27 results about "Aspartate kinase" patented technology

The invention relates to an Escherichia coli engineering strain for producing 2, 3-pentanediol as well as a construction method and application of the Escherichia coli engineering strain, and the method comprises the following steps: constructing recombinant plasmids pACYC-IlvA *-ThrA * BC for expressing an IlvA * gene of threonine deaminase, a ThrA gene of asparaginic acid kinase I, a Thr B gene of homoserine kinase and a ThrC gene of threonine synthetase; the method comprises the following steps: constructing a recombinant plasmid pRSF-Ilv2C-Aro10 for expressing an Ilv2C gene of acetohydroxy acid synthetase and an Aro10 gene of propiophenone dehydrogenase; and chemically converting the recombinant plasmid pACYC-IlvA *-ThrA * BC and the recombinant plasmid pRSF-Ilv2C-Aro10, and transferring the recombinant plasmid pACYC-IlvA *-ThrA * BC and the recombinant plasmid pRSF-Ilv2C-Aro10 into escherichia coli so as to obtain the escherichia coli engineering strain for producing the 2, 3-pentanediol. According to the escherichia coli engineering strain, the biosynthesis of 1.61 g / L of 2, 3-pentanediol is realized by using L-threonine and 40 g / L of glucose.

The invention provides a lysine artificial operon and application thereof. The gene sequence of the artificial operon comprises a promoter, a lysC gene for coding aspartate kinase, an asd gene for coding aspartate semialdehyde dehydrogenase, a dapA gene for coding dihydropicolinic acid synthetase and a dapB gene for coding dihydropicolinic acid reductase, the ddh gene is used for coding diaminopimelate dehydrogenase; the lysA gene is used for coding diaminopimelate decarboxylase; except for the first lysC gene, sequences of RBS ribose binding sites of other genes are RBS ribose binding sites translated after gene transcription by intercepting 20-26nt DNA sequences at the upstream of a coding region ATG (adenosine triphosphate); the RBS ribose binding site is connected in front of the corresponding gene.

The invention provides a beta-alanine producing strain as well as a construction method and application thereof. According to the bacterial strain, an acetaldehyde dehydrogenase gene adhE, a lactic dehydrogenase gene ldhA, an acetokinase gene ackA, a pyruvate dehydrogenase gene poxB, a DNA binding transcription inhibition factor coding gene lacI, an aspartate kinase thrA, a pantothenic acid synthase gene panC, an alanine synthesis transaminase gene cycA and an alanine synthesis transaminase gene yfbQ are knocked out from an E.coli W3110 genome, and the bacterial strain is obtained. A phosphoenolpyruvate carboxylase gene ppc, a pyridine nucleotide transhydrogenase coding gene pntAB, an aspartic acid transaminase gene aspC, an aspartic acid decarboxylase gene panD derived from pseudomonas aeruginosa and a beta-alanine transporter gene NCgl0580 derived from corynebacterium glutamicum are integrated at the same time, and the bacillus subtilis is used for producing beta-alanine and has the advantage of high fermentation yield.

The invention relates to aspartate kinase for relieving feedback inhibition of L-threonine and application of aspartate kinase, and belongs to the field of enzyme engineering and metabolic engineering. The mutant is obtained by carrying out E253K and / or K507E mutation on the basis of wild type aspartate kinase as shown in SEQ ID NO.1, the enzyme activity of the mutant is not obviously changed under the condition that the concentration of L-threonine is 0-12 mmol / L, and the feedback inhibition effect of L-threonine on the mutant is relieved. The method can be widely applied to synthesis of essential amino acids including L-threonine, L-tryptophan, L-isoleucine, L-lysine, L-leucine, L-valine, L-methionine, L-phenylalanine and the like.

The invention relates to the technical field of microorganisms, and particularly discloses a construction method and application of a recombinant microorganism. According to the construction method of the recombinant microorganism, compared with an original strain, the expression quantity of threonine operon leader peptide of the recombinant microorganism is reduced, and the expression quantities of glyceraldehyde-3-phosphate dehydrogenase mutant, aspartate kinase / homoserine dehydrogenase I mutant, homoserine kinase and threonine synthase are increased; the glyceraldehyde-3-phosphate dehydrogenase mutant is characterized in that glycine at the 188th site in a glyceraldehyde-3-phosphate dehydrogenase protein sequence is mutated into serine; the aspartate kinase / homoserine dehydrogenase I mutant is characterized in that glycine at the 433rd site in an aspartate kinase / homoserine dehydrogenase I protein sequence is mutated into arginine; the starting strain is escherichia coli. According to the construction method disclosed by the invention, a novel recombinant microorganism with improved threonine yield can be obtained, and a novel method is provided for fermentation production of threonine.

The invention discloses genetically engineered corynebacterium glutamicum and an application of the genetically engineered corynebacterium glutamicum in preparation of ectoine, and the genetically engineered corynebacterium glutamicum contains an L-diaminobutyric acid transaminase gene ectB, an L-diaminobutyric acid acetyltransferase gene ectA and an ectoine synthetase gene ectC, the invention relates to an aspartic acid transferase gene aspB, an aspartic acid kinase gene lysC and an aspartic acid semialdehyde dehydrogenase gene asd, wherein the nucleotide sequences of the gene aspB, the gene lysC and the gene asd are respectively as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The invention further discloses a preparation method of the aspartic acid transferase gene aspB, the aspartic acid kinase gene lysC and the aspartic acid semialdehyde dehydrogenase gene asd. After the recombinant corynebacterium glutamicum constructed by the invention is fermented for 60 hours, the yield of ectoine can reach 50g / L. The genetically engineered corynebacterium glutamicum disclosed by the invention has important practical significance on industrial production and large-scale application of ectoine.

The present invention discloses a genetically engineered bacterium for producing homoserine and a construction method and application thereof. The method comprises enhancing the precursor and reducing power supply in the genetically engineered bacterium. pyc、ppc、 Ec thrA、tkt Gene, knockout or weakening of branching pathways gltA、 dapA、avtA、alaT、pqo , ddh , thrB Gene; Modification of transporter system lysE、 Ec rthA Gene; Introduce heterologous feedback-resistant aspartate kinase gene Sc lysC and homoserine dehydrogenase Bd home Gene; Introduction of heterologous NADPH production enzymes; Enhancement of osmotic pressure tolerance in genetically engineered bacteria cglK、 betP、grpE The present invention discloses a method for constructing a genetically engineered bacterium with improved L-homoserine production by utilizing the method, and a method for producing L-homoserine and other downstream products by utilizing the genetically engineered bacterium.

This invention provides an engineered bacterium for producing ectoine, its preparation method, and its application. The engineered bacterium is prepared using a method comprising the following steps: using *Corynebacterium glutamicum* as the starting strain, feedback inhibition by aspartate kinase LysC is relieved, the activity of the gene lysE encoding lysine efflux permease is reduced, and the ectoine synthesis gene cluster ectABC is expressed. Based on this, the activity of any one or at least two of the following—diaminobutyrate acetyltransferase EctA, pyruvate carboxylase Pyc, or aspartate kinase LysC—is enhanced, and / or the activity of any one or at least two of the following—maloquinone oxidoreductase Mqo or homoserine dehydrogenase Hom—is reduced to further increase ectoine yield.

The invention relates to the technical field of microbial fermentation, and particularly discloses a recombinant microorganism for producing threonine and application of the recombinant microorganism. Compared with an original strain, the recombinant microorganism for producing threonine has the advantages that the expression quantity of thrL is reduced, and the expression quantity of threonine / homoserine export protein mutants, aspartate kinase / homoserine dehydrogenase I mutants, homoserine kinase and threonine synthase is increased; the threonine / homoserine ex-transport protein mutant is characterized in that the second site in the protein sequence of threonine / homoserine ex-transport protein is mutated from proline to leucine; the aspartate kinase / homoserine dehydrogenase I mutant is characterized in that glycine at the 433rd site in an aspartate kinase / homoserine dehydrogenase I protein sequence is mutated into arginine; the starting strain is escherichia coli. The invention constructs a novel recombinant microorganism for producing threonine, and provides a novel method for improving the fermentation production efficiency of threonine.

The present invention provides a method for constructing a threonine-producing engineered bacterium. According to the present invention, a 2-methylcitrate synthase 1-inactivated strain (Corynebacterium) is applied to the production of threonine, and the production of threonine produced by the 2-methylcitrate synthase 1-inactivated strain is increased by about 42% compared with that produced by an unengineered strain. When the application of the 2-methylcitrate synthase 1-inactivated strain is further combined with enhanced expression of at least one of aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase, NAD kinase, fructose-1,6-bisphosphatase 2 and the like in the threonine synthesis pathway, the production of threonine is improved. The method provides a new way for large-scale production of threonine and has high application value.

The invention discloses a Corynebacterium glutamicum with high yield of ectoine and its application, belonging to the field of genetic engineering technology. The invention performs aspartate kinase LysC S301Y , Homoserine dehydrogenase Hom V59A and isocitrate dehydrogenase ICD start codon mutations, knocked out genes LysE, PCK and ODX, enhanced the expression of genes FBP, TKT, PYC and PPC, and overexpressed the ectABC gene cluster, thereby enhancing the accumulation of ectoine precursors and greatly promoting the expression of ectoine; it can feedback inhibit the synthesis of competitive products of ectoine and enhance the metabolic pathway of ectoine precursor conversion to ectoine; at the same time, heterologous ectoine key synthases were transferred, and through a more reasonable gene element sequence, the production of ectoine was significantly improved, laying the foundation for the efficient synthesis and further industrialization of ectoine.

Disclosed is use of a Glycine max aspartate kinase / homoserine dehydrogenase family gene GmAK-HSDH. The gene GmAK-HSDH encoding a GmAK-HSDH protein has the nucleotide sequence set forth in SEQ ID NO: 1. A constructed overexpression vector pBA002-GmAK-HSDH is transformed into a recipient material JACK by cotyledonary node transformation.

This invention provides an engineered bacterium for producing ectoine, its preparation method, and its application. The engineered bacterium is prepared using the following steps: using Corynebacterium glutamicum as the starting strain, feedback inhibition of aspartate kinase LysC is relieved, the activity of the gene lysE encoding lysine efflux permease is reduced, and the ectoine synthesis gene cluster ectABC is expressed; based on this, the activity of any one or at least two combinations of aspartate semialdehyde dehydrogenase Asd, aspartate transaminase AspB, or 6-phosphogluconate dehydrogenase Gnd is enhanced to further increase the yield of ectoine.

The invention provides an engineering bacterium for producing Ectoine as well as a preparation method and application thereof, and the engineering bacterium is prepared by the preparation method comprising the following steps: taking corynebacterium glutamicum as a starting strain, relieving feedback inhibition of aspartate kinase LysC, reducing activity of gene lysE for coding lysine efflux permease, and obtaining the Ectoine-producing engineering bacterium. And expressing an Ectoin synthetic gene cluster ectABC. On the basis, the activity of any one or a combination of at least two of diaminobutyric acid acetyltransferase EctA, pyruvate carboxylase Pyc or aspartate kinase LysC is enhanced, and / or the activity of any one or a combination of two of malic acid quinone oxidoreductase Mqo or homoserine dehydrogenase Hom is reduced, so that the yield of Ectoine is further improved.

The invention discloses an aspartate kinase mutant as well as a coding gene, a carrier, a recombinant bacterium and application thereof, and the aspartate kinase mutant is lysCS317A, and is obtained by mutating 317-site serine of a parent amino acid sequence of which the amino acid sequence is as shown in SEQ ID NO.1 into alanine. Feedback inhibition of L-lysine on aspartokinase can be effectively relieved, and the catalytic activity on aspartic acid is improved, so that synthesis of L-lysine is promoted. A new thought is provided for constructing a high-yield L-lysine recombinant strain.

The present invention belongs to the field of bioengineering, and specifically discloses an aspartate kinase mutant with improved enzyme activity and its application in the preparation of L-homoserine or its derivatives. The amino acid sequence of the mutant is compared to the wild-type aspartate kinase derived from Saccharomyces cerevisiae, and there is one mutation type among V299A, N317S, N485S, Q107R / N317S, T114S / Q510R, and T155A / K390E. In a specific implementation, the enzyme activity assay analysis of the induced expression and purification showed that the enzyme activity of the mutant was 1.7 to 3.8 times higher than that of the wild type. Therefore, the beneficial mutants provided by the present invention can lay a good foundation for the industrialized and efficient production of L-homoserine and downstream metabolites.

The application discloses a high-yield L-homoserine Corynebacterium glutamicum mutant strain and application thereof, and belongs to the technical field of fermentation engineering. The application takes Corynebacterium glutamicum ATCC13032 as a starting strain, knocks out the regulatory protein McbR, amino acid intracellular transport protein MetD, homoserine kinase, phosphoenolpyruvate carboxykinase, cysteine beta cleavage enzyme, O-acetylhomoserine sulfhydrylase, weakens isocitric acid dehydrogenase and dihydrodipicolinic acid synthase, and overexpresses pyruvate carboxylase gene mutant, aspartate kinase mutant, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, branched-chain amino acid transport protein BrnF, branched-chain amino acid transport protein BrnE, aspartate aminotransferase, bifunctional aspartate kinase / homoserine dehydrogenase 1 mutant, NADP + dependent glycerol-3-phosphate dehydrogenase. The recombinant bacteria provided by the application can reach 63.5g / L of L-homoserine yield after 96h of culture in a fermenter.

The invention belongs to the technical field of microbial metabolism and genetic engineering, and relates to construction and application of engineering bacteria for producing L-homoserine. The construction method of the high-yield L-homoserine engineering bacteria provided by the invention is characterized in that escherichia coli (E.coli Bio-67727) for producing threonine is used as an original strain; a homoserine kinase coding gene thrB and a homoserine O-succinyltransferase coding gene metA are knocked out, and the expression of aspartate kinase I ThrA, aspartate semialdehyde dehydrogenase Asdbs, aspartate kinase III LysCpa, glucokinase Glk and a homoserine extracellular transport protein RhtA is enhanced. The engineering bacteria constructed by the invention can accumulate 156.2 g / L of L-homoserine after being fermented for 80 hours. The engineering bacterium does not carry plasmids, induction is not needed in the fermentation process, control is easy, and application to industrial production is facilitated.

A recombinant Escherichia coli THRS-6 and a method for synthesizing L-threonine thereof. The present invention belongs to the technical field of bioengineering, and overexpresses aspartokinase thrA <supgt; mut < / supgt; and pyridine nucleotide transhydrogenase pntAB genes, aspartate dehydrogenase aspDH gene derived from Pseudomonas aeruginosa, hemoglobin vgb gene derived from Vitreoscilla sp., and transcriptional regulator irrE gene derived from Deinococcus radiodurans, and further modifies the recombinant Escherichia coli by knocking out ldhA genes, obtaining a recombinant Escherichia coli THRS-6 using glucose as a substrate. Fermentation is carried out using the THRS-6, and the yield and conversion rate of L-threonine are significantly improved.

This disclosure provides the construction of a LysC scanning library for aspartate kinase using Retrotron editing technology, the screening of mutants, and their applications, belonging to the fields of biotechnology and genetic engineering technology. Specifically, this disclosure constructs a mutant library of an in situ whole-genome deep mutant library of aspartate kinase using novel Retrotron editing technology. The mutants in the library can significantly increase the production of amino acids such as lysine, threonine, and isoleucine.

The present application relates to an aspartate kinase mutant and an amino acid production method using same.

The application discloses a recombinant corynebacterium glutamicum with high yield of tetrahydropyrimidine and application thereof, and belongs to the technical field of bioengineering. S301Y By overexpressing aspartate kinase Lyc and aspartate semialdehyde dehydrogenase Asd, the supply of precursor aspartate semialdehyde can be increased. By inserting a repressor protein BetI binding sequence in the dapA promoter region of dihydropyridine dicarboxylic acid synthetase and overexpressing the repressor protein BetI, the content of by-product L-lysine in the fermentation liquor can be greatly reduced. By inserting a repressor protein LmrA binding sequence in the gltA promoter region of citrate synthase and overexpressing the repressor protein LmrA, the imbalance between cell growth and acid fermentation is solved, the supply of precursors oxaloacetate and acetyl CoA is increased, the yield of tetrahydropyrimidine is greatly improved, the yield is as high as 45.5 g / L, and the yield rate is as high as 0.25 g / g glucose.

The invention discloses a construction method of a high-purity hydroxyl Ectoine producing strain, which comprises the following steps: by taking corynebacterium glutamicum as an original strain, carrying out integration overexpression on multiple copy numbers of aspartate kinase lysC and hydroxyl Ectoine operon EctABCD on a corynebacterium glutamicum genome, and taking high salt as environmental selection pressure, so as to obtain the high-purity hydroxyl Ectoine producing strain. And knocking out the salt resistance related gene in corynebacterium glutamicum to obtain the production strain. The production strain obtained according to the construction method disclosed by the invention is cultured by using a high-salt fermentation culture medium, so that the hydroxyl Ectoine with the purity of more than 99% can be obtained.

The invention provides an engineering bacterium for producing Ectoine as well as a preparation method and application thereof, and the engineering bacterium is prepared by the preparation method comprising the following steps: taking corynebacterium glutamicum as a starting strain, relieving feedback inhibition of aspartate kinase LysC, reducing activity of gene lysE for coding lysine efflux permease, and obtaining the Ectoine-producing engineering bacterium. And expressing an Ectoin synthetic gene cluster ectABC. On the basis, the activity of any one or a combination of at least two of diaminobutyric acid acetyltransferase EctA, pyruvate carboxylase Pyc or aspartate kinase LysC is enhanced, and / or the activity of any one or a combination of two of malic acid quinone oxidoreductase Mqo or homoserine dehydrogenase Hom is reduced, so that the yield of Ectoine is further improved.

The invention relates to a recombinant bacterium capable of producing ectoine, L-theanine and collagen III at high yield and application of the recombinant bacterium, and belongs to the technical field of genetic engineering. The recombinant bacteria obtained by introducing plasmids containing ectoine biosynthetase genes and plasmids containing gamma-glutamine synthetase genes and III type collagen genes into corynebacterium glutamicum ATCC 13032 can be used for simultaneously producing ectoine, L-theanine and III type collagen at high yield. Meanwhile, an aspartate kinase gene and an aspartate semialdehyde dehydrogenase gene are connected to a plasmid containing an ectoine biosynthase gene, so that the yield of ectoine is increased. The recombinant bacteria obtained by the transformation can be used for simultaneously synthesizing 25.1 g / L of ectoine, 9.82 g / L of L-theanine and 356 mg / L of III type collagen after being subjected to fermentation culture.

Disclosed are an aspartate kinase gene expression regulatory sequence and use thereof. By modifying positions 366-373 of a sequence represented by SEQ ID NO: 1, a polynucleotide formed by linking the sequence with a start codon GTG or TTG has transcriptional expression regulation activity, and these polynucleotides can improve the expression of the aspartate kinase encoding gene lysC, such that a large amount of L-lysine is accumulated.

The invention relates to the technical field of microorganisms, and particularly discloses application of a microorganism with reduced lysine decarboxylase activity in fermentation production of L-threonine. The research finds that the fermentation yield of threonine can be improved by reducing the activity of lysine decarboxylase, and further the application of the microorganism with reduced activity of lysine decarboxylase in fermentation production of L-threonine is provided. According to the microorganism, the activity of lysine decarboxylase is reduced; furthermore, the expression quantity of threonine operon leader peptide of the microorganism is reduced; the 433rd site of the protein sequence of the aspartate kinase / homoserine dehydrogenase I is mutated from glycine to arginine to form an aspartate kinase / homoserine dehydrogenase I mutant, and the expression quantity of the aspartate kinase / homoserine dehydrogenase I mutant is increased; the expression quantity of homoserine kinase and threonine synthase is increased. The invention provides a novel microorganism and an effective method for fermentation production of threonine.