34 results about "Ketoglutarate dehydrogenase" patented technology

A microorganism which has an L-amino acid producing ability and has been modified so that succinate dehydrogenase activity and α-ketoglutarate dehydrogenase activity are decreased is cultured in a medium to produce and accumulate an L-amino acid in the medium or cells of the microorganism, and the L-amino acid is collected from the medium or cells to produce the L-amino acid.

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 aims at providing a method for synthesizing alpha-ketoglutaric acid by a biological conversion method. According to the method, L-glutamic acid is used as a substrate; in a conversion culture solution obtained from kluyveromyces marxianus ATCC36534 through being cultured via a conversion culture substrate, alpha-ketoglutaricdehydrogenase inhibitors are added; the synthesis conversion culture is performed under the oscillation conditions of 25 to 30 DEG C and 200 to 250 r/min; after the synthesis conversion culture is completed, the alpha-ketoglutaric acid is obtained through separating and purifying the synthesis conversion solution; the L-glutamic acid is converted into alpha-ketoglutaric acid by yeast cells in the growth state; the alpha-ketoglutaricdehydrogenase inhibitors are added; the further metabolism consumption is blocked, so that a great amount of alpha-ketoglutaric acid is accumulated in the culture medium; when the feeing concentration of the substrate L-glutamic acid is 50g/L, the mol conversion rate can reach 83.2 percent. The low-value L-glutamic acid is converted into high-value alpha-ketoglutaric acid; the large-scale industrial application can be realized; a production process has the advantages of short period, high conversion rate, small environment pollution and the like.

The invention belongs to the technical field of bioengineering, and discloses corynebacterium glutamicum for high yield of L-glutamine as well as a construction method and an application of corynebacterium glutamicum. The activity of alpha-ketoglutarate dehydrogenase and/or glutamic acid exporter protein in cells of the corynebacterium glutamicum for highly producing L-glutamine is lost, and adenylation modification of glutamine synthetase is removed. According to the corynebacterium glutamicum for highly producing L-glutamine disclosed by the invention, L-glutamine can be accumulated in a culture medium or cells, so that the yield of L-glutamine is increased to a great extent. The experiments show that the corynebacterium glutamicum disclosed by the invention is an L-glutamine high-yieldstrain; compared with an unmodified strain, the strain has the advantages that the capacity of producing L-glutamine is enhanced, L-glutamine can be effectively accumulated, the yield of L-glutamine is increased, the conversion rate is relatively high, a foundation is laid for industrial production of L-glutamine, and the strain has a wide industrial application prospect.

The invention discloses a tomato bacterial wilt resistance gene Slalpha-KGDH E2 and application thereof. The Slalpha-KGDH E2 codes an alpha-ketoglutarate dehydrogenase E2 subunit, the nucleotide sequence of the gene is shown as SEQ ID NO: 1, and the protein sequence coded by the gene is shown as SEQ ID NO: 2. Reduction of the expression quantity of the gene or partial base mutation can enable tomato plants to lose resistance to tomato bacterial wilt. After ralstonia solanacearum is inoculated into disease-resistant plants, the expression quantity of the Slalpha-KGDH E2 gene is remarkably increased compared with that of a control group; and functional verification shows that the Slalpha-KGDH E2 gene has a key positive regulation effect on the resistance of tomatoes to ralstonia solanacearum, the resistance of tomatoes to bacterial wilt can be improved by overexpression of the Slalpha-KGDH E2 gene, and a new way is provided for breeding of new tomato disease-resistant varieties, so that the Slalpha-KGDH E2 gene has good application value.

The invention discloses corynebacterium glutamicum and corynebacterium crenatum, as well as a method for improving the yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum. According to the method, dtsR1 genes of corynebacterium glutamicum and corynebacterium crenatum are knocked out; then the transcriptional level of alpha-ketoglutarate dehydrogenase complex (oxoglutarate dehydrogenase complex, ODHC) regulated by the dtsR1 genes is greatly lowered, and alpha-ketoglutaric acid flows to arginine; meanwhile, Tween-80, oleic acid or a mixture of Tween-80 and oleic acid is added to a fermentation medium, so as to make up for nutrition loss caused by loss of the dtsR1 genes, and at the early logarithmic growth stage during the fermentation culturing, Tween-40 is added to induce the transcription level of the genes in the pentose phosphate pathway to be greatly improved. Thus, sufficient NADPH is provided for the synthesis of arginine, and other complicated molecular breeding methods for improving supply of NADPH and other accumulation methods for improving content of alpha-ketoglutaric acid in the tricarboxylic acid cycle pathway are eliminated.

The present invention provides a method and pharmaceutical composition for preventing or treating the development of syndromes related to dysfunctional energy metabolism, such as neuropathy, spontaneous nocturnal muscle cramps associated with neuropathy, seizuring, diabetes mellitus; pediatric hypoglycemia; myopathy; muscle fatigue; muscle spasms; somnolence; reduced mental acuity; exercise intolerance and myocardial insufficiency, which are due to cofactor deficiencies associated with the pyruvate dehydrogenase complex and the alpha-ketogluterate dehydrogenase (a-ketogluterate dehydrogenase) complex in humans or other mammals in need thereof. In particular, a method of preventing or treating at least one syndrome related to defective glucose metabolism in humans or other mammals is provided comprised of administering a combination of enzyme cofactors containing therapeutically effective amounts of thioctic acid, niacinamide, pantothenate, riboflavin and thiamine. Also provided is a pharmaceutical composition comprised of a carrier and the combination of cofactors.

Provided are a recombinant strain for preparing a terpenoid, and method for constructing the recombinant strain. Also provided is a recombinant bacterium 1, the recombinant bacterium 1 being a recombinant bacterium obtained in order to improve the enzymatic activity of α-ketoglutarate dehydrogenase in escherichia coli or the mutant thereof. The method for improving the enzymatic activity of α-ketoglutarate dehydrogenase in escherichia coli or the mutant thereof is replacing the original regulating element of the ketoglutarate dehydrogenase gene (sucAB) in escherichia coli or the mutant thereof with any of the following regulating elements: artificial regulating element M1-46, M1-37, and M1-93. Also provided are a plurality of recombinant bacteria. By improving the enzymatic activity of α-ketoglutarate dehydrogenase, succinic acid dehydrogenase and transaldolase therein and improving the ability of a cell to synthesize NADPH and ATP, the efficiency of the MEP pathway and the production capacity of terpenoid are improved.

The invention provides construction of high-yield trans-4-hydroxyproline sucA gene knockout bacteria and discloses a biosynthesis method of trans-4-hydroxy-L-proline through gene knockout. According to the method, the gene suc A is knocked out on the basis of escherichia coli BL21(DE3) delta put A, the process that alpha-oxoglutarate generates succinic acid or an alpha-oxoglutarate dehydrogenase complex is utilized for catalyzing in the TCA circulation process is disturbed, meanwhile, the trans-4-hydroxy-L-proline hydroxylase gene (hyp) is inserted, and then plasmid pUC19-Ptrp2-hyp-vgb is converted; hydroxylase replaces functions of alpha-ketoglutaricdehydrogenase, 'anaplerosis' is conducted on the TCA circulation process, and it is guaranteed that hydroxyproline is generated while the TCA circulation is conducted. The invention further discloses application of recombinant escherichia coli to production of trans-4- hydroxyproline, it is indicated by the flask shaking fermentation result that the yield of hydroxyproline obtained after mediumoptimization is 1344.1 mg/L and is about 5.53 times that before optimization; compared with original bacteria which are converted into the plasmid pUC19-Ptrp2-hyp-vgb in the same mode, advantages are achieved on the aspect of production of hydroxyproline.

Disclosed are Corynebacterium L-glutamic acid producing L-glutamic acid deficient in α-ketoglutarate dehydrogenase, a method for producing L-glutamic acid by using the bacterium, coding derived from Corynebacterium L-glutamic acid producing A gene for an enzyme with KGDH activity, a recombinant DNA containing the gene, a coryneform bacterium carrying the recombinant DNA, and a method for producing L-lysine using a bacterium carrying the recombinant DNA and having the ability to produce L-lysine .

A coryneform bacterium which has an L-glutamic acid-producing ability and grows at least at the same growth rate as a non-mutated strain or a wild-type strain and has intracellular α-ketoglutarate dehydrogenase activity which is less than half that of the non-mutated or wild-type strain, and is obtained by introducing a mutation into a coding region or an expression control region of the chromosomal odhA gene encoding the E1o subunit of the α-ketoglutarate dehydrogenase complex.

The invention belongs to the field of gene engineering, and discloses a construction method of a synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum. The construction method comprises the following steps of (1) expressing heterogenous pentose transport protein in the corynebacterium glutamicum; (2) expressing heterogenous xylose isomerase and xylulokinasein the corynebacterium glutamicum; (3) generating alpha-ketoglutarate from generated 5-xylulosephosphate through a phosphopentose pathway and a glycolysis pathway, and carrying out weakened expressionon alpha-ketoglutarate dehydrogenase, so that more alpha-ketoglutarate flows to the synthesis of glutamic acid; and (4) modifying glutamic acid secretory protein to ensure that the corynebacterium glutamicum does not respond to external biotin pressure any more, and continuously secreting the glutamic acid out of cells. According to the constructed synthetic path from the xylose to the glutamic acid, the corynebacterium glutamicum can utilize the xylose to produce the glutamic acid in a conventional synthetic culture medium, and the synthesis from the xylose to the glutamic acid can also be conducted in a high-biotin environment such as lignocellulose.

The invention discloses a glutamic acid production strain ands a preparation method and application thereof, and belongs to the field of bioengineering. The method comprises the following steps: fusing protoplast between corynebacterium glutamicum A (the preservation number is CGMCC NO.1. 10760) and corynebacterium glutamicum B (the preservation number is CGMCC NO.1. 1735), and performing mutationand regeneration so that the FM19-007 strain (CGMCC No. 18678) is obtained. The strain is characterized in that the strain is short-rod-shaped, spore-free and flagellate-free when observed under a microscope, gram staining is positive, the cell size is 0.7-1 * 1.2-3 [mu] m, and alpha-ketoglutarate dehydrogenase is lost; the glutamate dehydrogenase activity is high; glutamic acid is not used, glutamic acid feedback inhibition is relieved, and the capacity of secreting glutamic acid to the outside is achieved; in the fermentation process, obvious morphological change and cell membrane permeability change (forming cell leakage) occur at the decay stage. The mutant strain is fermented to produce glutamic acid, the yield of glutamic acid is 22%, the yield is increased by 51.72%, and the yieldof glutamic acid is remarkably increased.

The invention discloses an Escherichia coli strain for producing succinic acid with glycerol as well as a construction method and use. The Escherichia coli strain for producing succinic acid with glycerol has the preservation number of CGMCC No. 7999. The construction method comprises the following steps: knocking out the phosphoenolpyruvate carboxylase gene of Escherichia coli, and performing over-expression of key enzymes (isocitrate lyase and malate synthetase) of the glyoxylate cycle to obtain an evolved starting strain; by virtue of evolution, obtaining an evolved strain which can grow in an M9 basic salt medium with glycerol as unique carbon source and has enhanced glyoxylate cycle flux; knocking out the succinate dehydrogenase gene, and performing over-expression of phosphoenolpyruvate carboxylase and alpha-ketoglutaric dehydrogenase systems to obtain an Escherichia coli strain for producing succinic acid by using glycerol. The strain grows rapidly and has a high cell density, so that the rate of producing succinic acid with glycerol is increased.

Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

The invention relates to a screening method of high-activity Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylase. The method includes the steps of: constructing a bacterial strain with deletion of alpha-ketoglutaric acid dehydrogenase encoding gene and isocitric acid lyase encoding gene; amplifying Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylase encoding gene through error-prone PCRto obtain a mutant thereof, and connecting the mutant to an expression vector; and converting the expression vector to the bacterial strain with deletion of alpha-ketoglutaric acid dehydrogenase encoding gene and isocitric acid lyase encoding gene and screening a bacterial strain, having high growth speed, on a culture medium containing alpha-ketoglutaric acid and corresponding substrates; extracting a plasmid therefrom, and amplifying a hydroxylase encoding gene mutant. The method achieves high-throughput screening on the high-activity Fe<2+> and alpha-ketoglutaric acid-dependent hydroxylaseand accelerates application process of the enzyme.

The invention relates to the technical field of genetic engineering and biological fermentation, and particularly discloses a recombinant microorganism capable of efficiently utilizing methanol and application of the recombinant microorganism. The invention provides a recombinant microorganism which has increased expression and/or enzymatic activity of methanol dehydrogenase and dihydroxyacetone synthetase and reduced expression quantity of a formaldehyde dissimilatory gene frmAB and expression and/or enzymatic activity of 6-phosphofructokinase compared with an original strain, and the recombinant microorganism has the advantages that the expression quantity of the methanol dehydrogenase and the dihydroxyacetone synthetase is increased, and the expression quantity of the formaldehyde dissimilatory gene frmAB and the expression quantity of 6-phosphofructokinase are reduced; the starting strain is escherichia coli. Specifically, after a formaldehyde dissimilatory gene frmAB, a 6-phosphofructokinase gene pfkAB and an alpha-ketoglutarate dehydrogenase gene sucA are knocked out at the same time, and a 3-phosphoglyceraldehyde dehydrogenase gene gapA is inhibited, the utilization rate of methanol can be further improved, and accumulation of intermediate formaldehyde is reduced.

The invention relates to a genetically engineered bacterium for producing 4-hydroxyisoleucine and application of the genetically engineered bacterium, and belongs to the field of metabolic engineering. According to the strain, TUIE03 is taken as a host, and expression of an alpha-ketoglutarate dehydrogenase E1 subunit encoding gene sucA is weakened while an isoleucine dioxygenase encoding gene idois overexpressed. After the strain is fermented for 24-48h, the yield of the 4-hydroxyisoleucine reaches 32.5-62.4 g/L, and the L-isoleucine is not detected in the whole fermentation process, which indicates that the L-isoleucine is completely converted. The fermentation process adopted by the method is simple, precursors L-isoleucine and alpha-ketoglutaric acid do not need to be added, and the method is easy to control, low in production cost and beneficial to popularization and application of industrial production.