47 results about "Aspartate kinase" patented technology

An L-amino acid is produced by culturing a Methylophilus bacterium which can grow by using methanol as a main carbon source and has L-amino acid-producing ability, for example, a Methylophilus bacterium in which dihydrodipicolinate synthase activity and aspartokinase activity are enhanced by transformation through introduction into cells, of a DNA coding for dihydrodipicolinate synthase that does not suffer feedback inhibition by L-lysine and a DNA coding for aspartokinase that does not suffer feedback inhibition by L-lysine, or a Methylophilus bacterium made to be casamino acid auxotrophic, in a medium containing methanol as a main carbon source, to produce and accumulate an L-amino acid in culture, and collecting the L-amino acid from the culture.

This invention relates to a nucleic acid fragment encoding a plant 5-methyltetra-hydropteroyltriglutamate-homocysteine methyltransferase or methionine synthase. The invention also includes chimeric genes, a first encoding a plant methionine synthase (MS) gene, a second encoding a plant cystathionine γ-synthase (CS) gene, a third encoding feedback-insensitive aspartokinase (AK) or bifunctional feedback-insensitive aspartokinase-homoserine dehydrogenase (AK-HDH), which is operably linked to a plant chloroplast transit sequence, and a fourth encoding a methionine-rich protein, all operably linked to plant seed-specific regulatory sequences. Methods for their use to produce increased levels of methionine in the seeds of transformed plants are provided.

Disclosed is a strain of Escherichia sp., capable of producing O-acetyl homoserine in high yield, with the introduction and enhancement therein of the activity of: homoserine acetyl transferase, aspartokinase and homoserine dehydrogenase; and at least one enzyme selected from a group consisting of phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate semi-aldehyde dehydrogenase. Also, a method of producing O-acetyl homoserine using the strain is provided.

The invention provides a method of producing tetrahydropyrimidine by fermenting recombinant corynebacterium glutamicum. The recombinant corynebacterium glutamicum is obtained by the steps of performing overexpression in corynebacterium glutamicum to relieve an aspartokinase gene lysC of feedback inhibition; then replacing a promoter of a dihydropyrimidine dicarboxylic acid synthetase in the recombinant bacteria to weaken the activity of the dihydropyrimidine dicarboxylic acid synthetase; and then transferring tetrahydropyrimidine synthetic path related gene ectABC into the recombinant bacteria to obtain the recombinant corynebacterium glutamicum. The recombinant corynebacterium glutamicum provided by the invention can use different cheap raw materials to produce tetrahydropyrimidine by fermentation under a low salt condition, and cheap corn slurry can be also used as a nutritional component which replaces expensive yeast powder, so that the cost of the raw materials is further lowered. Meanwhile, the recombinant corynebacterium glutamicum provided by the invention solves the problem of biosafety, simplifies the post-extraction process and has a good market application prospect.

This invention relates to four chimeric genes, a first encoding lysine-insensitive aspartokinase (AK), which is operably linked to a plant chloroplast transit sequence, a second encoding lysine-insensitive dihydrodipicolinic acid synthase (DHDPS), which is operably linked to a plant chloroplast transit sequence, a third encoding a lysine-rich protein, and a fourth encoding a plant lysine ketoglutarate reductase, all operably linked to plant seed-specific regulatory sequences. Methods for their use to produce increased levels of lysine or threonine in the seeds of transformed plants are provided. Also provided are transformed plants wherein the seeds accumulate lysine or threonine to higher levels than untransformed plants.

The present invention relates to a microorganism producing L-methionine precursor, O-succinylhomoserine, 1) the homoserine O-succinyltransferase activity (EC2.3.1.46) is introduced and enhanced, wherein the homoserine O-succinyltransferase activity is feed back resistant to methionine; and 2) the aspartokinase or homoserine dehydrogenase activity (EC2.7.2.4 or 1.1.1.3) is enhanced, and a method of producing L-methionine precursor using the microorganism.

Disclosed are a nucleic acid molecule of Corynebacterium glutamicum origin, having an improved promoter activity, which is operably linked to operon encoding aspartate kinase and aspartate semialdehyde dehydrogense, a vector containing the same, a transformant transformed with the vector, and a method for the production of L-lysine using the transformant.

The invention discloses construction and applications of a corynebacterium glutamicum mutant strain for producing L-homoserine, and belongs to the technical field of fermentation engineering. Corynebacterium glutamicum ATCC 13032 is taken as a starting strain to knock out regulatory protein McbR, homoserine kinase, transport protein MetD, phosphoenolpyruvate carboxykinase; the expression of isocitrate dehydrogenase is down regulated; transport protein BrnFE, aspartic semialdehyde dehydrogenase and homoserine dehydrogenase are overexpressed; and the expression of aspartate kinase, pyruvate carboxylase and the aspartate kinase I derived from escherichia coli are enhanced. Shake flask culture can be performed on the mutant strain for 48 h, and the yield of L-homoserine can reach 8.8 g / L.

The present invention discloses a genetically engineered bacterium of corynebacterium glutamicum producing L-isoleucine. ddh gene encoding diaminopimelate dehydrogenase is knocked out in a genome of astarting strain. A nucleotide sequence of the ddh gene is shown as SEQ ID No:2; a lysC operon encoding an aspartokinase is inserted, the lysC operon contains a lysC mutant gene, a 5'-end promoter anda 3' end termination thereof, and a nucleotide sequence of the lysC mutant gene is shown as SEQ ID No:1; and besides, the starting strain is C. glutamicum H5 delta argG delta alaT::ilvC with a preservation number of CCTCC NO:M2016609. The present invention also discloses a preparation method and an application of the genetically engineered bacterium. The production of the L-isoleucine by the genetically engineered bacterium is increased by 5%, synthesis of a by-product lysine of a metabolic branch is reduced by 45.24%, production cost is lowered, and the genetically engineered bacterium has abroad industrial application prospect.

Threonine is an essential amino acid for humans and in the animal feed industry where its levels in feed rations can significantly impact the cost of production of important meat sources, such as swine and poultry. Threonine as well as essential amino acids lysine and methionine are all synthesized via the aspartate family pathway. Aspartate kinase (AK) is the first enzyme in the pathway, and catalyzes the ATP-dependent phosphorylation of aspartate to form ss-aspartyl phosphate. AK constitutes the main regulatory step controlling the metabolic flux through the pathway, and is subject to end product inhibition by Lys and / or Thr. The current invention provides a method to produce a transgenic high free threonine soybean via the overexpression of feedback-resistant AK enzymes in developing soybean plants and seeds. These modifications provide a method to enhance both plant nitrogen metabolism and crop growth performance.

The present invention provides a double gene construct which includes a nucleic acid sequence with a code having active enzyme of aspartokinase (AK) and a nucleic sequence with a code having active enzyme of dihydropyridine dicarboxylic acid synthase. The construct can differently express two genes in plant or part of plant, and can generate lysine and threonine with a level more than five times of the level of wild amino acid.

Provided is a method for efficiently producing a 3-amino-4-hydroxybenzoic acid-type compound by culturing a coryneform bacterium that has a gene encoding a mutated aspartokinase not subject to feedback inhibition, and that is transformed with a recombinant vector containing a DNA encoding a protein having an activity to form 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde.

Process for the production of -lysine by constructing a recombinant microorganism which has a deregulated lysine 2,3-aminomutase gene and at least one deregulated gene selected from the group (i) which consists of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, succinyl-CoA synthetase, methylmalonyl-CoA mutase, provided that if aspartokinase is deregulated as gene (i) at least a second gene (i) other than aspartokinase has to be deregulated, and cultivating said microorganism.

Process for the production of -lysine by constructing a recombinant microorganism which has a deregulated lysine 2,3-aminomutase gene and at least one deregulated gene selected from the group (i) which consists of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diamino-pimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, succinyl-CoA synthetase, methylmalonyl-CoA mutase, provided that if aspartokinase is deregulated as gene (i) at least a second gene (i) other than aspartokinase has to be deregulated, and cultivating said microorganism.

Provided is a method for efficiently producing a 3-amino-4-hydroxybenzoic acid-type compound by culturing a coryneform bacterium that has a gene encoding a mutated aspartokinase not subject to feedback inhibition, and that is transformed with a recombinant vector containing a DNA encoding a protein having an activity to form 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde.

Belonging to the technical field of bioengineering, the invention relates to an aspartokinase mutant, an engineering bacterial strain and a preparation method of the mutant. The high enzyme activity aspartokinase mutant has an amino acid sequence formed by substitution, deletion or addition of 2-10, further 2-6, and even further 2-3 amino acid residues to an exogenous aspartokinase with an amino acid sequence shown as SEQ ID NO:2, and has the functions of aspartokinase. Preferably, the sequence is formed by substitution, deletion or addition of 2 amino acid residues. In particular, preferably,site-specific saturation mutation is carried out to the 372nd and 379th amino acids in the amino acid sequence, and a high throughput screening method is utilized to select the high enzyme activity aspartokinase mutant. The invention further utilizes the electrotransformation method to transfer a pEC-AK recombinant shuttle expression vector of the aspartokinase mutant that has high enzyme activity and can relieve or weaken Lys feedback inhibition effect into Corynebacterium pekinense, finally the recombinant Corynebacterium pekinense M372I-T379S engineering bacterial strain can be obtained, and fermentation product analysis is conducted on the engineering bacterial strain.

The present application relates to an aspartokinase mutant, a microorganism including the mutant, and a method for production of an aspartate-derived L-amino acid or homoserine derivative, using the microorganicm.

The invention relates to a method for constructing recombinant escherichia coli. The method comprises the following steps: knocking out the coding gene lysC of aspartokinase, the coding gene panC of pantothenate synthetase and the coding gene ptsG of glucose transporter EIICBGlc in the escherichia coli; and overexpressing the panD gene with a PL promoter to obtain the recombinant escherichia coli. The invention further provides a method for producing beta-alanine by fermenting the recombinant escherichia coli constructed by the method. The recombinant escherichia coli constructed by the method is used for fed-batch fermentation in an improved inorganic salt culture medium, and has a yield of beta-alanine being 18.4g / L and a production strength being 0.61g / L.h, but the yield of beta-alanine of escherichia coli B0016-050 which is not improved by the method is only 5.2mg / L.

A method for producing L-threonine using a microorganism is provided. In the method, additional one or more copies of each of the phosphoenolpyruvate carboxylase (ppc) gene and the threonine operon are integrated into a particular site of the chromosomal DNA of a microorganism, while its inherent ppc gene and threonine operon remain. Accordingly, two or more ppc genes and threonine operons are included in the chromosomal DNA of the microorganism to thereby enhance the expression of the ppc gene encoding an enzyme to convert phosphoenolpyruvate to a threonine biosynthesis precursor, oxaloacetete, and the genes encoding enzymes involved in the synthetic pathway of threonine from oxaloacetate, including thrA (aspartokinasel-homoserine dehydrogenase), thrB (homoserine kinase), and thrC (threonine synthase), thereby markedly increasing L-threonine productivity.

A method for producing L-threonine using a microorganism is provided. In the method, additional one or more copies of each of the phosphoenolpyruvate carboxylase (ppc) gene and the threonine operon are integrated into a particular site of the chromosomal DNA of a microorganism, while its inherent ppc gene and threonine operon remain. Accordingly, two or more ppc genes and threonine operons are included in the chromosomal DNA of the microorganism to thereby enhance the expression of the ppc gene encoding an enzyme to convert phosphoenolpyruvate to a threonine biosynthesis precursor, oxaloacetete, and the genes encoding enzymes involved in the synthetic pathway of threonine from oxaloacetate, including thrA (aspartokinasel-homoserine dehydrogenase), thrB (homoserine kinase), and thrC (threonine synthase), thereby markedly increasing L-threonine productivity.

Disclosed are a nucleic acid molecule of Corynebacterium glutamicum origin, having an improved promoter activity, which is operably linked to operon encoding aspartate kinase and aspartate semialdehyde dehydrogense, a vector containing the same, a transformant transformed with the vector, and a method for the production of L-lysine using the transformant.

The present invention relates to a transgenic plant comprising one or more plant cells transformed with exogenous nucleic acid encoding a Dunaliella salt-inducible or salt-responsive protein selected from the group consisting of elongation initiation factor 3 (eIF3), NADPH dependent quinone reductase (QOR), aldo-keto reductase (AKR), bifunctional aspartate kinase-homoserine reductase (AK-HSD) and mitochondrial import membrane translocase subunit (TIM9), or a fragment, homolog or variant thereof. The transgenic plant has increased tolerance to salt as compared to a corresponding non-transgenic plant. The present invention further relates to nucleic acids, vectors and constructs encoding the Dunaliella salt-inducible or salt-responsive proteins, and to a method of producing a transgenic plant having an increased tolerance to salt, a method of modifying a plant capacity to survive salt shock, and a method of modifying plant recovery after exposure to salt stress, by introducing the nucleic acids, constructs and / or vectors into one or more cells of the plant.

Belonging to the field of bioengineering, the invention relates to construction and application of C.glutamicum subspecies lactofermentum with high L-methionine yield. The methionine high-yielding recombinant strain C.glutamicum subspecies lactofermentum QW102 / pJYW-4-hom<m>-lysC<m>-brnFE provided by the invention is preserved in China Center for Type Culture Collection with a preservation number of CCTCC M2014232. The recombinant strain has the genetic characteristics of: expression of transport protein BrnFE, expression of aspartokinase fragment lysC and homoserine dehydrogenase fragment hom, and deletion of thrB and mcbR genes on a genome at the same time. According to the invention, by a genetic engineering method, a recombinant strain with high methionine yield is obtained. The invention also provides a method for high yield of L-methionine by the recombinant strain.

A coryneform bacterium harboring an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and comprising an enhanced DNA sequence coding for a dihydrodipicolinate reductase, an enhanced DNA sequence coding for dihydropicolinate reductase, an enhance DNA sequence coding for dihydropicolinate synthase, an enhanced DNA sequence coding for diaminopimelate decarboxylase and an enhanced DNA sequence coding for aspartate aminotransferase; a method for producing L-lysine comprising the steps of cultivating the coryneform bacterium in an appropriate medium to allow L-lysine to be produced and accumulated in a culture of the bacterium, and collecting L-lysine from the culture; and a recombinant DNA usable for production of the coryneform bacterium.

Loline alkaloids (LA), which are 1-aminopyrrolizidines with an oxygen bridge, are produced by Epichloë (anamorph=Neotyphodium) species, endophytes of grasses. LA are insecticidal, thus helping protect host plants from insect herbivory. Suppression subtractive hybridization PCR was used to isolate transcripts up-regulated during loline alkaloid production in cultures of Neotyphodium uncinatum. Subtracted cDNAs were cloned, and a λ-phage cDNA library from an LA-expressing N. uncinatum culture was screened with subtracted cDNA. In BLAST searches, several cDNAs identified had sequence similarities to aspartate kinases, and another with O-acetylhomoserine-(thiol)lyase. Differential expression of these two genes in LA-producing cultures of N. uncinatum was confirmed, and in a survey of 23 isolates from 21 Neotyphodium and Epichloë species these two genes strictly correlated with LA production. Two nucleic acid molecules encoding two loline alkaloid gene clusters have been identified.

The ability and speed with which a coryneform bacterium can produce L-lysine are improved when the coryneform bacterium contains an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized. This is accomplished by successively enhancing the DNA coding for dihydrodipicolinate reductase, the DNA coding for dihydrodipicolinate synthase, the DNA coding for diaminopimelate decarboxylase, and the DNA coding for diaminopimelate dehydrogenase.

The invention discloses a method for culturing a transgenic alfalfa with high sulfur-containing amino acid content and special materials thereof. The method for culturing transgenic alfalfa with changed amino acid content provided by the invention introduces aspartokinase encoding gene and adenylylsulfate reductase encoding gene into receptor alfalfa to obtain transgenic alfalfa with amino acid content changed. The aspartokinase is a protein shown as 65-512th site in an amino acid sequence SEQ ID No.2; and the adenylylsulfate reductase is a protein shown as 65-330th in an amino acid sequence SEQ ID No.4. The methionine content of the transgenic alfalfa with changed amino acid content cultivated by the invention is significantly higher than namely 1.5 times of that of the receptor alfalfa of an untransformed Baoding alfalfa (wild type); cysteine content of a transgenic pCAMBIAK-APR line is significantly higher than namely 1.2 times that of the wild type; aspartic acid content of a transgenic pCAMBIAK-APR line is significantly higher than namely 1.4 times that of the wild type; and lysine content of a transgenic pCAMBIAK-APR line is significantly higher than namely 1.5 times that of the wild type.