25 results about "Isomerase Gene" patented technology

The method for preparing D-mannose through catalysis can effectively reduce the amount of by-products glucose and fructose, and greatly improves the conversion rate of D-mannose. Specifically, engineering bacteria for expressing isoamylase genes and engineering bacteria for expressing alpha-glucan phosphorylase genes, glucophosphate mutase genes, difunctional enzyme glucophosphate isomerase / mannose 6-phosphate isomerase genes and mannose 6-phosphate phosphatase genes are utilized, starch or starch derivatives are used as substrates, and the starch or starch derivatives are used as substrates. A phosphate buffer solution and Mg < 2 + > are added, a preliminary catalysis system is constructed, after the reaction is completed, an incompletely-reacted substrate and maltodisaccharide exist in the reaction system, the incompletely-reacted substrate and maltodisaccharide can be hydrolyzed into glucose by glucoamylase, and the polyphosphoglucokinase can be used for catalyzing the reaction of the polyphosphoglucokinase under the assistance of sodium hexametaphosphate. All glucose is converted into phosphorylated glucose, then D-mannose is generated, and by-products in a system are removed while the yield is increased.

The invention discloses recombinant escherichia coli with high yield of N-acetylneuraminic acid and application of the recombinant escherichia coli, and relates to the technical field of biological genetic engineering. The invention relates to a recombinant escherichia coli, which is characterized in that the escherichia coli is taken as a host, and free expression of an N-acetylmannosamine epimerase gene yihS from Streptomyces xiamenensis or an N-acetylmannosamine epimerase gene ce3 from Bacteroides polymorpha and an exogenous N-acetylneuraminic acid lyase gene nano A is carried out; and carrying out recombinant expression on N-acetyl hexosamine 1-kinase nahK, a UDP-N-acetyl glucosamine pyrophosphorylase gene glmU and a UDP-N-acetyl glucosamine-2-epimerase gene neuC in the other synthetic route of the ManNAc. According to the recombinant escherichia coli with high yield of N-acetylneuraminic acid, the yield of N-acetylneuraminic acid can reach 23.08 g / L under a shake flask fermentation condition; a two-stage batch feeding strategy is adopted, the yield of N-acetylneuraminic acid in a 5L fermentation tank reaches 71.25 g / L, the molar conversion rate of GlcNAc reaches up to 57.60%, and the method has the potential of industrial application.

The method for catalytically preparing D-mannose provided in the application can effectively reduce the amount of by-products glucose and fructose, and greatly improve the conversion rate of D-mannose. Specifically, the engineering bacteria expressing isoamylase gene and the engineering bacteria expressing alpha-glucan phosphorylase gene, glucose phosphomutase gene, bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase gene and mannose 6-phosphate phosphatase gene are used, starch or starch derivatives are used as substrates, phosphate buffer solution and Mg 2+ A preliminary catalytic system is constructed, and after the reaction is completed, there are unreacted substrates and maltodextrin in the reaction system, the saccharifying enzyme can hydrolyze the unreacted substrates and maltodextrin into glucose, the polyphosphate glucose kinase can convert all the glucose into phosphorylated glucose with the assistance of sodium hexametaphosphate, and then D-mannose is generated, thereby improving the yield and removing the by-products in the system.

The invention relates to the field of bioengineering and genetic engineering, and particularly provides a rapid digging method of isomerase genes. According to the method, on the basis of big data analysis and bioinformatics technologies, in combination with means of sequence alignment, gene expression analysis, function prediction and the like, rapid and accurate mining of isomerase genes is achieved, and powerful technical support is provided for development and application of the enzyme engineering field.

The invention discloses a genetically engineered bacterium for biosynthesis of inositol and application of the genetically engineered bacterium for biosynthesis of inositol, and the genetically engineered bacterium for biosynthesis of inositol is obtained by co-expressing a sweet orange inositol-3-phosphate synthase gene Csino3 and an escherichia coli inositol-1-monophosphate gene suhB in recombinant escherichia coli and then knocking out a glucose phosphate isomerase gene pgi. According to the technical scheme, the yield of inositol produced by the strain reaches 2.04 g / L and is increased by about 6.6 times compared with that before optimization, the inositol yield is greatly increased, the metabolic flux of glycolysis to glucose-6-phosphoric acid is effectively blocked, carbon metabolic flux is forced to be redirected to a pentose phosphate pathway, glucose-6-phosphoric acid is enriched, and a raw material is provided for inositol production. By blocking downstream branches of a glycometabolism pathway, enrichment of glucose-6-phosphate in cells is realized, so that sufficient precursor substances are provided for synthesis of inositol, and finally the yield of inositol is increased. The technical scheme is expected to be applied to construction of microbial cell factories, so that large-scale and low-cost production of inositol is realized, and theoretical support is provided for development of related industries.

The invention provides a strain for producing beta-carotene as well as a construction method and application of the strain, and belongs to the technical field of synthetic biology and genetic engineering. The construction method of the MYy107 strain disclosed by the invention comprises the following steps: step 1, integrating a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate diphosphate decarboxylase gene ERG19 and an isopentene diphosphate isomerase gene IDI1 on an RT08 bacterium, so as to obtain an RT10 strain; 2, a xylose reductase gene XYL1, a xylitol dehydrogenase gene XYL2, a xylose isomerase mutant XylA3 * and a xylulokinase gene Yl.Xk are integrated on the RT10 strain, and the MYy107 strain is obtained. According to the MYy107 strain constructed by the invention, the beta-carotene can be produced by using xylose and acetic acid at the same time, and the yield of the beta-carotene is remarkably improved and reaches 710.01 mg / L.

The present application relates to the field of bioengineering, and particularly provides a epimerase Epi20 gene, the sequence of the gene is shown as SEQ NO:1, the enzyme can effectively utilize cucurbitacin 6a to synthesize cucurbitacin 6d, and provides a source for cucurbitacin production.

The invention relates to a recombinant pichia pastoris strain for producing coupled xylitol by NADPH (nicotinamide adenine dinucleotide phosphate) dependent growth and a construction method of the recombinant pichia pastoris strain, and belongs to the technical field of microbial fermentation. The recombinant pichia pastoris strain provided by the invention contains an NADPH dependent xylitol dehydrogenase gene, a sugar phosphatase gene, a D-arabinol-4-dehydrogenase gene, a fructose-1, 3, 4-triazole-1, 3, 4-triazole-1, 3-triazole-1, 3-triazole-1, 3-triazole-1, 3- The gene sequence is as follows: a 1, 6-diphosphatase gene, a phosphoketolase gene, a phosphate transacetylase gene, a glucose-6-phosphate dehydrogenase gene, a 6-phosphogluconolactonase gene, a 6-phosphogluconate dehydrogenase gene and a ribulose-5-phosphate epimerase gene; gene for expressing xylulokinase is knocked out or down-regulated: NADH dependent type xylitol dehydrogenase gene, ribose-5-phosphate isomerase gene, glutamate dehydrogenase gene, phosphofructokinase gene and phosphoglucose isomerase gene. The yield of xylitol produced by fermentation of the strain is far higher than that in the prior art.

The application discloses a platycodon grandiflorum phosphoglucoisomerase gene PgGPI and an application of a product coded by the platycodon grandiflorum phosphoglucoisomerase gene PgGPI, and belongs to the technical field of platycodon grandiflorum genes. The platycodon grandiflorum phosphoglucoisomerase gene PgGPI is cloned from the platycodon grandiflorum, and a nucleotide sequence of the platycodon grandiflorum phosphoglucoisomerase gene PgGPI is shown as SEQ ID NO. 1. The PgGPI gene is successfully expressed in Escherichia coli, and it is verified through an enzymatic experiment that the PgGPI has catalytic activity and can effectively isomerize glucose-6-phosphate into fructose-6-phosphate, thereby laying a solid foundation for further exploring a biosynthesis mechanism of the platycodon grandiflorum polysaccharide. In addition, by means of the PgGPI gene and gene engineering technology, it is expected to significantly improve the content of the polysaccharide substance in the platycodon grandiflorum.

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a D-psicose-3-epimerase mutant, which is obtained by performing point mutation on one site in the 79th, 115th, 141st, 193rd or 202nd amino acid of a D-psicose-3-epimerase parent; specifically, a mutation primer for point mutation is designed, a vector carrying a wild-type D-psicose-3-epimerase gene is used as a template to perform point mutation and construct a plasmid vector containing the mutant, and then the plasmid vector is transformed into a host cell to obtain the D-psicose-3-epimerase mutant; and the D-psicose-3-epimerase mutant is applied to the production of psicose. The thermal stability and catalytic activity of the D-psicose-3-epimerase mutant are significantly improved, and the D-psicose-3-epimerase mutant has good industrial application value.

The invention provides an engineering bacterium for producing D-pantothenic acid as well as a construction method and application of the engineering bacterium. The engineering bacterium expresses an acetolactate synthase large subunit gene ilv2, an acetolactate synthase small subunit gene ilv6, a keto acid reductoisomerase gene ilvC, a dihydroxy acid dehydratase gene ilvD, a hydroxymethyltransferase gene ecm31, a keto pantoic acid reductase gene panE, an L-aspartic acid decarboxylase gene panD and a pantothenic acid synthase gene panC. According to the method, mitochondria is selected as a targeting compartment, and modular combination optimization approach positioning is adopted, so that the yield of D-pantothenic acid is greatly increased, compared with the yield of an original strain without spatial tissue optimization, the yield is increased by more than one time, the metabolic flux bottleneck in a traditional cytoplasm synthesis mode is broken through, and efficient synthesis of D-pantothenic acid is realized.

The present application relates to the field of bioengineering and genetic engineering, and particularly provides a rapid mining method of isomerase genes. The method is based on big data analysis and bioinformatics technology, combined with sequence alignment, gene expression analysis and function prediction, etc. means, realizes the rapid and accurate mining of isomerase genes, and provides strong technical support for the development and application of enzyme engineering field.

The application provides a saccharomyces cerevisiae engineering bacterium for producing squalene through endogenous and artificial synthetic pathways and an application thereof, the saccharomyces cerevisiae engineering bacterium takes ZS00 strain as a starting strain, overexpresses acetyl coenzyme A acetyltransferase gene, hydroxymethylglutaryl coenzyme A synthase gene and N-terminal truncated hydroxymethylglutaryl coenzyme A reductase gene, and effectively improves the yield of squalene. Heterologous expression of mevalonate kinase gene and mevalonate pyrophosphate decarboxylase gene, overexpression of the key gene squalene synthase gene for promoting the conversion of precursors into squalene effectively promotes the synthesis of squalene. The introduction of isopentenol utilization pathway genes and their mutants, through the regulation of IU pathway key substrate, overexpression of isopentenyl pyrophosphate isomerase gene and farnesyl pyrophosphate synthase gene, the final strain can effectively accumulate squalene to 687.93 mg / L. The application realizes the breakthrough of squalene yield and efficiency through complementation and synergistic effect.

The invention belongs to the technical field of bioengineering, and particularly relates to a kluyveromyces marxianus engineering strain for efficiently expressing bovine beta-lactoglobulin and application of the kluyveromyces marxianus engineering strain. According to the invention, disulfide bond forming enzyme genes ERO1 and ERV2 of a disulfide bond isomerization pathway of pichia pastoris and disulfide bond isomerase genes PDI1 and MPD1 are introduced into a kluyveromyces marxianus chromosome interface, so that a disulfide bond synthesis pathway is enhanced, and folding of beta-lactoglobulin and formation of disulfide bonds are promoted; the glucose phosphate isomerase gene PGI1 of a glycolytic pathway is knocked out, and NADPH and synthesis of an amino acid precursor are improved; through fermentation condition optimization, the secretory expression beta-lactoglobulin yield of beta-lactoglobulin A and B recombinant expression engineering strains reaches 5.1 g / L and 7.3 g / L. The kluyveromyces marxianus engineering strain is used for preparing two subtypes of beta-lactoglobulins, is high in yield, short in fermentation period and low in cost, and can be applied to the fields of food, nutrient supplements, medical care and the like.

The invention provides a platycodon grandiflorum 1-deoxy-D-xylulose-5-phosphate reductoisomerase PgDXR1 gene, the nucleotide sequence of the platycodon grandiflorum 1-deoxy-D-xylulose-5-phosphate reductoisomerase PgDXR1 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of a corresponding code is as shown in SEQ ID NO. 2. The invention further provides a plant overexpression vector containing the PgDXR1 gene and a genetically engineered host cell containing the PgDXR1 gene. In addition, the invention also provides an application of the PgDXR1 gene in regulation and control of synthesis of plant triterpenoid saponin. The invention provides the platycodon grandiflorum PgDXR1 gene related to synthesis of the triterpenoid saponin of the platycodon grandiflorum for the first time, and an expression product of the gene can positively regulate synthesis of the triterpenoid saponin; the invention provides new evidence for perfecting the synthetic route of the plant triterpenoid saponin, has important theoretical and practical significance for improving the quality of traditional Chinese medicinal materials, and provides a new way for regulating and controlling the content of the triterpenoid saponin of platycodon grandiflorum by utilizing genetic engineering.

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant foal foal xylose as well as a construction method and application thereof. The recombinant foal foal capable of improving the bacterial cellulose yield and the xylose utilization amount is constructed by taking the foal foal foal as an original strain and overexpressing a xylose isomerase gene xylA, a xylulokinase gene xylB, a phosphopentose pathway key gene TKL and a structural gene BcsD. Under the fermentation conditions of pure xylose and xylose and glucose, the bacterial cellulose yields of the bacterial strain can reach 9.51 g / L and 8.1 g / L respectively and are increased by 553.23% and 44.75% respectively compared with original bacteria, and the highest xylose consumption of the bacterial strain under the fermentation conditions of pure xylose can reach 11.18 g / L. According to the method, the utilization rate of xylose is remarkably increased, the production efficiency of bacterial cellulose is improved, and a feasible solution is provided for replacing efficient conversion of traditional biomass resources with glucose as a carbon source.

The invention belongs to the technical field of gene engineering, and particularly relates to a method for synthesizing D-chiro-inositol through biotransformation and a synthetic strain of the D-chiro-inositol, and the strain is obtained by co-expressing an inositol dehydrogenase iolG gene and a myose isomerase iolI gene from bacillus subtilis in escherichia coli; according to the invention, a key enzyme inositol dehydrogenase is further modified to construct an inositol dehydrogenase mutant Q122V / D187Y, and the inositol dehydrogenase mutant Q122V / D187Y is obtained by mutating glutamine at the 122th site of inositol dehydrogenase iolG into valine and mutating aspartic acid at the 187th site of inositol dehydrogenase iolG into tyrosine. The strain co-expressed by the inositol dehydrogenase iolG mutant Q122V / D187Y and the inositol isomerase iolI is used for whole-cell transformation of D-chiral inositol, the DCI yield reaches 24.1 g / L within the production period of 4 h, and the production rate is 6.025 g / L / h.

This invention belongs to the field of genetic engineering technology, specifically relating to a method for biotransformation synthesis of D-chiral inositol and its synthetic strain, wherein the strain is Bacillus subtilis. Bacillus subtle The inositol dehydrogenase (iolG) gene and the inositol isomerase (iolI) gene were co-expressed in *E. coli*. Further modification of the key enzyme, inositol dehydrogenase, resulted in the construction of the inositol dehydrogenase mutant Q122V / D187Y. This mutant Q122V / D187Y was obtained by mutating glutamine at position 122 of inositol dehydrogenase (iolG) to valine and aspartic acid at position 187 to tyrosine. A strain co-expressing the inositol dehydrogenase (iolG) mutant Q122V / D187Y and the inositol isomerase (iolI) was used for whole-cell transformation of D-chiral inositol. Within a 4-hour production cycle, the DCI yield reached 24.1 g / L, with a production rate of 6.025 g / L / h.

This invention provides a group of recombinant *E. coli* strains capable of synthesizing D-allulose, wherein the recombinant *E. coli* strains include the following modifications: (a) introduction and expression of dTDP-glucose-3-epimerase gene, NDP sugar hydrolase gene, allosugar-1 phosphate-isomerase gene, and acid phosphatase gene; (b) enhanced expression of glucose-1-phosphate thymidine transferase gene, glucose-1-phosphate thymidine transferase 1 gene, and glucose-1-phosphate thymidine transferase 2 gene. This disclosure produces recombinant *E. coli* strains capable of high-yield D-allulose production, increasing the yield of the target compound while reducing the yield of by-products, thus solving a bottleneck problem in industrial production and enabling large-scale production of D-allulose.

Provided are an engineered bacterium producing 3'-sialyllactose, a construction method and application thereof. The engineered bacterium has the ability to produce 3'-sialyllactose, and has an introduced exogenous UDP-N-acetylglucosamine-2-epimerase gene (neuC), a sialic acid synthase gene (neuB), an N-acetylneuraminic acid cytidyltransferase gene (css) and an alpha-2,3-sialyltransferase gene (ST) in its genome or a recombinant plasmid carried thereby. The engineered bacterium has a higher ability to produce 3'-sialyllactose without the need for antibiotics.

The application discloses a chalcone isomerase gene of Lilium regale LrCHI1 The nucleotide sequence is shown as SEQ ID NO:1, and the protein encoded by the amino acid sequence shown as SEQ ID NO:2. LrCHI1 The application proves that the gene has the function of improving the plant resistance to fungi through reverse genetics technology research. LrCHI1 The gene is constructed into a plant expression vector and is introduced into tobacco to be overexpressed, and the transgenic tobacco plant has strong anti-invasion ability to fungi, and experimental results show that the transgenic tobacco with overexpression has high-level resistance to the invasion of Phoma exigua and Alternaria solani. LrCHI1 ​

This invention relates to a genetically engineered strain for synthesizing retinal, its construction method, and its applications. The construction method involves introducing DNA fragments of the β-carotene 15,15'-oxygenase gene, isopentenyl pyrophosphate isomerase gene, phytoene dehydrogenase gene, geranyl-geranyl pyrophosphate synthase gene, and phytoene synthase gene into *Rhizopus cylindrica* to obtain a genetically engineered strain. This strain enhances the expression of β-carotene 15,15'-oxygenase, isopentenyl pyrophosphate isomerase, phytoene dehydrogenase, geranyl-geranyl pyrophosphate synthase, and phytoene synthase in *Rhizopus cylindrica*. By selecting *Rhizopus cylindrica* as a host, introducing key synthetic genes, and achieving stable genome integration, this method yields an engineered strain capable of efficiently synthesizing retinal using glucose as a carbon source, simplifying the production process, reducing costs, and improving industrial applicability.

The application discloses an N-acetylglucosamine 2-epimerase mutant and a preparation and application thereof. The mutant enzyme comprises an amino acid sequence shown in SEQ ID No. 3. The preparation method comprises the following steps: taking a recombinant carrier containing a Sumo enhancing element and a wild-type N-acetylglucosamine 2-epimerase gene as a template, and performing a PCR reaction by using a mutant primer to obtain a mutant gene fragment; transforming the mutant gene fragment into an engineering bacterium to obtain a recombinant bacterium for expressing N-acetylglucosamine 2-epimerase mutant protein; culturing the recombinant bacterium, collecting and crushing the bacterium body, centrifuging the bacterium crushing liquid to obtain supernatant, and obtaining a crude enzyme liquid containing the mutant enzyme. The application improves the thermal stability of the wild-type enzyme by point mutation of specific amino acid sites, and the substrate affinity and catalytic efficiency of the mutant are superior to those of the wild-type enzyme. The mutant can be used for efficiently catalyzing preparation of lactulose, and has the advantages of less by-products and higher product yield.

The invention provides a method for synthesizing D-psicose from glucose by modifying escherichia coli. The modification comprises the following steps: (1) introducing an exogenous D-psicose-6-phosphate epimerase gene alsE with an SUMO tag and an exogenous D-psicose-6-phosphate phosphatase gene a6PP with an SUMO tag, and constructing a metabolic pathway for synthesizing D-psicose from glucose; (2) a phosphofructokinase A gene pfkA, a phosphofructokinase B gene pfkB and a UDP-galactose-4-epimerase gene galE are knocked out, so that side metabolic shunt is reduced, accumulation of a D-psicose synthesis precursor is increased, and the synthesis efficiency of D-psicose is improved; according to the present invention, a 6-glucose phosphate isomerase gene pgi and a phosphogluconate dehydratase gene edd are knocked out, and exogenous NADP + dependent glutamate dehydrogenase gene gdh1 and NAD + dependent glutamate dehydrogenase gene gdh2 are introduced so as to regulate intracellular cofactor balance and restore cell growth, such that the bacterial strain can effectively synthesize the target product while the bacterial strain has good growth performance;

This invention discloses the cloning and application of a mannuronic acid C5-isomerase gene related to high-temperature response in kelp, belonging to the field of biotechnology. This invention screens out a mannuronic acid C5-isomerase (MC5E) gene related to high-temperature stress response in kelp; based on this, specific primers are designed to clone the gene from kelp. MC5E The cDNA sequence of the gene was obtained; soluble recombinant protein was successfully expressed using a prokaryotic expression system, and high-purity MC5E protein was purified; 1H NMR spectroscopy analysis showed that this recombinant protein can convert mannuronic acid (M) in alginate to guluronic acid (G), increasing the G content in alginate; quantitative real-time PCR detection showed that under high temperature stress, kelp larvae... MC5E The significantly elevated transcription level of the gene indicates its involvement in kelp's response to high-temperature stress.