29 results about "Pyruvic dehydrogenase" patented technology

The invention relates to a repetitive structural unit recombinant protein cpE2 and a preparation method and application thereof. The recombinant protein cpE2 of the repetitive structural unit has self-assembly performance and is formed by modifying an E2 subunit of a pyruvate dehydrogenase compound and dihydrofatty acid amide transacetylase. Specifically, the C end of the E2 protein is arranged at the N end through a connecting peptide by utilizing a circulating recombination principle, so that the recombinant protein cpE2 is constructed. The engineered cpE2 protein cage can be self-assembled into a cage-shaped structure, linking groups can be respectively brought into the protein cage through N-terminal or C-terminal modification of cpE2, the problem that a modifier or a drug is difficult to put into the protein cage by a traditional E2 protein is solved, meanwhile, the specific particle size of the cpE2 protein cage can be controlled through the length of a linking peptide, and the protein cage has a good application prospect. And efficient carrying of goods is achieved.

The invention discloses a production strain for fermenting L-histidine and application of the production strain, and belongs to the technical field of biological engineering. According to the invention, escherichia coli E.coli CICC 10243 is taken as an original strain, histidine synthesis operon genes hisDCB-cg2302-cg2301-HA-impA-FI, hisN and hisEG from corynebacterium glutamicum are integrated on a genome of the escherichia coli E.coli CICC 10243, a pyruvate dehydrogenase AceE mutant coding gene is introduced, and an escherichia coli transcription factor SlyA coding gene slyA is inactivated. The novel strain E.coli His08 for fermentation production of L-histidine is prepared and fermented in a 5L fermentation tank for 60 h, the histidine yield reaches 58.39 g / L, the sugar-acid conversion rate is 0.15 g / g, and the strain E.coli His08 has important application value.

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 application belongs to the field of biological analysis, and provides a method for precise metabolic flow analysis of TCA cycle based on isotope tracing technology combined with high-resolution mass spectrometry. By optimizing mass spectrometry parameters and liquid chromatography method, one injection within 6 min is realized, and high-throughput, high-sensitivity and high-resolution detection is realized on 11 biomarkers related to TCA cycle metabolic pathways. At the same time, the application innovatively uses LC-MS / MS to decipher 13 C the stepwise position-specific transfer from glucose to subsequent metabolites by glycolysis and TCA cycle, thereby calculating 13 C the integrated steady-state analysis of key metabolic rates of position-specific transfer from sequential precursors to their products (pyruvate dehydrogenase, beta-oxidation, pyruvate carboxylase, isocitrate dehydrogenase and pyruvate cycle). This technology has wide applicability and can potentially characterize mitochondrial metabolism of any tissue or cell.

The application discloses a kind of modified bacteria for producing L-alanine, compared with wild type bacteria, the genome is integrated with pyruvate dehydrogenase alaD coding gene of bacillus stearothermophilus origin and the coding gene of mutant alaE (A48S / A149D) of alanine export protein alaE, and the expression of gene ldhA, mgsA, pflB, adhE, ackA and frdB in chassis bacterial genome is interrupted.The application also discloses a kind of method for quickly constructing recombinant escherichia coli for producing L-alanine, the method is suitable for multiple escherichia coli subspecies including MG1655, BL21 and ATCC8739, has universality and popularization and application value.

The present application relates to a repetitive structural unit recombinant protein cpE2 and its preparation method and application. The repetitive structural unit recombinant protein cpE2 has self-assembly performance and is transformed from the E2 subunit of the pyruvate dehydrogenase complex and the dihydrolipoamide transacetylase. Specifically, the present application utilizes the principle of cyclic recombination to place the C-terminus of the E2 protein at the N-terminus through a connecting peptide, thereby constructing the recombinant protein cpE2. The engineered cpE2 protein cage of the present application can self-assemble into a cage-like structure, and the connecting group can be brought into the interior of the protein cage by modifying the N-terminus or C-terminus of cpE2, respectively, thereby overcoming the problem that it is difficult for traditional E2 proteins to place modifiers or drugs into the protein cage. At the same time, the specific particle size of the cpE2 protein cage can be controlled by the length of the connecting peptide to achieve efficient transportation of goods.

The invention discloses an artificial synthesis method and application of malonyl coenzyme A. An artificial synthesis route for synthesizing malonyl-CoA by taking beta-alanine (beta-ala) as a precursor is constructed by heterologous expression of aminotransferase and malonyl-CoA reductase: firstly, under the catalysis of transaminase, amino of beta-ala is transferred to alpha-keto acid (such as pyruvic acid, oxaloacetic acid or alpha-ketoglutaric acid and the like), and then the malonyl-CoA is obtained; an intermediate product 3-oxopropionic acid and corresponding amino acid are formed; and the malonyl-CoA is generated from the 3-oxopropionic acid under the action of the malonyl-CoA reductase. According to the method, the defects existing in a natural malonyl-CoA synthesis route, such as low carbon utilization rate, energy substance ATP consumption, greenhouse gas CO2 release, pyruvate dehydrogenase (PDH) of route enzymes and acetyl-CoA carboxylase (ACC) which are strictly regulated and controlled, are overcome, and high yield of products taking malonyl-CoA as a precursor, including light flavomycin, octanoic acid, phloroglucinol, pentadecheptaene, natamycin, spinosad and the like, is realized.

The invention relates to the technical field of microorganisms, in particular to lactobacillus brevis X4-4 and application of the lactobacillus brevis X4-4 in fermentation. The preservation number of the lactobacillus brevis X4-4 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 31284. The activity of pyruvate dehydrogenase, malic acid-lactase, N-acetyltransferase and acid protease of the strain is remarkably higher than that of existing common lactobacillus, malic acid and protein in food raw materials can be efficiently utilized, and beneficial products can be generated. The lactobacillus brevis X4-4 has the specific advantages that 1) the lactobacillus brevis X4-4 can survive in a large quantity in an environment with relatively low pH (Potential of Hydrogen); (2) malic acid is converted into lactic acid which participates in pyruvic acid metabolism and arginine synthesis, so that the irritative sour feeling of the raw materials is reduced; (3) protein can be hydrolyzed into small molecule peptide under an acidic condition, so that the utilization rate of the protein is increased; and 4) the method can be used for single fruit raw materials or high-protein raw materials, and can also be used for fruit and protein composite raw materials.

The application discloses a pyruvate dehydrogenase E2 inhibitor and a preparation method thereof. The application also discloses a bactericide containing the pyruvate dehydrogenase E2 inhibitor. The application occupies the binding site of lipoic acid in the active cavity by designing an analogue of lipoic acid, so as to hinder the catalytic reaction and achieve the purpose of inhibiting the growth of organisms. Based on this, the application takes the pyruvate dehydrogenase system in microorganisms as a target, designs and synthesizes a novel and efficient pyruvate dehydrogenase E2 (PDHc-E2) inhibitor, which can be used for developing a novel and efficient bactericide and is widely applied to the prevention and treatment of bacterial brown spot bacteria of rice or bacterial leaf blight bacteria of rice.

The invention discloses a genetically engineered bacterium for producing 1, 3-butanediol and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The invention discloses a genetically engineered bacterium for producing 1, 3-butanediol, which is characterized in that JM109 (DE3) is used as a starting strain, and a citrate synthase gene gltA is weakened; the method comprises the following steps: knocking out a D-lactic dehydrogenase gene ldhA and a pyruvate dehydrogenase-ubiquinone gene poxB; according to the invention, a phosphoketolase gene xfp and phosphotransacetylase genes pta, PhaA, PhaB, B1d *, yqhd, fdh1, nadk, pntA and pntB are overexpressed. When the genetically engineered bacterium constructed by the invention is used for producing the 1, 3-butanediol, the conversion rate is high, the economical efficiency is good, the sustainability is realized, and the industrial production is easy.

The invention discloses a construction method of escherichia coli for efficiently synthesizing glycollic acid, and belongs to the technical field of biochemical engineering. According to the invention, citric acid synthase gltA and aconitic acid synthase acnA and acnB are overexpressed in escherichia coli cells, pyruvate dehydrogenase transcription inhibition factor pdhR is knocked out, transhydrogenase pntA and pntB are overexpressed, and transhydrogenase stA is knocked out, so that the constructed recombinant escherichia coli can produce glycollic acid by using glucose in a short time. The reaction cost is lower, the yield is higher, the concentration of glycollic acid produced after fermentation in a 5L fermentation tank for 48 hours is 81 g / L, the conversion rate is 0.62 g / g, and the production intensity is 1.69 g / L / h.

The invention discloses an engineering strain for increasing supply of cofactors and application of the engineering strain in improving production of epsilon-polylysine, and belongs to the technical field of microbial genetic engineering. The method comprises the following steps: by taking streptomyces albus for producing epsilon-polylysine as an original strain, simultaneously connecting a nicotinic acid ribose phosphate transferase gene pncB, a phosphoenolpyruvate carboxylase gene pck and a pyruvate dehydrogenase gene pdh in the strain with a pIB139 vector by utilizing a homologous recombination method to obtain a recombinant plasmid vector pIB139-pncB-pck-pdh, and introducing the recombinant plasmid vector into a competent cell to obtain the recombinant plasmid vector pIB139-pncB-pck-pdh. The engineering bacterium BP3 is obtained. The shake flask epsilon-PL yield of the engineering bacterium BP3 reaches 1.61 g / L and is improved by 30.89% compared with that of an original strain; the yield of fed batch fermentation reaches 37.21 g / L, the yield reaches 4.85 g / (L.d), and the maximum thallus concentration DCW reaches 43.47 g / L.

The invention discloses a construction method and application of an engineering bacterium for increasing the yield of alpha-ketoglutaric acid. Wild E.coli.W3110 serves as a chassis strain, lactic dehydrogenase ldhA, pyruvate dehydrogenase poxB and formic acid C-acetyltransferase pflB are knocked out firstly, and the flow direction of pyruvic acid to lactic acid, acetic acid and formic acid is blocked; meanwhile, the promoter PBBaJ23109 is used for replacing a natural promoter of the sucA gene, so that alpha-ketoglutaric acid dehydrogenase is weakened, and the decomposition way of alpha-ketoglutaric acid is weakened; the phosphoenolpyruvate carboxylase ppc is overexpressed, and the flow direction of phosphoenolpyruvate to oxaloacetic acid is enhanced; isocitrate dehydrogenase icd from escherichia coli is overexpressed, so that synthesis of a product is promoted; besides, pyruvate dehydrogenase derived from saccharomyces cerevisiae is introduced and coded by genes PDB1, PDA1 and LAT1, so that an important precursor, namely pyruvic acid, is promoted to flow to acetyl coenzyme A; the engineering strain with high yield of alpha-ketoglutaric acid is finally obtained, the yield of 75.1 g / L can be achieved in a 5L fermentation tank through a fed-batch fermentation method for 40 hours, and the conversion rate is 39.6%.

The present invention relates to recombinant Modified Vaccina Virus Ankara (MVA) encoding Epstein-Barr virus (EBV) antigens, wherein surface glycoprotein 350 (EBV gp350) and glycoprotein gH (EBV gH) are fused to subunits of self-assembling multimeric protein particle PdhC (acetyltransferase of pyruvate dehydrogenase (PDH) complex) or DPS (DNA binding protein from starved cells).

The invention discloses a preparation method of yoghourt with high diacetyl content, which comprises the following steps: carrying out ultrasonic-enzymolysis synergistic pretreatment on fruits containing citric acid to release natural citric acid so as to meet the citric acid concentration required for activating a pyruvate dehydrogenase system of streptococcus thermophilus and starting a citric acid metabolic pathway of lactobacillus bulgaricus; under the condition of high-concentration citric acid, a streptococcus thermophilus pyruvate dehydrogenase system is activated, and pyruvic acid generated by lactose metabolism can be promoted to be quickly converted into alpha-acetolactic acid; meanwhile, citric acid lyase of lactobacillus bulgaricus can decompose citric acid into acetyl coenzyme A, the acetyl coenzyme A is further converted into alpha-acetolactic acid, the alpha-acetolactic acid can produce diacetyl, and the generation rate of the diacetyl is increased through the combined action of the alpha-acetolactic acid and the alpha-acetolactic acid; and by combining the metabolic dynamic characteristics of the gradient temperature control fermentation precise adaptive strain, the synthesis and accumulation of diacetyl are promoted. The yoghurt prepared by the method disclosed by the invention is high in diacetyl content, mellow and smooth in flavor, pure in fragrance and suitable for industrial production and preparation.

The present invention discloses a genetically engineered bacterium producing 1,3-butanediol and its application, belonging to the field of genetic engineering technology. A genetically engineered bacterium producing 1,3-butanediol disclosed in the present invention, with JM109 (DE3) as the starting strain, weakens the citrate synthase gene gltA; knocks out the D-lactate dehydrogenase gene ldhA and the pyruvate dehydrogenase ubiquinone gene poxB; overexpresses the phosphoketolase gene xfp, the phosphotransacetylase gene pta, PhaA, PhaB, Bld*, yqhd, fdh1, nadk, pntA, pntB gene. The genetically engineered bacterium constructed using the present invention produces 1,3-butanediol with high conversion rate, good economy, sustainability and ease of industrial production.

This application relates to a microorganism in which the activity of pyruvate dehydrogenase complex subunit E1 is increased, and to a method for producing O-acetylhomoserine or its derivatives using the same.

An engineered strain that optimizes the mannose metabolic pathway and produces high amounts of spinosad is developed. The strain is based on Saccharopolyspora spinosa. manB Genes and leuA Gene blocking, and aceF Gene overexpression is obtained; manB The gene refers to the phosphomannose mutase gene; leuA The gene refers to the 2-isopropylmalate synthase gene; aceF The gene refers to the pyruvate dehydrogenase gene. The present invention starts with the precursor supply pathway of Saccharopolyspora spinosa, takes the mannose metabolic pathway as the optimization object, reduces the competitive utilization of the substrate glucose, and promotes more glucose metabolic flow to the pyruvate metabolic pathway; then reduces the competitive utilization of acetyl-CoA, improves the ability to synthesize acetyl-CoA from pyruvate, increases the accumulation of the precursor substance acetyl-CoA, and finally achieves the promotion of the efficient biosynthesis of spinosad in the engineered strain.

This application relates to a microorganism in which the activity of pyruvate dehydrogenase complex dihydrolipoyllysine residue acetyltransferase is improved, and to a method for producing L-amino acids using the same.

Disclosed in the present invention are an artificial synthesis method for malonyl-CoA and the use of same. By means of heterologous expression of an aminotransferase and a malonyl-CoA reductase, an artificial synthesis pathway using β-alanine (β-ala) as a precursor for synthesizing malonyl-CoA is constructed, which comprises: first, under the catalysis of the aminotransferase, β-ala transferring an amino group to an α-keto acid (such as pyruvic acid, oxaloacetic acid or α-ketoglutaric acid), so as to form an intermediate product 3-oxopropanoic acid and a corresponding amino acid; and 3-oxopropanoic acid then generating malonyl-CoA under the action of the malonyl-CoA reductase. The pathway solves the defects of a natural malonyl-CoA synthesis pathway, such as low carbon utilization rates, consuming an energy substance ATP, releasing greenhouse gas CO2, and pathway enzymes pyruvate dehydrogenase (PDH) and acetyl-CoA carboxylase (ACC) therein being strictly regulated, thereby achieving high yields of products using malonyl-CoA as a precursor, including flaviolin, caprylic acid, phloroglucinol, pentadecaheptaene, natamycin, spinosad, etc.

Disclosed herein are immune effector cells for use in adoptive cell transfer that have chemically- or genetically-inhibited PDHB (Pyruvate dehydrogenase E1 subunit beta) expression or activity. Also disclosed are methods of inhibiting or ablating PDHB expression in immune effector cells ex vivo and methods of using these cells to treat subjects with cancer. In some embodiments, the immune effector cells are further treated with a TIMS inhibitor or genetically engineered to ablate TIM3 expression. In some embodiments, the immune effector cells are further treated with a LAGS inhibitor or genetically engineered to ablate LAG3 expression. In some cases, the PDHB gene is disrupted by insertion of the gene encoding the chimeric receptor into the PDHB gene loci of the cell. Therefore, disclosed herein is a chimeric cell expressing a chimeric receptor.

The invention discloses a genetically engineered bacterium for producing succinic acid by efficiently utilizing corn straw hydrolysate and application of the genetically engineered bacterium, and belongs to the technical field of gene recombination and metabolic engineering. The invention discloses a genetically engineered bacterium. On the basis of a strain BW20, NADH supply is improved by inactivating a pyruvate dehydrogenase composite regulatory factor PdhR and overexpressing a transhydrogenase gene stA, succinic acid transport is promoted by oxygen responding to a metabolic switch, and supply of coenzyme A, acetyl phosphate and ATP is improved by refilling expression of a phosphate acetyl transferase gene Pta with a low-expression-intensity constitutive promoter. The conversion rate of succinic acid produced by fermentation of the genetically engineered bacterium in an anaerobic bottle reaches 1.05 g / g (total sugar), and the yield of succinic acid produced by metabolism of corn straw hydrolysate in aerobic-anaerobic two-stage fed-batch fermentation reaches 92.4 g / L. The genetically engineered bacterium disclosed by the invention can be used for efficiently metabolizing cellulose hydrolysate to produce succinic acid.

Methods of improving survival and eliminating treatment-limiting toxicities of dichloroacetate (DCA) in patients with pyruvate dehydrogenase complex deficiency (PDCD).

This invention relates to the field of agricultural biotechnology, specifically to a method and strain for constructing an engineered strain of *Hookworm Copper-Gropping Bacteria* capable of producing methylmalic acid using both organic and inorganic carbon sources. This application involves knocking out the restriction endonuclease encoding gene of the type I restriction modification system in the genome of wild-type *Hookworm Copper-Gropping Bacteria* H16. 、 Promoters adapted for methylmalate synthase expression in *Hypoxonometra* were screened, and methylmalate synthases with high catalytic activity in *Hypoxonometra* were obtained, capable of effectively catalyzing methylmalate synthesis in *Hypoxonometra*. A methylmalate transporter protein that promotes methylmalate excretion was also screened, as well as a gene encoding the *Hypoxonometra* endogenous pyruvate dehydrogenase complex subunit that promotes methylmalate synthesis when sodium gluconate is used as a carbon source.

The application discloses a fermentation L-histidine production strain and application thereof, and belongs to the technical field of bioengineering.The application takes E.coli CICC 10243 as a starting strain, integrates histidine synthesis operon genes hisDCB-cg2302-cg2301-HA-impA-FI, hisN and hisEG from corynebacterium glutamicum on the genome, introduces a pyruvate dehydrogenase AceE mutant coding gene, and inactivates the E.coli transcription factor SlyA coding gene slyA.The application prepares a new strain E.coli His08 for fermentation production of L-histidine, and the histidine yield reaches 58.39 g / L in a 5L fermenter for 60 h, and the sugar acid conversion rate is 0.15 g / g, so the application has important application value.

Methods of improving survival and eliminating treatment-limiting toxicities of dichloroacetate (DCA) in patients with pyruvate dehydrogenase complex deficiency (PDCD).

The invention discloses construction and application of recombinant kluyveromyces marxianus for producing L-lactic acid, and belongs to the technical field of microorganisms. According to the invention, through a metabolic engineering modification strategy, a pyruvate decarboxylase coding gene, a cytochrome oxidase gene and a pyruvate dehydrogenase gene are knocked out, and three copies of lactic dehydrogenase derived from lactobacillus plantarum are integrated, so that the L-lactic acid high-yield strain FMME-LA05 is obtained. The L-lactic acid yield can reach 130 g / L or above at the shake flask level, the L-lactic acid yield can reach 140 g / L or above in a 5 L fermentation tank, the optical purity can reach 99% or above, and the method has good genetic stability and is suitable for industrial production.

The invention relates to the field of biological pharmacy, in particular to product development and application of a targeted gender differential expression gene PDHB in fat reduction and obesity treatment. The invention provides a novel drug target, namely PDHB (pyruvate dehydrogenase E1 component beta subunit), which is closely related to obesity and sex difference, and is used for preparing or screening drugs capable of specifically reducing fat and treating obesity for visceral fat obesity (especially male) by improving mitochondrial energy metabolism.

Provided herein is a method of treating a patient having pyruvate dehydrogenase complex deficiency (PDCD), including administering to the patient an amount of triheptanoin effective to treat PDCD in the patient.