of biological engineering .A gene of N-acetylgutamate kinase coming from Corynebacteriaum crenatum SYPA5-5 is subjected to site-directed mutation, so that the amino acid sequence encoded by the gene is mutated from isoleucine I to valine V at the 74th site .Catalysis efficiency (Kcat / Km) of the N-acetylgutamate kinase mutant is improved to 843 mM<-1>min<-1> from 513 mM<-1>min<-1> of a wild type, and is 1.64 times of the catalysis efficiency before mutation .Heat stability of the mutant is also improved .The N-acetylgutamate kinase mutant better meets production requirements of arginine, and a foundation is laid for efficient synthesis of L-arginine.

The invention discloses a high-producing strain for the cyclic lipopeptide antibiotic--himastatin and a construction method thereof. The high-producing strain for himastatin is constructed by inactivating the hmtA gene or hmtB gene of S. himastatinicus sp.Nov, so the high-producing mutant strain S. himastatinicus sp.Nov for himastatin is obtained, wherein the nucleotide sequence of the hmtA gene is as shown in SEQ ID No. 1 and the nucleotide sequence of the hmtB gene is as shown in SEQ ID No. 3. According to the invention, the hmtA-inactivated mutant strain S. himastatinicus sp.Nov Ju2014 and the hmtB-inactivated mutant strain S. himastatinicus sp.Nov Ju2015 are respectively obtained by inactivating the Mer-family transcriptional regulation gene hmtA and the acetyl glutamic acid kinase gene hmtB at the upper stream of the biosynthesis gene cluster of the cyclic lipopeptide antibiotic--himastatin of S. himastatinicus sp.Nov. The himastatin output of the mutant strain S. himastatinicus sp.Nov Ju2014 and the mutant strain S. himastatinicus sp.Nov Ju2015 is more than 10 times that of a wild strain S. himastatinicus sp.Nov. Thus, successful construction of the two high-producing mutant strains for the cyclic lipopeptide antibiotic--himastatin enables acceleration of the industrialization process of himastatin to be possible and hopeful.

The invention discloses a method for knocking out argCJBD to increase the yield of acetylglucosamine of recombinant Bacillus subtilis, belonging to the field of genetic engineering. The invention uses the recombinant Bacillus subtilis BSGN6-PxylA-glmS-P43-GNA1 as the starting strain, and knocks out the gene encoding acetylglutamate semialdehyde dehydrogenase (argC) and the gene encoding ornithine acetyltransferase through homologous recombination (argJ), acetylglutamate kinase encoding gene (argB) and acetylornithine transaminase encoding gene (argD), block the pathway of host bacteria producing arginine from glutamic acid, and promote the conversion of glutamic acid into glutamic acid The reaction of amide, and glutamine is the amino donor for the synthesis of acetylglucosamine, thereby increasing the production of acetylglucosamine. During the shake flask fermentation process using semi-synthetic medium, the yield of acetylglucosamine of the recombinant Bacillus subtilis with knockout of argCJBD reached 6.4 g / L, which was 16.3% higher than that before knockout. It laid the foundation for further metabolic engineering of Bacillus subtilis to produce glucosamine.

The invention provides an N-acetyl glutamic acid kinase mutant capable of relieving the feedback inhibition effect of L-arginine and obviously increasing the catalytic activity and thermal stability and belongs to the field of bioengineering. A molecular docking technique and the structure analysis for N-acetyl glutamic acid kinase (CcNAGK) from corynebacterium crenatum CGMCC NO:0890 are firstly adopted for confirming that the E19 locus plays an important role in inhibiting the feedback of arginine and three key amino residue loci I74, F91 and K234 located on a substrate combining bag have significant impacts on enzymatic catalytic activity and heat stability; the encoded amino acid is replaced and the gene argB4 after compound mutation is introduced into the corynebacterium crenatum highly yielding arginine through pDXW10, so that the compounding rate of the arginine in corynebacterium crenatum can be obviously increased; lastly, after fermentation for 98h in a 5L fermentation tank, the yield of arginine is increased to 61.2g / L from original 43.2g / L, and the yield of the L-arginine is increased by 41.8%.

It is intended to provide a polypeptide having (i) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by substitution of one or more amino acids in the amino acid sequences of from the 20- to the 38-th positions from the N end, or (ii) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by substitution of one or more amino acids in the amino acid sequences of from the 20- to the 38-th positions from the N end and deletion, substitution or addition of one or more amino acids in the amino acid sequence of from the first to the 19-th positions or from the 39-th to the 294-th positions, and having an N-acetylglutamate kinase activity.

The invention relates to a method for improving the yield of arginine by the mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase. L-arginine is one of semi-essential basic amino acids for a human body, and has various particular physiology and pharmacology functions. High-yield arginine mutant strain C. crenatum SYPA5-5 is compounded into the arginine through a circulating way, andN-acetyl glutamic acid kinase (NAGK) is a key enzyme in the compounding way and is subject to the feedback inhibition of the product arginine. By using an overlapping PCR (Polymerase Chain Reaction) technique, a GAT (glycerol phosphate acyl transferase) codon used for coding aspartic acid is used for substituting a GGA (General Gonadotropic activity) codon used for coding glycine at the site 287 in the coding NAGK albumen, and the NAGK which has high activity and arginine with obviously reduced by the feedback inhibition can be obtained after the mutation. An argBSD gene is brought into the Corynebacterium crenatum of the high-yield arginine through pJCl-tac, and the expression volume of the key enzyme is further improved. The final acid yield is increased to 36.3g / L from original 28g / L, and the yield of the L-arginine is increased by 29.7 percent.

The invention relates to a recombination strain for producing trans-4-hydroxy-L-proline and building and application of the recombination strain and belongs to the technical field of genetic engineering. The recombination strain for producing the trans-4-hydroxy-L-proline is identified as escherichia coli HJ which is preserved in China Center for Type Culture Collection on September 17th, 2015, and the preservation number of the escherichia coli HJ is CCTCC No. M2015550. The recombination strain has the advantages that wild glutamate kinase controlled by feedback regulation is used to enhance expression, three to-be-expressed genes and expression elements such as related initiators are integrated to the chromosome of the escherichia coli, the to-be-expressed genes can be constantly replicated along with the replication of the chromosome, extremely-high genetic stability is kept, the problem of plasmid loss is solved, effective fermentation cycle is prolonged, and the yield of hydroxyproline is increased.

The invention provides an N-acetyl glutamic acid kinase mutant capable of relieving the feedback inhibition effect of L-arginine and obviously increasing the catalytic activity and thermal stability and belongs to the field of bioengineering. A molecular docking technique and the structure analysis for N-acetyl glutamic acid kinase (CcNAGK) from corynebacterium crenatum CGMCC NO:0890 are firstly adopted for confirming that the E19 locus plays an important role in inhibiting the feedback of arginine and three key amino residue loci I74, F91 and K234 located on a substrate combining bag have significant impacts on enzymatic catalytic activity and heat stability; the encoded amino acid is replaced and the gene argB4 after compound mutation is introduced into the corynebacterium crenatum highly yielding arginine through pDXW10, so that the compounding rate of the arginine in corynebacterium crenatum can be obviously increased; lastly, after fermentation for 98h in a 5L fermentation tank, the yield of arginine is increased to 61.2g / L from original 43.2g / L, and the yield of the L-arginine is increased by 41.8%.

The invention discloses an N-acetylgutamate kinase mutant with heat stability improved remarkably and belongs to the field of bioengineering .The gene of N-acetylgutamate kinase from Corynebacterium crenatum SYPA5-5 is subjected to site-specific mutagenesis, so that lysine K at the site 234 of the encoded amino acid sequence is mutated into threonine T .the half-life period of the N-acetylgutamate kinase mutant at 37 DEG C is prolonged to be 112 h from 36 h of a wild type N-acetylgutamate kinase mutant, which is 3.11 times of the length before mutation .The N-acetylgutamate kinase mutant can better meet the production requirement of arginine, and lays a foundation for efficient synthesis of L-arginine.

The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, and / or alleviating a limitation such that yield is increased, or a combination of these and that have been transformed using a novel vector construct including a synthetic isocitrate dehydrogenase (icdh) gene that modulates nitrogen use in plants. The invention also relates to stacking the icdh gene with other exogenous or heterologous genes that modulate nitrogen use in the plant, including a N-acetylglutamate kinase gene. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants or are modulated by nitrogen conditions.

The invention discloses engineering bacteria for producing trans-4-hydroxy-L-proline and a construction method and an application thereof. The construction method of the engineering bacteria provided by the invention includes the steps of A1) and A2): A1) introducing a L-proline-4-hydroxylase gene, a glutamate-5-kinase gene and a glutamate-5-semialdehyde dehydrogenase gene into a receptor cell; and A2) knocking an alpha-ketoglutaricdehydrogenase gene, an isocitratlyase gene or a proline dehydrogenase gene of the receptor cell out, or replacing a pyruvic oxidase gene of the receptor cell with an acetylcoenzyme A synthetase gene; and carrying out a reaction of the recombinant cell and a substrate to obtain the trans-4-hydroxy-L-proline. Experiments prove that the production method of the trans-4-hydroxy-L-proline can be used for production of the trans-4-hydroxy-L-proline.

The invention discloses an N-acetylgutamate kinase mutant with improved catalytic efficiency and thermal stability and belongs to the field of biological engineering. Gene of N-acetylgutamate kinase from Corynebacterium crenatum SYPA5-5 is subjected to site-specific mutagenesis so that coding amino acid sequence of this gene is mutated at 91-th site into histidine H by phenylalanine F. The N-acetylgutamate kinase mutant described herein is 2.12 times as high as a wild one in catalytic efficiency, and has half-life period that is 2.78 times as high as pre-mutation period. The N-acetylgutamate kinase mutant obtained herein is more satisfactory to the need for production of arginine and lays basis for efficient synthesis of L-arginine.

The invention discloses an N-acetylgutamate kinase mutant with improved catalysis efficiency and belongs to the field of biological engineering .A gene of N-acetylgutamate kinase coming from Corynebacteriaum crenatum SYPA5-5 is subjected to site-directed mutation, so that the amino acid sequence encoded by the gene is mutated from isoleucine I to valine V at the 74th site .Catalysis efficiency (Kcat / Km) of the N-acetylgutamate kinase mutant is improved to 843 mM<-1>min<-1> from 513 mM<-1>min<-1> of a wild type, and is 1.64 times of the catalysis efficiency before mutation .Heat stability of the mutant is also improved .The N-acetylgutamate kinase mutant better meets production requirements of arginine, and a foundation is laid for efficient synthesis of L-arginine.