30 results about "Fumarase" patented technology

The invention provides malic acid-production gene engineering bacteria Escherichiacoli BA043. The malic acid-production gene engineering bacteria is named as BA043 (Escherichiacoli BA043) and has a preservation registration number CCTCC NO: M2014034. The invention also provides a construction method of the malic acid-production gene engineering bacteria and a method for fermentation production of malic acid. Through knockout or inactivation of fumarase and fumarate reductase and through over coexpression of exogenous pyruvate carboxylase and nicotinic acid phosphoribosyl transferase, recombinant escherichia coli can reduce by-product pyruvic acid generation by glucose metabolism growth so that a malic acid yield and production intensity are greatly improved.

The invention provides a gene engineering bacterial strain for producing L-malic acid as well as a construction method and application thereof. In the invention, key phosphoenolpyruvate carboxykinase(PEPCK) related to the L-malic acid, malate dehydrogenase (MDH) and fumarase (FumC) are transformed to obtain the gene engineering bacterial strain; a deactivation mutation is carried out on the fumCto cause the interruption of a metabolic flux at the converting position from the malic acid to the fumaric acid so as to accumulate the target product L-malic acid; before the mdh, the promoter of aninner source pepck gene is added to generate the metabolic flux biased towards the malic acid; in addition, through the inducement of monofluorine sodium acetate, the activity of the PEPCK is improved, and the product feedback inhibition is greatly reduced. The bacterial strain applied to the fermentative production of the L-malic acid so as to obviously improve the yield of L-malic acid. The invention has advantages of simple technology, obvious effect and low cost and can satisfy the demand of markets.

The invention belongs to the field of biochemical engineering, and particularly relates to a method for constructing escherichia coli genetic engineering bacteria for producing fumaric acid. The method for constructing the escherichia coli genetic engineering bacteria mainly comprises the following steps of: inactivating or knocking fumarase serving as key enzyme for converting fumaric acid into malic acid in a tricarboxylic acid (TCA) cycle out, and knocking a key gene arcA for inhibiting the TCA cycle out. Further, key enzyme genes in paths of succinic acid, lactic acid and formic acid can also be knocked out, so the fumaric acid can be accumulated under the anaerobic condition.

The invention relates to engineered Escherichia coli capable of high-yield production of fumaric acid; specifically, fumarase coded genes fumA and fumC of Escherichia coli are knocked off, and maleic cis-trans isomerase gene from Serratia marcescens is converted to obtain the engineered Escherichia coli. The invention also discloses a method of synthesis of catalyzing fumaric acid from maleic acid by using the engineered Escherichia coli. The method includes culturing the engineered Escherichia coli by fermenting, subjecting the engineered Escherichia coli to high-density inductive expression when OD600 reaches 40-80, and applying the obtained fermentation broth to biochemical catalysis of maleic acid in presence of Engineered Escherichia coli to obtain fumaric acid. The genes fumA and fumC in the Escherichia coli are knocked off by using Red homologous recombination, a metabolic pathway of its fumaric acid to L-malic acid is cut off, the maleic cis-trans isomerase from Serratia marcescens is then expressed, and the obtained genetically-engineered Escherichia coli can efficiently convert maleic matrix to synthesize high-purity fumaric acid, there is nearly no L-malic acid byproduct synthesized, and basis is provided for the industrialized production of fumaric acid by whole-cell process catalysis of maleic acid.

The invention discloses a construction method and use of a fumaric acid producing T.glabrata candida glabrata engineering strain and belongs to the field of fermentation engineering. In the invention, a recombinant T.glabrata FMME045 strain is obtained by overexpression of a malic dehydrogenase (RoMDH) gene and a fumarase (RoFUM1) gene from rhizopus oryzae in a uracil deficient (Ura) T.glabratadeltaura3 strain of a pyruvic acid producing strain T.glabrata in a free expression mode. When the strain is used for fermentation production of fumaric acid, the fumaric acid yield after the fermentation is performed for 60 hours is increased by 6 times to 35mg/L compared with that of a starting strain. Thus, a new approach is provided for producing fumaric acid by a microbial fermentation process. The method has a bright application prospect.

The invention discloses a method of utilizing a yarrowia lipolytica strain having a reductive TCA path to aerobically synthesize succinic acid. The yarrowia lipolytica engineering strain heterogeneously expresses key enzymes: fumaric reductase (FRD) and fumarase (FUM) in the reductive TCA path, and theoretical yield is substantially increased when compared with that of conventional oxidative TCA paths. The process of electrodialysis or acidizing ultrafiltration is omitted in a step of separating and purifying succinic acid, and no ammonium sulfate which is a byproduct is generated, so that themethod has considerable application value and prospect.

The invention discloses a recombinant escherichia coli efficiently transforming fumaric acid into L-asparagine as well as a construction method and an application thereof. Fumarase encoding genes fumA, fumB, fumC in an ammonium-tolerant escherichia coli BEW308 are inactivated, and then L-aspartase encoding and L-asparaginase encoding genes are inserted into the positions of the fumarase encoding fumAC genes, thereby obtaining the recombinant escherichia coli having no malic acid by product, less L-aspartic acid accumulated, and a major product L-asparagine. The invention also discloses a construction method and an application of the bacterial strain. The recombinant escherichia coli can realize constitutive high-activity expression of L-aspartase and L-asparaginase, and ultimately achieve transformation of fumaric acid into L-asparagine.

The invention discloses a genetically engineered bacterium synthesizing L-asparaginate. The genetically engineered bacterium is classified and named Escherichia coliDeltafumABCDeltaarcADeltaptsGDeltagltADeltaargG/pAA, and the preservation number of the genetically engineered bacterium is CCTCC NO:M2019170. The construction process of the genetically engineered bacterium comprises the steps that aspawn running strain fumarase encoding gene, a DNA transcription binding regulatory factor encoding gene, a glucose transport related gene, a citrate synthase encoding gene and an argininosuccinate synthase encoding gene are knocked out; an aspartase encoding gene and an asparagine synthetase A encoding gene are cloned to an expression plasmid, the recombinant plasmid is transformed into recombinant escherichia coli after gene knockout, and a target strain is obtained. The genetically engineered bacterium is synthesized into the L-asparaginate through fermentation, achieves a route for biosynthesizing and preparing the L-asparaginate by completely using glucose as a raw material, is efficient, green and environmentally friendly, and has economy.

The invention discloses a method for fermenting and producing L-malic acid with a raw material of citric acid broth. According to the method, citric acid broth is carbon source, through fermentation fumarase is generated, and by utilizing broth of fumarase and an enzyme conversion method, sodium fumarate generates the L-malic acid. After conversion of 20 to 26 hours, malic acid content reaches 78-85g/L, and conversion rate reaches 81.2%-85.5%. The method of the invention has the advantages of simple technology, low energy consumption, cheap raw material and substantial saving of production cost.

The invention provides a construction method for producing fumaric acid based on pichia stipitis synthetic strain fermented xylose. The method comprises the following steps: using pichia stipitis having a natural xylose metabolic capability as a host; deleting genes PSfum1 and PSfum2 that encode fumarase in tricarboxylic acid cycle of pichia stipitis, to acquire fumarase deficient mutants; heterologously expressing a modified fumaric reduction synthesis module on the basis of the acquired mutants, using xylose to ferment yeast synthesis strains of fumaric acid; on the basis of the yeast synthesis strains, expressing translocator YMAE that is modified with codons and originated from Chestnut wine fission yeasts, to acquire pichia stipitis synthesis strains for producing fumaric acid by fermented xylose; efficiently fermenting the acquired strains in a fully synthesized fermentation culture medium to produce fumaric acid by fermentation of xylose; cultivating by fermentation for 3-4 days at a temperature of 30 degrees centigrade with a stirring speed of 150-200rpm. The content of fumaric acid is greater than 4g/L.

The invention discloses aspergillus oryzae capable of reducing accumulation of a by-product in a synthesis process of aspergillus oryzae and an application of the aspergillus oryzae, and belongs to the field of genetic engineering. According to the invention, on the basis of A.oryzae GAA, over-expression of a fumarase coding gene derived from saccharomyces cerevisiae, a pyruvate carboxylase codinggene derived from rhizopus oryzae, a succinic acid-fumaric acid mitochondria transport protein coding gene derived from saccharomyces cerevisiae is implemented, so that conversion of fumaric acid andpyruvic acid in cytoplasm and mitochondria towards the malic acid and material exchange of cytoplasm and mitochondria are further enhanced. As for the finally obtained recombinant bacterium A.oryzaeCMPFS, a yield of L-malic acid undergoing shake-flask fermentation for 102h reaches 105.3g/L, concentration of succinic acid is 3.8g/L and concentration of fumaric acid is 0.21g/L; therefore, the concentration of the by-product is obviously reduced and product yield is improved; and a foundation is laid for production of the L-malic acid which is higher in purity through further metabolic transformation.

The invention discloses a strain for maleic acid whole-cell catalysis synthesis of L-aspartic acid and a method. The strain is named as Escherichia coli delta fumABC delta gltA delta argG/pMA, the preservation number of the strain is CCTCC NO:M 2019171, and the method comprises the construction processes that knocking out fumarase encoding gene, a citrate synthase encoding gene and an argininosuccinate synthase encoding gene in an original strain, meanwhile, aspartase encoding gene mutation N217K-T233R-V367G and maleic cis-trans isomerase encoding gene mutation G8A-G179A are cloned to expression plasmids to form recombinant plasmids, the recombinant plasmids are converted into the strain with knockout genes, and the target strain is obtained. According to the method for genetically engineered bacterium whole-cell catalysis synthesis of L-aspartic acid, the path for catalysis synthesis of L-aspartic acid by adopting maleic acid as raw materials is achieved, the production cost is lowered, efficient catalysis is achieved, and economy is achieved.

The invention relates to a method for producing malic acid by fermentation of citric acid mother liquor. In the method, with the citric acid mother liquor as a carbon source, a strain capable of producing fumarase by fermentation produces fumarase, and fermentation liquor is used as a raw material to convert sodium fumarate. After 20 to 25 hours of conversion, the content of L-malic acid reaches about 1.1 mol/L, and the conversion rate is about 94 percent. In the invention, performed separately, the fumarase production and conversion both can be performed under more proper conditions; the process is simple; energy consumption is small; the raw material is cheap; and the substrate conversion rate is high.

The invention discloses a gene-defective-type Escherichia coli strain containing a resveratrol synthase system and a malonyl coenzyme-A synthase system and a construction method thereof, as well as amethod for increasing resveratrol accumulation amount. A recombinant plasmid composed of the resveratrol synthase system and the malonyl coenzyme-A synthase system provided by the invention includes Rgtal, Pc4cl, Ahsts, accBC and PTDH sequences as well as two suitable vector fragments. The biological method capable of increasing resveratrol accumulation amount provided by the invention comprises the following steps: transforming the recombinant plasmids composed of the resveratrol synthase system and the malonyl coenzyme-A synthase system, namely pCDF-Rgtal-Pc4cl-Ahsts and pACYC-accBC-PTDH, into Escherichia coli strains with fumarinase gene deficiency (delta-fumB) so as to obtain engineering strains; and then, carrying out culturing with an optimized medium. Thus, yield of resveratrol withthe optimized medium can be up 22.62 mg/L, which is increased by 5.73 times compared with yield of original strains.

The invention belongs to the technical field of detection of argininosuccinic acid lyase, and discloses a reagent for detecting argininosuccinic acid lyase in serum and a preparation method thereof. The reagent consists of R1 and R2, wherein R1 consists of a buffer solution, nicotinamide adenine dinucleotide, fumarase, malic dehydrogenase, an enzyme protective agent, a preservative, a surfactant and ethylenediaminetetraacetic acid, and R2 consists of a buffer solution, disodium arginine succinate, ethylenediaminetetraacetic acid and hydrazine sulfate. The method comprises: adding 10ul of a sample, adding 200ul of R1, mixing the sample and R1 uniformly, and carrying out incubation at 37 DEG C for 2-3min; and adding 50ul of R2, mixing the components uniformly, carrying out incubation at 37 DEG C for 1 min, then continuously measuring the absorbance in 3min, and calculating an absorbance change rate. According to the invention, fumarase, another product generated by catalyzing argininosuccinic acid by ASAL, is detected, and the amount of ASAL is detected by a full-automatic biochemical instrument by utilizing the characteristic that NAD+ has an absorption peak at a 340nm position.

The invention provides a preparation method of L-malic acid. The preparation method comprises steps as follows: firstly, fumaric acid and calcium carbonate react to generate calcium fumarate, and generated calcium fumarate is converted into calcium malate under the action of fumarase; secondly, calcium malate and carbonate react in an aqueous solution, and a malate solution is generated; finally, the generated malate solution is converted into a malic acid solution with an electrodialysis method, and L-malic acid is obtained. According to the method, sulfuric acid is not utilized, no byproducts are produced, the method is environment-friendly, an extraction process is simple, intermediate products can be recycled, the cost is saved, and the enzymatic conversion rate is higher than 99.9%.