114 results about "Carboxysome" patented technology

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 present application relates to a mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant 5' untranslated region (5'UTR), a polyhydroxyalkanoate (PHA)-producing microorganism containing the gene, and a PHA production method using the same.

The invention belongs to the technical field of biological engineering, and discloses a schizochytrium limacinum engineering strain co-expressing a PDC-ACC gene, a construction method and application, and the genetic engineering strain is obtained by taking schizochytrium limacinum as an original strain and expressing a pyruvate decarboxylase compound gene PDC and an acetyl-coenzyme A carboxylase gene ACC in yarrowia lipolytica. The PDC and ACC genes in the schizochytrium limacinum genetic engineering strain disclosed by the invention are cloned to yarrowia lipolytica, namely Po1f. The yield of traditional fermented grease is 43.90 g / L. The engineering strain performs overexpression on PDC and ACC genes, so that the grease content of the schizochytrium limacinum engineering strain is increased by 53.13% and reaches 67.22 g / L, and the DHA content is increased by 36.97% and is increased from 40.71% to 55.76%.

The invention discloses a genetically engineered bacterium for high yield of L-alanine as well as a construction method and application of the genetically engineered bacterium, a dominant strain is obtained by knocking out an alanine racemase gene dadX and integrating coding sequences of alanine dehydrogenase from different sources, and the shake flask yield is 10.8 g / L; the activity of a corresponding mutant M18L / E75A / A78S / I266V is 4.3 times that of a wild type; 6 by-product related genes are knocked out, meanwhile, a multi-copy strain integrated with alanine dehydrogenase is constructed, and the yield of E.coli A-LB6 in a 5L fermentation tank is increased to 133.3 g / L. Through a dynamic regulation and control strategy, an oxygen-sensitive promoter Pomp * is adopted to replace a phosphoenolpyruvate carboxylase promoter, so that succinic acid byproducts are reduced by 85.8%, and the yield reaches 143.5 g / L after 48 hours of aerobic and anaerobic two-stage fermentation. And finally, by regulating and controlling the ATP synthetase gene cluster atp promoter, the yield of L-alanine in a 50 L fermentation tank reaches 151.3 g / L, and the saccharic acid conversion rate reaches 95.2%. The combination of polygene knockout and dynamic metabolism regulation effectively enhances the biosynthesis ability of alanine, and provides an efficient engineering strain for industrial production.

The present disclosure relates to a genetically modified microorganism satisfying some of predetermined conditions. The predetermined conditions include: (I) succinate dehydrogenase activity or fumarate reductase activity being reduced or inactivated relative to a wild-type microorganism; (II) lactate dehydrogenase activity being reduced or inactivated relative to the wild-type microorganism; (III) the genetically modified microorganism having modified phosphoenolpyruvate carboxylase activity showing resistance to feedback inhibition by aspartic acid in wild-type phosphoenolpyruvate carboxylase activity, or exogenous phosphoenolpyruvate carboxylase activity having higher resistance to feedback inhibition by aspartic acid than that of the wild-type phosphoenolpyruvate carboxylase activity shown by the wild-type microorganism; and (IV) pyruvate:quinone oxidoreductase being reduced or inactivated relative to the wild-type microorganism.

The application provides a recombinant Yarrowia lipolytica capable of producing palmitoleic acid at a high yield, a construction method and application thereof, and belongs to the technical field of bioengineering. Yarrowia lipolytica The recombinant Yarrowia lipolytica is obtained by knocking out peroxisome biogenesis factor 10 and triacylglycerol lipase 4 in the genome of Yarrowia lipolytica and inserting an acetyl-CoA carboxylase, a Δ9 desaturase, an acetyl-CoA diacylglycerol acyltransferase, a glycerol-3-phosphate acyltransferase, a lysophosphatidylcholine acyltransferase and a phosphatidylcholine:diacylglycerol acyltransferase gene expression cassette. Experiment proves that the recombinant Yarrowia lipolytica can efficiently ferment and produce palmitoleic acid, and realizes efficient synthesis of the natural product palmitoleic acid from plants in Yarrowia lipolytica.

The present invention provides a mutant acetyl-CoA carboxylase (ACC) protein, a nucleic acid and an application thereof, and in particular, relates to a mutant acetyl-CoA carboxylase (ACC) protein, a nucleic acid and an application thereof in plant breeding. In particular, the present invention provides a mutant acetyl-CoA carboxylase (ACC) protein, and the mutant acetyl-CoA carboxylase (ACC) protein is compared with the parent acetyl-CoA carboxylase (ACC) protein in the region corresponding to SEQ ID Mutations occur at any one or several of the following amino acid positions in the amino acid sequence shown in No. 1: 2125, 2097, 2139, 2194, 2186, 2273, 2168, 1975, 1954, 1864, 2211, 2187, 2123, and 2126; plants with the acetyl-CoA carboxylase (ACC) mutation have high herbicide resistance and have very broad application prospects in breeding herbicide-resistant plants.

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 present application relates to the field of plant genetic breeding, and particularly relates to a kind of evaluation method of asparagus yield. Including the following steps: S1, obtaining young plant period asparagus;S2, measures i variety of young plant period asparagus following parameters: photosynthetic rate A, ribulose-1,5-bisphosphate carboxylase / oxygenase activity B, leaf dry weight per unit area C, acid invertase activity D and root crown ratio E;The measurement sample of the acid invertase activity D is the tender stem of young plant period asparagus at 10-30cm from top;S3, according to the formula, the evaluation value is calculated, and the evaluation value is used to evaluate the yield of asparagus. The evaluation value obtained by using the above measurement index and calculation formula, and the significance level obtained by SPSS software is used to evaluate the yield of asparagus. The yield potential of asparagus can be comprehensively evaluated from the genetic point of view in a short time. Using the evaluation method of the present application, high-yield asparagus varieties can be selected in a short time without tracking for many years.

This invention aims to create a spatially isolated local reaction microenvironment for Rubisco by introducing a complete β-carboxysome microcompartment system. This design goal avoids oxygen inhibition, laying the foundation for ultimately achieving efficient and specific CO2 fixation. It provides a novel approach to overcome the fundamental problem of low efficiency of carbon-fixing enzymes in cytosols due to intense oxygen competition. By introducing the complete carboxysome system as a module, engineered yeast strains with high carboxylase activity and significantly improved ethanol production were obtained, providing a solid foundation for building next-generation yeast cell factories that produce high-yield ethanol using CO2 as a raw material.

Herein is reported a method for producing C-terminally biotinylated FcRn comprising the steps of cultivating a mammalian cell comprising a deoxyribonucleic acid encoding FcRn and E. coli biotin-[acetyl-CoA-carboxylase] ligase (BirA) in a biotin containing medium, and recovering C-terminally biotinylated FcRn from the cell or the cultivation medium, wherein the deoxyribonucleic acid encoding FcRn and E. coli BirA is stably integrated into the genome of the mammalian cell and comprises in 5′- to 3′-direction a first expression cassette encoding class I major histocompatibility complex-like protein (α-FcRn) comprising a HisAvi-tag at the C-terminus, a second expression cassette encoding β2-microglobulin (β2m), a third expression cassette encoding class I major histocompatibility complex-like protein (α-FcRn) comprising a HisAvi-tag at the C-terminus, a fourth expression cassette encoding β2-microglobulin (β2m), and a fifth expression cassette encoding E. coli biotin-[acetyl-CoA-carboxylase] ligase.

The invention discloses a recombinant bacterium for producing mevalonic acid by taking glucose and acetone as co-substrates as well as a construction method and application of the recombinant bacterium, and belongs to the technical field of genetic engineering. The problems that an existing mevalonic acid biosynthesis mode is low in carbon atom economy, limited in theoretical yield and the like are solved. According to the invention, acetone carboxylase, acetoacetyl coenzyme A synthetase, 3-hydroxy-3-methylglutaryl coenzyme A reductase and hydroxymethyl glutaryl-CoA synthetase are subjected to heterologous expression in escherichia coli with an acetyl coenzyme A acetyltransferase gene atoB and a histidine protein kinase gene atoS knocked out, and self carbonic anhydrase is over-expressed; and a new way for synthesizing MVA by using glucose and acetone as a co-substrate is constructed. According to the method, one molecule of CO2 can be fixed in the biosynthesis of mevalonic acid, the yield of carbon atoms in the synthesis process of mevalonic acid is increased, the problem of low yield of carbon atoms in the synthesis process of mevalonic acid by taking glucose as a single substrate is solved, and a new thought is provided for constructing a carbon neutralization type biological manufacturing technology.

The invention provides recombinant Escherichia coli for synthesizing heme and derivatives thereof, and the recombinant Escherichia coli is used for knocking out genes such as pyruvate oxidase poxB, pyruvate formate lyase pflB and porphyrinoperoxidase yfeX and enhancing genes such as phosphoenolpyruvate carboxylase ppc, glycerol transporter glpF and 3-phosphoglycerol dehydrogenase glpD, so that the heme and the derivatives thereof can be synthesized. After genes such as exogenous 5-aminolevulinic acid synthase hemA and ferrous chelating enzyme hemH are introduced, the heme and the derivatives thereof can be synthesized by taking glycerol as a raw material, and the heme has an application prospect in the field of biological manufacturing.

The invention discloses a compound enzyme preparation-containing nutritional feed for regulating and controlling intramuscular fat of black pigs and a preparation method of the compound enzyme preparation-containing nutritional feed, and relates to the technical field of livestock and poultry breeding feeds. The invention discloses a black pig skeletal muscle cell fat directional transport feed which is prepared from the following components in parts by weight: 850-920 parts of basal feed, 5-12 parts of fat directional deposition compound enzyme group, 25-40 parts of amino acid-enzymatic synthesis substrate combination and 2-6 parts of lipolytic enzyme specific inhibitor. According to the black pig skeletal muscle cell fat directional transport feed, fat precursor directional transport is realized through the compounded fat directional deposition compound enzyme group, a fat synthesis pathway targeting black pig skeletal muscle cells and lipoprotein lipase; acetyl-coenzyme A carboxylase and fatty acid synthetase catalyze fat synthesis step by step, only intramuscular fat generation pathways are activated, subcutaneous and abdominal fat deposition paths are blocked, the technical defects that fat deposition of an existing single enzyme preparation is non-directional, and the body fat rate is caused to be abnormally increased are overcome, meanwhile, an exclusive amino acid-enzymatic synthesis substrate combination is matched, and the body fat synthesis efficiency is improved. Sufficient adaptive substrates are supplied to enzyme group catalytic reaction, and the problem that a single enzyme preparation is insufficient in catalytic efficiency is solved.

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 present invention provides a class of new mutant proteins for increasing malic acid yield. Specifically, the present invention provides a class of new pyruvate carboxylase mutant protein and malic acid transporter mutant proteins or combinations thereof, a preparation method therefor and use thereof in improving malic acid 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 invention belongs to the field of insecticidal and acaricidal agents, and relates to an insecticidal and acaricidal composition. The composition contains an active component A and an active component B, and the weight part ratio of the active component A to the active component B is (1: 99)-(99: 1); the active component A is selected from a compound I; the active component B is selected from inhibitors influencing the growth of chitin synthetase 1 (CHS1) mites, mitochondrial adenosine triphosphate (ATP) synthetase inhibitors, mitochondrial electron transfer complex (III) inhibitors, mitochondrial electron transfer complex (I) inhibitors, acetyl-CoA carboxylase inhibitors and mitochondrial electron transfer complex (II) inhibitors; a trifluoroethyl sulfide type acaricide or other types of insecticidal and acaricide; the composition disclosed by the invention has the advantages of obvious synergism, resistance delay and the like, and can be used for preventing and treating various pests.

The application belongs to the technical field of biology, and discloses a strain for efficiently fixing carbon dioxide and improving succinic acid conversion rate and a construction method thereof. The application discloses a genetically engineered bacterium, which is obtained by overexpressing carbonic anhydrase genes, phosphoenolpyruvate carboxylase genes and phosphoenolpyruvate carboxykinase genes in a succinic acid-producing strain. ‑ The carbonic anhydrase can enhance the conversion efficiency of CO2 to HCO3 , and the phosphoenolpyruvate carboxylase and the phosphoenolpyruvate carboxykinase can realize efficient fixation of C3 and CO2 into C4 intermediates. Experiments prove that the genetically engineered bacterium can effectively produce succinic acid by using glucose, and has a relatively high sugar acid conversion rate (up to 79.80%).

The composition of the present invention is suitable for use in preparing emulsion-type meat analogue, preferably as a sausage type in a casing, which comprises rubisco protein and kappa-carrageenan in relative amounts between 1:5 to 30:1. Generally, the composition comprises 0.5-30 wt % rubisco and 0.1-15 wt % carrageenan relative the finished meat analogue product, and vegan protein other than rubisco preferably in an amount of 0.5-30 wt % relative to the meat analogue product. The meat analogue may contain the composition described in an amount of between 2-40 wt % with 30-90 wt % water and 2-40 wt % plant-based lipid.

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.

The application provides a pyroglutamic acid transaminase mutant and application thereof, the mutant is obtained by mutation on the basis of wild-type pyroglutamic acid transaminase, and specific mutation includes at least one of the following sites: (1) the 13th isoleucine is mutated into arginine; (2) the 35th valine is mutated into arginine; (3) the 183rd asparagine is mutated into aspartic acid; and the mutant is suitable for industrial enzymatic production of L-aspartic acid and the process of amino acids taking L-aspartic acid as a precursor; especially, the I13R / V35R / N183D mutant obtained by modification has high positive catalytic activity and strict direction selectivity, and completely loses the reverse reaction activity of decomposing L-aspartic acid; the mutant is overexpressed with phosphoenolpyruvate carboxylase in L-threonine production strain THRS-6, and a high-efficiency L-threonine synthesis metabolic pathway is constructed, and the yield of L-threonine is significantly improved when glucose is used as a substrate.

The invention provides a multi-enzyme composition and a method for carbon sequestration synthesis of 3-hydracrylic acid, the multi-enzyme composition is composed of acetyl-CoA ligase, acetyl-CoA carboxylase, malonyl-CoA reductase and polyphosphoric acid-AMP phosphotransferase, and can be used for producing 3-hydracrylic acid, and the specific method is that acetic acid which is cheap and easy to obtain is used as a substrate; the 3-hydracrylic acid is prepared through carbon sequestration by means of in-vitro multienzyme driven by the light engine, the yield of the 3-hydracrylic acid is increased by adjusting the adaptability of the system, and the method is low in production cost, simple in step and environmentally friendly.

The present application relates to the technical field of genetic engineering, and particularly relates to a phosphoenolpyruvate carboxylase mutant and application thereof. The phosphoenolpyruvate carboxylase mutant is obtained by a plurality of mutations in the amino acid sequence of the phosphoenolpyruvate carboxylase. The present application provides a plurality of mutation sites of the phosphoenolpyruvate carboxylase, and further finds that after the mutation of the sites of the phosphoenolpyruvate carboxylase in the strain, the yield of amino acids such as glutamic acid, glutamine and proline is significantly improved, which has important significance in the field of improving the strain to produce glutamic acid and its derivatives.

The invention discloses a phosphoenolpyruvate carboxylase mutant and application thereof, which is characterized in that an enzyme with an amino acid sequence as shown in SEQ ID NO.1 is used as a parent, and the 935th site or 155th site of the parent is mutated. The thermal stability of the phosphoenolpyruvate carboxylase is improved.

The invention belongs to the technical field of plant molecular biology, and particularly discloses a diatom RNA extraction method and a method for evaluating influence of pollutants on diatom gene expression. The invention firstly provides a diatom RNA extraction method, RNA is extracted by virtue of a cell homogenate physical crushing method and according to an improved TRIzol method, the release amount of RNA is high, and the purity reaches the standard. The invention also provides a method for evaluating the influence of pollutants on gene expression of diatom, which is based on the diatom RNA extraction method, takes Tubulin as a reference gene, takes one or more of iron redox protein, ferrithionein, ketoacyl coenzyme A thiolase, ethanol dehydrogenase, lipoprotein and acetyl coenzyme A carboxylase as target genes, and evaluates the influence of pollutants on gene expression of diatom. The relative expression level of a target gene is detected through qPCR and can be used for evaluating the acute toxicity of pollutants. The method effectively solves the problems that diatom RNA is difficult to extract, the diatom gene expression level is difficult to detect through qPCR, and the molecular mechanism of pollutant action is difficult to evaluate.

The invention relates to the technical field of bioengineering, and provides a ppc mutant and application thereof in fermentation production of L-valine. According to the invention, the ppc gene is subjected to point mutation, so that the expression activity of phosphoenolpyruvate carboxylase is reduced, bypass pathway competition is weakened, pyruvic acid precursor supply is enhanced, and the production capacity and biomass of the valine engineering strain are further improved. Fermentation results show that the method can obviously improve the yield of valine, the yield is improved from 1.3 g / L to 1.49 g / L, and the yield is improved by 14.6%. Therefore, the method has application value.

The invention provides a genetic engineering strain for efficiently producing beta-alanine, an escherichia coli strain is modified by adopting a genetic engineering means, and a multi-plasmid system is constructed, so that a metabolic pathway is optimized and the product synthesis efficiency is improved. The method comprises the following steps: firstly, by constructing a recombinant plasmid pET-PanD, introducing aspartic acid decarboxylase (L17F / G24R mutant) with mutation so as to promote efficient conversion of aspartic acid to beta-alanine; meanwhile, phosphoenolpyruvate carboxylase (PPC) is overexpressed through the recombinant plasmid pRSF-PPC, generation of oxaloacetic acid (OAA) is increased, and then the synthesis path of carbon flow to beta-alanine is improved. Besides, NADH dependent type aspartate dehydrogenase (AspDH) from pseudomonas aeruginosa is overexpressed by adopting a pBAD-AspDH plasmid, so that the dependence on NADPH is reduced, and the conversion efficiency is further improved.

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.