16 results about "Pyruvate synthesis" patented technology

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 invention provides a preparation method of pyruvic acid-1-13C, and relates to the technical field of chemical drugs, and the preparation method comprises the following steps: carrying out reaction and post-treatment on 13C-carbon dioxide and a Grignard reagent to obtain pyruvic acid-1-13C; wherein the Grignard reagent comprises a Grignard reagent obtained by reacting magnesium with acetyl halide. The technical problems that in the prior art, a pyruvic acid-1-13C synthesis method has safety risks, raw materials are not easy to obtain, the number of reaction reagent types is large, the number of process steps is large, and the yield is low are solved, the technical effects that safety and environmental protection are achieved, the raw materials are easy to obtain, the process is simple, and the synthesis route is short are achieved, and the obtained pyruvic acid-1-13C is high in purity and high in yield. And clinical test requirements can be met.

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.

Disclosed are genetically engineered Schizosaccharomyces pombe strains capable of fermenting glucose to produce malic acid. The S. pombe strains produce an exogenous malate dehydrogenase (MDH) enzyme and optionally produce an exogenous pyruvate carboxylase (PYC) enzyme or an exogenous phosphoenolpyruvate carboxylase (PPC) enzyme. Additionally, the S. pombe strains may be further engineered to produce a fumarase selectively located in mitochondria and / or have an inactivated mitochondrial citrate transporter, so that the resulting S. pombe strains are capable of producing malic acid as the predominant C4 dicarboxylic acid. Also disclosed are methods of producing malic acid with the S. pombe strains.

The application discloses genetically engineered bacteria for producing alpha-ketoglutaric acid and application. The genetically engineered bacteria are obtained by weakening the expression of alpha-ketoglutaric acid dehydrogenase gene odhA, weakening the expression of glutamate transporter protein gene cg1434, weakening the expression of glutamate synthase key subunit gene gltB, weakening the expression of lactic acid dehydrogenase gene ldh, weakening the expression of glutamate dehydrogenase gene gdh, weakening the expression of serine / threonine protein kinase gene pknG, enhancing the expression of pyruvate carboxylase gene pyc, introducing and expressing glutamate oxidase gene lgox at the site of lactic acid dehydrogenase ldh gene, and enhancing the expression of strong catalase gene katA on the basis of a Corynebacterium glutamicum starting strain. The genetically engineered bacteria can efficiently produce alpha-ketoglutaric acid under the condition of limited ammonia culture, and have a good application prospect.

Aspects of the present disclosure relate to the discovery that antagonizing pyruvate carboxylase and glutaminase inhibits cell proliferation and extracellular matrix production. Thus, methods and compositions disclosed herein may be used to treat fibrotic disease and cancer by inhibiting cell proliferation and extracellular matrix production.

The present invention discloses an L-alanine-producing genetically engineered strain, as well as a method of construction and use thereof, and pertains to the field of bioengineering. According to the present invention, through enhancing the glycolysis pathway or / and introducing a gene for thermostable alanine dehydrogenase, a genetically engineered strain capable of high-yield production of alanine under a high temperature condition of 42° C. to 55° C. can be constructed. Moreover, through knocking out alanine racemase genes, optical purity of L-alanine can be significantly increased. When the original strain possesses a lactate synthesis pathway, blocking this lactate synthesis pathway can augment the proportion of a pyruvate synthesis pathway, resulting in an additionally increased yield of L-alanine. The present invention overcomes the problems of fermentation at a low temperature, high cost and the like, which arise from the use of conventional L-alanine production techniques, enables production of L-alanine by fermentation at a high temperature of 42° C. to 55° C. with a yield of 95 g / L or higher, and is of high value to industrial application.

The present application relates to a kind of synthetic polyunsaturated fatty acid binding phospholipid yeast engineering bacteria and construction method and application, belong to microbial engineering technical field, the engineering bacteria is with Yarrowia lipolytica as bottom cell, knock out the Mhy1, Iro1, IDH1, CEX1 gene in its genome for generating mycelium, synthesis of triglyceride, citric acid metabolism, citric acid efflux;Phospholipid synthesis, pyruvate synthesis, polyunsaturated fatty acid synthesis and acyltransferase CDS1, OPI3, Mce2, LplA, △9Elo, △8Des, △5Des, △17Des, △5Elo, △15Des, LPCAT3 gene are sequentially connected together, as an expression module is integrated in the URA site of genome.The yeast engineering bacteria of the present application obtains polyunsaturated fatty acid binding phospholipid in one step efficiently, and phospholipid yield is 151.19 mg / g, and in binding fatty acid, EPA content is 13.2%.

This application discloses the application of the pyc gene and its mutants in the preparation of lysine, belonging to the field of genetic engineering technology. The technical problem this application aims to solve is: how to increase the amino acid yield of microorganisms. To solve the above technical problem, this application provides a protein obtained by mutating serine (S) at position 1059 of pyruvate carboxylase to proline (P) and related biological materials. This application also provides the application of said protein and related biological materials in constructing recombinant bacteria and preparing L-amino acids. This application has obtained a mutant pyruvate carboxylase (pyc) that increases the lysine yield in target microorganisms through extensive experiments. S1059P The results of the recombinant bacterial validation showed that, compared with the original target microorganism and recombinant microorganisms that overexpress wild-type pyruvate carboxylase (e.g., recombinant Corynebacterium glutamicum), the L-lysine production of recombinant Corynebacterium glutamicum expressing this mutant pyruvate carboxylase was significantly increased.

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 pyruvate carboxylase (PC) serving as a biomarker for detecting endometriosis and application of the pyruvate carboxylase (PC) to treatment of the endometriosis. The biomarker can be used for detecting the expression level of PC in an endometrial tissue sample through an immunohistochemical method, and when the IHC score is greater than or equal to 4, a subject is judged to be at a high risk of endometriosis or suffered from the endometriosis. Meanwhile, the invention provides an application of drugs (including specific siRNA, ZY-444, octyl gallate and the like) for inhibiting PC expression in treatment of endometriosis, and a corresponding treatment method. The invention provides a new biomarker, target spot and scheme for detection and treatment of endometriosis, and has important clinical application value.

The invention discloses construction and application of an issatchenkia orientalis strain for producing succinic acid, and belongs to the technical field of microorganisms. Through a metabolic engineering means, a pyruvate decarboxylase coding gene on an issatchenkia orientalis genome is knocked out, and pyruvate carboxylase, malate dehydrogenase, fumarase and fumarate reductase are integrally expressed; a glycerol dehydrogenase coding gene on a genome is knocked out, dicarboxylic acid outer transport protein from schizosaccharomyces pombe is integrally expressed, carboxylic acid inner transport protein JEN2-1 and JENE2-2 on the genome are knocked out at the same time, and fumarate reductase and / or malic acid dehydrogenation are / is overexpressed. The succinic acid yield of the modified yeast strain in the fermentation process is remarkably increased and reaches 125.77 g / L, and the production intensity is 1.75 g / L / h. The engineering strain provided by the invention has good industrial application potential in efficient succinic acid production.

The application discloses pyruvate carboxylase (PC) as a biomarker for detecting endometriosis and application of the biomarker in treating endometriosis. The biomarker can be used for detecting the expression level of PC in an endometrial tissue sample by an immunohistochemical method, and when the IHC score is greater than or equal to 4, the subject is determined to be at high risk of or suffering from endometriosis. Meanwhile, the application provides application of a drug (including specific siRNA, ZY-444, octyl gallate and the like) for inhibiting the expression of PC in treating endometriosis and a corresponding treatment method. The application provides a new biomarker, target and scheme for detecting and treating endometriosis, and has important clinical application value.

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 present application provides a genetically engineered L-malic acid producing yeast strain, wherein the strain has or has enhanced malate transporter activity and has or has enhanced NADPH-dependent malate dehydrogenase (EC 1.1.1.82) activity, optionally further has or has enhanced at least one of the following activities: (i) pyruvate carboxylase (EC 6.4.1.1) activity, (ii) phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activity, (iii) phosphoenolpyruvate carboxylase activity, and (iv) biotin transporter activity; as well as methods of making the same, methods of using the same to produce L-malic acid, and applications thereof.

The application discloses an Escherichia coli pentanediamine response promoter and application. Pyruvate carboxylase in corynebacterium glutamicum is synthesized from scratch and is heterologously expressed in Escherichia coli, and a pentanediamine response promoter PgrcA and lysine decarboxylase cadA in Escherichia coli are copied by PCR, and a PcadR-PgrcA-pyc-trc-cadA-rrnB recombination plasmid is constructed. The plasmid grows well in Escherichia coli with high lysine yield, gene expression is stable, glucose is efficiently utilized, can respond to pentanediamine, the fermentation cycle is short, and byproducts are few. It can be seen from the fermentation result that the pentanediamine yield of the recombined Escherichia coli KAGPC is significantly improved compared with a control group.