369results about "Racemaces/epimerases" patented technology

The invention provides recombinant escherichia coli for producing chondroitin sulfate A and application of the recombinant escherichia coli, and relates to the technical field of genetic engineering. According to the recombinant escherichia coli disclosed by the invention, chondroitin synthase, chondroitin 4-O-sulfotransferase, N-acetyl glucosamine epimerase, UDP-glucose dehydrogenase and chondroitin sulfate degrading enzyme are overexpressed; wherein the amino acid sequence of the chondroitin sulfate degrading enzyme is as shown in SEQ ID NO. 5. The recombinant escherichia coli is inoculated into a culture medium for fermentation culture, the average molecular weight of the obtained chondroitin sulfate A is 800Da, and the yield is about 12.5 g / L. Therefore, low-molecular-weight chondroitin sulfate A can be obtained and the yield of chondroitin sulfate A can be increased by fermenting the recombinant escherichia coli disclosed by the invention.

This invention discloses a D-allulose-3-epimerase mutant, its host cell, and its application in the synthesis of allulose, belonging to the field of genetic engineering technology. This invention utilizes a mutant derived from Ruminococcus CAG55 (… Ruminococcus sp. Simultaneous mutations at positions 47, 72, 114, and 221 of wild-type D-allulose-3-epimerase (CAG55) yielded a series of D-allulose-3-epimerase mutants, significantly improving their catalytic activity and stability. The mutants obtained by simultaneous mutations at positions 47, 72, 114, and 221 exhibited the highest catalytic activity. The D-allulose-3-epimerase mutants provided by this invention have significant application value in the production of D-allulose.

The invention belongs to the technical field of biotechnology and biocatalysis, and particularly relates to a method for preparing D-tagatose by catalyzing isomerization of D-fructose. The invention provides a method for preparing D-tagatose by catalyzing isomerization of D-fructose, which comprises the following steps: by taking a wet cell obtained by inducible expression of a recombinant genetically engineered bacterium containing a D-tagatose-4-epimerase gene as a catalyst and D-fructose as a substrate, when the concentration of the substrate is higher than 300g / L, continuously supplementing the substrate and enzyme to prepare the D-tagatose by adopting a strategy of continuously supplementing the substrate and enzyme. The substrate inhibition is reduced, the conversion rate of the D-tagatose is increased, and the final conversion rate of the D-tagatose is increased from 19.5% to 24.2%.

The invention discloses a production method of fucosylated oligosaccharide. The core of the method is as follows: the GDP-D-rhamnose is converted into the GDP-L-fucose by using the GDP-D-rhamnose-3, 5-epimerase; the GDP-D-rhamnose is obtained by converting the GDP-D-mannose-4, 6-dehydratase and the GDP-4-keto-6-deoxy D-mannose reductase by taking the GDP-D-mannose as a substrate, and the GDP-D-rhamnose is obtained by taking the GDP-D-mannose as a substrate. The invention relates to a method for producing a fucosylated oligosaccharide, which is characterized in that a GDP-D-mannose-4, 6-dehydratase, a GDP-4-keto-6-deoxyD-mannose reductase, a GDP-D-rhamnose-3, 5-epimerase and a fucosyltransferase are expressed in a genetically modified host cell, and the fucosylated oligosaccharide can be produced by the way mentioned above.

Provided is a heat-resistant D-allulose-3-epimerase. A polypeptide shown in any one of the following (1) to (3):(1) a polypeptide consisting of an amino acid sequence having one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G and F111W in SEQ ID NO:1;(2) a polypeptide consisting of the amino acid sequence having substitution, addition, insertion or deletion of one or several amino acid residues in the amino acid sequence described in (1) above, having D-allulose-3-epimerase activity and retaining one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W; and(3) a polypeptide having a 90% or more sequence identity to the amino acid sequence described in (1) above, having D-allulose-3-epimerase activity and retaining one or two or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W.

The invention belongs to the technical field of gene engineering and protein engineering, and particularly relates to thermal-stable D-psicose 3-epimerase as well as a preparation method and application thereof. According to the invention, a series of D-psicose 3-epimerase DAE mutants are constructed, and six mutants which not only can retain good catalytic activity, but also have high thermal stability are obtained through screening. The catalytic temperature of the three mutants A19D / A223P / V241I is increased by 25 DEG C compared with that of a wild type, while the three mutants A19D / A223P / V241I still show the optimal thermal stability, the catalytic activity of the three mutants is improved by 17.0%, and the three mutants show good industrial suitability. The D-psicose 3-epimerase DAE mutant with improved thermal stability provided by the invention is beneficial to improving the production efficiency of the DAE in industrial application of D-psicose, and provides powerful industrial enzyme guarantee for large-scale and low-cost preparation of D-psicose.

The invention relates to a NeuC mutant, a host cell containing the NeuC mutant and a method for producing sialic acid by fermentation. The invention discloses a NeuC mutant, a coding nucleic acid thereof, a vector, a host cell containing the vector, and a method for producing sialic acid through fermentation by using the host cell. Compared with wild NeuC, the NeuC mutant can obviously improve the yield of sialic acid. The sialic acid yield can reach 61 g / L after high-density fermentation culture is performed for 60 h in a 3L fermentation tank.

Provided are an Aeromonas dhakensis mutant strain with deletion of a surface polysaccharide synthesis gene, a complemented strain, a construction method therefor, and the use thereof. The mutant strain is obtained by deleting one or more of ugd, ugd2, and cap1J from the Aeromonas dhakensis wild strain A. dhakensis C160501.

The invention belongs to the field of genetic engineering and biological treatment, and provides an eukaryotic expression vector, gram-negative bacteria containing the eukaryotic expression vector and application of the eukaryotic expression vector in preparation of tumor drugs. By selecting and combining a phage-derived promoter, a specific phage RNA polymerase gene, a membrane rupture protein gene and an eukaryotic expression drug protein gene, an eukaryotic expression vector is divided into three modules: (1) a phage-derived RNA polymerase gene transcription module; (2) a membrane rupture protein gene expression module; and (3) a eukaryotic drug protein gene expression module. Through organic combination of the three modules, the eukaryotic drug delivery system is constructed, and the system and method for stably and efficiently performing eukaryotic drug protein expression, drug delivery and drug killing are realized.

The invention discloses a recombinant escherichia coli strain, a preparation method and application thereof, and a synthesis method of oryzanol A, and relates to the technical field of bioengineering. The recombinant escherichia coli strain takes E.coli BL21 (DE3) as a chassis cell, and is modified as follows: (a) knockout of a gene pfkA; or, the gene pfkA is knocked out, and an untranslated region of the 5'end of the gltA gene is mutated from the sequence as shown in SEQ ID NO: 1 to the sequence as shown in SEQ ID NO: 2; and (b) inserting exogenous genes agmA, agmB, agmC, agmE and agmF, so as to obtain the gene. The recombinant Escherichia coli strain is high in growth speed and short in fermentation period (within 72 hours), a culture medium which is cheap and easy to obtain can be adopted, and compared with an existing Escherichia coli system, the fermentation yield of oryzanol A is obviously increased.

The invention relates to the technical field of microbial metabolism engineering, and discloses a genetically engineered bacterium for high-yield L-histidine as well as a construction method and application of the genetically engineered bacterium. A nucleoside triphosphate pyrophosphate hydrolase coding gene mazG gene and a flagellin synthesis gene flhC gene are knocked out at the same time, an o-aminobenzoic acid phosphoribosyltransferase coding gene trpD gene and a ribulose phosphate 3-isomerase coding gene rpe gene are knocked down, and the recombinant protein is obtained. A ribose-5-phosphate isomerase gene coding rpiA gene and a lysine exponential coding gene lysE gene are overexpressed, and a promoter of 6-phosphoglucose isomerase pgi is replaced by a PfliC promoter, so that key substances for synthesis of L-histidine can be effectively accumulated, the fermentation yield of L-histidine is greatly increased, and compared with an initial strain, the yield of L-histidine is increased by 33.3%.

The invention provides a group of recombinant Escherichia coli for forming D-psicose. The recombinant Escherichia coli comprises the following multiple modifications: (a) introducing and expressing a dTDP-glucose-3-epimerase gene, an NDP glycohydrolase gene, an allose-1 phosphate-isomerase gene and an acid phosphatase gene; and (b) enhancing the expression of the glucose phosphate mutase gene, the expression of the glucose 1-phosphate thymine transferase 1 gene and the expression of the glucose 1-phosphate thymine transferase 2 gene. The recombinant Escherichia coli capable of producing D-psicose at high yield is prepared, the yield of by-products is reduced while the yield of a target object is increased, the bottleneck problem in industrial production is solved, and the recombinant Escherichia coli can be used for mass production of D-psicose.

The present disclosure relates to microorganisms for psicose biosynthesis. Methods of producing the disclosed microorganisms and methods of producing psicose are also provided.

One embodiment of the present invention provides a psicose epimerase expression cassette comprising a polynucleotide encoding a psicose epimerase and a promoter operably linked thereto and modulating the expression of the psicose epimerase in a Corynebacterium strain. The promoter consists of a base sequence as shown in a sequence 1, a base sequence as shown in a sequence 9, a base sequence as shown in a sequence 10 or a base sequence as shown in a sequence 11. The psicose epimerase expression cassette disclosed by the embodiment of the invention is almost not started in a normal environment for culturing a recombinant corynebacterium strain, and does not induce growth inhibition of the recombinant corynebacterium strain. Therefore, the recombinant corynebacterium strain transformed by the psicose epimerase expression cassette of one embodiment of the present invention can normally grow and proliferate to the logarithmic phase, and stably and highly express psicose epimerase after the logarithmic phase.

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 invention relates to the technical field of gene engineering, in particular to a D-psicose-3-epimerase mutant and application of the D-psicose-3-epimerase mutant in synthesis of psicose. According to the present invention, the 59th site, the 106th site and the 188th site of the wild type D-psicose-3-epimerase derived from Brevibacillus thermophilus (Brevibacillus thermophilus) are subjected to simultaneous mutation to obtain the D-psicose-3-epimerase mutant, such that the efficiency of producing D-psicose by using fructose as the raw material is significantly improved, the reaction of catalyzing 500 g / L fructose to produce D-psicose is performed for 4 h, and the yield of the D-psicose is significantly improved; the conversion rate is up to 36.5%; according to the present invention, the coexpression binding enzyme strain is constructed by coupling glucose isomerase, such that the intermediate transfer efficiency and the enzyme stability are improved, the reaction for producing D-psicose through catalysis of 500 g / L glucose is performed for 6 h, and the conversion rate can achieve 30.5%; and the activity of the immobilized enzyme can still be kept at 80% after the immobilized enzyme is repeatedly used for 10 batches.

The present disclosure provides metabolic signatures and genetic markers for tracking enhanced nitrogen utilization efficiency phenotypes in tobacco plants and for introgressing enhanced nitrogen utilization efficiency phenotypes into tobacco plants. The disclosure also provides tobacco plants comprising enhanced nitrogen utilization efficiency and methods to the creation of tobacco plants comprising enhanced nitrogen utilization efficiency. The disclosure also provides recombinant polynucleotides and polypeptides for enhancing nitrogen utilization efficiency in modified tobacco plants and tobacco plants comprising the provided recombinant polynucleotides and polypeptides.

The present invention belongs to the technical field of Aspergillus niger genetic engineering, and specifically relates to an Aspergillus niger with high mycelial protein production, a bacterial agent, an application and a method for high mycelial protein production. An ΔABSU The mycelial protein content of the strain increased by about 107.3% compared with the wild type, from 27.49% to 47.00%. An ΔABSU The hyphae of the strain are completely dispersed and no longer aggregate into balls, which is conducive to the recovery and film-forming of hyphae protein. AnΔ ABSU The growth rate of the strain was significantly accelerated, and the biomass reached 16.5g / L, which was 113.1% higher than that of the wild type. This strain has broad application prospects in microbial protein production.

The invention belongs to the field of escherichia coli metabolic processes, and particularly relates to a method for producing D-psicose from escherichia coli through glucose fermentation. Escherichia coli is used as a chassis host bacterium, a pgi gene, an SUMO-alsE fusion gene and an a6PP gene are subjected to overexpression at the same time, then a pfkA gene, a pfkB gene and a rpiA gene are knocked out in sequence, and a recombinant bacterium capable of producing D-psicose through glucose fermentation is obtained; the recombinant bacteria are used for fermentation, and glucose is converted to generate D-psicose.

The invention relates to a construction method and application of recombinant escherichia coli for increasing the yield of lactoyl-N-tetrasaccharide, and belongs to the field of microbial genetic engineering. According to the invention, three genes tauB, nikD and livM screened by using a transcriptome technology are proved to be capable of promoting the generation of LNT and LNTri II. An engineering strain MG-LNT-09 is used as a starting strain, the biosynthesis efficiency of endogenous transporter genes tauB, nikD, livM, LNT and LNTri II derived from Escherichia coli K-12 derivative bacteria MG1655 is remarkably improved through single plasmid overexpression, the yield of the LNT in shake-flask culture is as high as 7.77 g / L, and the yield of the LNTri II is as high as 3.03 g / L. The optimized strain MG3WB of which the tauB gene is integrated to the hlyE site obtains the maximum LNT titer of 40.35 g / L in a 5L bioreactor without using any antibiotics or plasmids, has a good industrial prospect, and lays a foundation for further industrial production and synthesis of other complex breast milk oligosaccharides.

The present invention relates to the technical field of biotechnology, particularly to an enzyme combination, a genetically modified bacterium, and its use in the production of D-allulose. The present invention relates to Bacillus subtilis co-expressing glucose isomerase and D-allulose 3-epimerase from a specific source. The resulting modified bacterium is fermented to obtain a crude enzyme solution, which is then isomerized using a high concentration of glucose as a substrate. After separation, purification, and concentration, fructoglucose syrup and D-allulose syrup are obtained. The enzyme combination provided by the present invention has a high catalytic rate relative to the substrate, significantly improving the conversion rate of D-allulose. At the same time, the present invention reduces the production costs of D-allulose by using inexpensive glucose as a raw material, and simultaneously produces fructoglucose syrup while producing D-allulose, thereby saving on the input of the fructoglucose syrup production line and significantly improving economic benefits.

The invention belongs to the technical field of bioengineering and enzyme engineering, and discloses a strain for producing D-psicose-3-epimerase, thallus immobilized particles and a catalytic method, the strain is classified and named as Bacillus halotolans, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 26, 2022, the preservation number is CGMCC No.26285, and the preservation number is CGMCC No.26285. The invention further discloses a preparation method of the D-psicose-3-epimerase for producing the D-psicose-3-epimerase for producing the D-psicose-3-epimerase for producing the D-psicose-3-epimerase for producing the D-psicose-3-epimerase. The thalli are loaded on the supporting carrier through the cross-linking agent, slow drying is achieved in the cross-linking drying process, and the obtained thalli immobilized particles have the advantages of being large in mechanical strength and insoluble in water.

The invention discloses an N-acetyl glucosamine 2-epimerase mutant as well as preparation and application of the N-acetyl glucosamine 2-epimerase mutant. The mutant enzyme comprises an amino acid sequence as shown in SEQ ID No.3. The preparation method comprises the following steps: by taking a recombinant vector containing a Sumo enhancing element and a wild type N-acetyl glucosamine 2-epimerase gene as a template, carrying out PCR (Polymerase Chain Reaction) by adopting a mutation primer to obtain a mutant gene segment; transforming the mutant gene segment into an engineering bacterium to obtain a recombinant bacterium for expressing the N-acetyl glucosamine 2-epimerase mutant protein; culturing recombinant bacteria, collecting and crushing thalli, centrifuging thallus crushing liquid, and taking supernatant, so as to obtain crude enzyme liquid containing the enzyme mutant; the thermal stability of the wild-type enzyme is improved through point mutation of specific amino acid sites, and the substrate affinity and the catalytic efficiency of the mutant are superior to those of the wild-type enzyme. The mutant can be used for efficient catalytic preparation of lactulose, and has the advantages of few byproducts, high product yield and the like.

Disclosed herein are genetically engineered yeast cells capable of producing xylitol and comprise an exogenous polynucleotide sequence encoding a ZWF enzyme at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:15, 16, and 23. The genetically engineered yeast cell may additionally be engineered to overexpress a native RPE enzyme, express an exogenous XPDH enzyme, express an exogenous XKS enzyme, express an exogenous XDH enzyme, overexpress a native X5PP enzyme, and / or express an exogenous X5PP enzyme.

The invention belongs to the technical field of gene engineering, and in particular relates to a D-psicose-3-epimerase mutant subjected to Spytag / SpyCatcher cyclization modification and an application of the D-psicose-3-epimerase mutant. The preparation method comprises the following steps: carrying out multi-site simultaneous mutation on 109th aspartic acid and 160th serine of wild type D-psicose-3-epimerase to obtain a mutation intermediate, respectively connecting a Spytag tag and a SpyCatcher tag to the N end and the C end of the mutation intermediate, and carrying out cyclization modification, so as to obtain the wild type D-psicose-3-epimerase, wherein the Spytag tag and the SpyCatcher tag are respectively connected to the N end and the C end of the mutation intermediate; when fructose is used as a raw material to produce D-psicose and host bacteria are escherichia coli, the reaction of catalyzing 600 g / L fructose to produce D-psicose is performed for 4 h, and the conversion rate can reach 35.7%; when the host bacteria are bacillus subtilis, the reaction for catalyzing 500 g / L fructose to produce D-psicose is performed for 4 h, and the conversion rate reaches 36.5%; compared with a wild type D-psicose-3-epimerase strain of bacillus subtilis, the conversion rate of the strain is improved by 13.5%.

Disclosed herein are genetically engineered Kluyveromyces sp. cells capable of producing D-allulose. The genetically engineered Kluyveromyces sp. cells comprise an exogenous polynucleotide sequence encoding an allulose-6-phosphate 3-epimerase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or100% identical to at least one of SEQ ID NOs:249-256, 258, and 259; and an exogenous polynucleotide sequence encoding an allulose-6-phosphate phosphatase enzyme at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:87, 89, 190, 123, 105, 107, 115, 83, 95, 113, 117, 119, 121, 127, 131, 137, 145, 169, 173, 179, and 183.

The present invention relates to a method for producing cytidine 5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac, 1) from low-cost substrates N-acetyl-D-glucosamine (GlcNAc), pyruvate, cytidine and polyphosphate in a single reaction mixture with a set of optionally immobilized or optionally co-immobilized enzymes comprising N-acylglucosamine 2-epimerase (AGE), an N-acetylneuraminate lyase (NAL), an N-acylneuraminate cytidylyltransferase (CSS), a uridine kinase (UDK), a uridine monophosphate kinase and a polyphosphate kinase 3 (PPK3). Further, said process may be adapted to produce Neu5Acylated i.e. sialylated biomolecules and biomolecules including a saccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a glycolipid, a glycan, an antibody, and a glycoconjugate, in particular, an antibody drug conjugate, and a carbohydrate conjugate vaccine, or a flavonoid.

Disclosed are genetically engineered microbial cells for the production of oligosaccharides comprising a galactose-β1,4-glucose moiety at their reducing end, wherein said microbial cells are able to produce said oligosaccharides in the absence of exogenously added lactose, and a method of producing said oligosaccharides using said microbial cells.

Disclosed herein is a genetically engineered yeast cell capable of producing xylitol, the genetically engineered yeast cell characterized by a genetic modification resulting in overexpression of a natural enzyme having xylitol-5-phosphate phosphatase (X5PP) activity and / or an exogenous polynucleotide sequence encoding an enzyme having xylitol-5-phosphate phosphatase (X5PP) activity. The genetically engineered yeast cell may additionally be engineered to overexpress a native RPE enzyme, to express an exogenous XPDH enzyme, to express an exogenous XKS enzyme, and / or to express an exogenous XDH enzyme.

The present invention discloses a D - psicose 3 - epimerase, its recombinant vector, strain and application, belonging to the technical field of functional enzymes. The present invention firstly provides a D - psicose 3 - epimerase derived from halotolerant Bacillus sp. ( Halotolerant Bacillus ), SYNY - 019. The protein sequence of the D - psicose - 3 - epimerase is as shown in SEQ ID No.1, and the nucleotide sequence obtained after codon optimization of this protein sequence is as shown in SEQ ID No.2. This D - psicose 3 - epimerase can be used for efficiently catalyzing the synthesis of D - psicose from D - fructose.