122 results about "Recombineering" patented technology

The invention relates to a diacylglycerol acyltransferase capable of synthesizing triglyceride as well as a coding gene and application of the diacylglycerol acyltransferase, and belongs to the field of enzymology, the nucleotide sequence of the gene for coding the diacylglycerol acyltransferase is as shown in SEQ ID NO.1, and the amino acid sequence of the diacylglycerol acyltransferase is as shown in SEQ ID NO.2. The invention also provides a recombinant plasmid and a recombinant engineering bacterium containing the gene of the enzyme. Diglyceride and an acyl donor are used as substrates, and triglyceride is catalytically synthesized through an acyl transfer reaction by using the enzyme. The invention provides an efficient tool for realizing enzymatic biosynthesis and large-scale production of natural triglyceride.

The invention belongs to the field of biotechnology and environmental governance, and particularly relates to a manganese peroxidase mutant based on high-throughput screening modification and application of the manganese peroxidase mutant to straw degradation. Aiming at the problems of low natural secretion amount, insufficient specific activity, complex preparation process and the like of the existing manganese peroxidase, the method comprises the following steps: firstly, constructing engineering bacteria based on an escherichia coli exocrine system, constructing a mutant library through error-prone PCR in combination with a directed evolution technology, and performing high-throughput screening by utilizing an ABTS colorimetric method; the recombinant manganese peroxidase mutant with high catalytic activity is obtained, and the enzyme activity is improved by 1.29 times compared with that of a wild type. Furthermore, recombinant engineering bacteria are constructed on the basis of a bacillus subtilis exocrine system, and efficient secretion of the mutant enzyme is achieved. The mutant enzyme preparation is used for wheat straw degradation, and the lignin degradation rate reaches 41.37% (improved by 30.09% compared with that of a wild type). The method is simple and convenient in process, low in cost and remarkable in effect, and has a wide industrial application prospect.

The invention discloses a recombinant engineering bacterium for expressing fetuB and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the recombinant engineering bacteria, pichia pastoris serves as host bacteria, after fetuB genes are introduced, efficient expression of the fetuB in the pichia pastoris is achieved by optimizing codons and improving fermentation conditions, and the expression quantities of CBS7435 under the shake-flask culture condition are 320 + / -25 mg / L, SMD116 under the shake-flask culture condition is 280 + / -30 mg / L, and SuperMan5 under the shake-flask culture condition is 580 + / -45 mg / L. After fermentation conditions are optimized, the culture yield of a fermentation tank can reach 27.35 g / L. The fetuin B disclosed by the invention is good in biological activity, high in expression, simple in operation process and suitable for large-scale industrial production.

The invention relates to the field of protein, in particular to a preparation method of recombinant human albumin and a product thereof. The preparation method comprises the following steps: constructing recombinant engineering bacteria which can silence or weaken the expression of one or more genes of PEP4, YPS1, MKC7, PRC1 and PRB1 through a gene editing technology while expressing recombinant human albumin, and silence or weaken the expression of one or more genes of PMT1, PMT2 and PMT4 at the same time; and then, carrying out low O-glycosylation fermentation culture on the obtained engineering bacteria, and carrying out separation and purification to obtain a high-purity recombinant human albumin product. According to the present invention, the protein degradation and the O-glycosylation pathway are cooperatively regulated, and the specific fermentation process is combined, such that the yield of the obtained recombinant human albumin is significantly improved, the characteristics of low immunogenicity and low glycosylation level are provided, and the performance optimization and the clinical application expansion of the recombinant human albumin product can be significantly promoted.

The invention relates to an artificial efficient nitrogen fixation system and a preparation method and application thereof. The artificial efficient nitrogen fixation system contains recombinant engineering bacteria constructed by an artificial combination module and nitrogen fixation chassis microorganisms; wherein the artificial combination module sequentially comprises a constitutive promoter element Pc and a coding gene of methyltransferase selected from nitrogen-fixing pseudomonas stutzeri A1501 in the direction from upstream to downstream. According to the recombinant engineering bacteria constructed by introducing the artificial combination module into the nitrogen-fixing chassis microorganism, the nitrogenase activity of the nitrogen-fixing chassis microorganism can be remarkably improved, the nitrogen-fixing capacity of the nitrogen-fixing chassis microorganism is remarkably improved, and the recombinant engineering bacteria have a relatively good application prospect in the aspect of promoting plant growth.

Provided herein are methods and compositions for truncating and, optionally, joining polynucleotides. The methods bypass inefficient cloning methods and the requirement for expensive enzymes by using in vivo homologous recombination in conjunction with specific endonuclease digestion. The methods may further be used to assemble long fragments of DNA from existing DNA constructs or libraries or to generate novel DNA libraries.

The invention relates to euphausia superba-derived diacylglycerol acyltransferase as well as a coding gene and application thereof, and belongs to the field of enzymology, the nucleotide sequence of the gene for coding the enzyme is as shown in SEQ ID NO.1, and the amino acid sequence of the enzyme is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the euphausia superba-derived diacylglycerol acyltransferase. The invention also provides a recombinant plasmid and a recombinant engineering bacterium containing the gene of the enzyme. Diglyceride and an acyl donor are used as substrates, and triglyceride is catalytically synthesized through an acyl transfer reaction by using the enzyme. The invention provides an efficient tool for realizing enzymatic biosynthesis and large-scale production of natural triglyceride.

The present invention provides a Klebsiella pneumonia KPC-Pal recombinant protein, which is composed of a KPC peptide segment extracted from the Klebsiella pneumonia KPC-2 protein and a Pal peptide segment extracted from the Klebsiella pneumonia Pal protein connected by a polypeptide linker, wherein the amino acid sequence of the Klebsiella pneumonia KPC-2 protein is SEQ ID NO: 3, and the amino acid sequence of the Klebsiella pneumonia Pal protein is SEQ ID NO: 4. The present invention also provides its expression vector and recombinant engineered bacteria, as well as its application in the preparation of a preparation for diagnosing, preventing or treating Klebsiella pneumonia infection. The Klebsiella pneumonia KPC-Pal recombinant protein provided by the present invention can effectively stimulate the body to cause a protective immune response, thereby resisting the lethal infection of Klebsiella pneumonia.

This invention discloses a method for preparing self-healing concrete based on microbial gene programming, belonging to the field of building materials technology. The method includes: constructing a recombinant engineered strain incorporating a urease gene cluster and a carbonic anhydrase gene; preparing a spore suspension and protecting it with a three-layer microencapsulation system of sodium alginate / chitosan / gelatin; preparing nutrient-releasing microspheres encapsulating calcium lactate and urea in a polylactic acid-glycolic acid copolymer; and mixing cement, standard sand, water, fly ash, microencapsulated spore particles, nutrient-releasing microspheres, nano-silica, and modified bentonite to prepare self-healing concrete. This invention enhances the urease activity of the strain through genetic engineering, protects the spores using a three-layer microencapsulation technology, and combines a nutrient-releasing system and nano-synergists to achieve efficient self-repair of concrete cracks.

The invention discloses a gene CsANS1 for coding anthocyanin synthetase and application of the gene CsANS1, and belongs to the technical field of biology. The nucleotide sequence of the gene CsANS1 is as shown in SEQ ID NO. 1. The gene CsANS1 or related biological materials thereof can be used for synthesizing anthocyanin and quercetin in vitro. Recombinant engineering bacteria verify that the anthocyanin synthetase CsANS1 not only has activity of catalyzing colorless anthocyanin to generate anthocyanin, but also has relatively strong activity of catalyzing quercetin to generate, so that the limitation of anthocyanin and quercetin production in the prior art is effectively solved. The bifunctional catalytic characteristic of the gene CsANS1 widens the application prospect of ANS enzyme in metabolic engineering, and is helpful for promoting the development and utilization of natural active ingredients in the fields of food, medicine, cosmetics and the like.

A process for engineering a host cell comprising the steps of a) integrating a first polynucleotide cassette including a first selection marker flanked by a first pair of recombination sites; b) removing the first selection marker by the action of a recombinase which recognises the first pair of recombination sites; c) integrating a second polynucleotide cassette including a second selection marker flanked by a second pair of recombination sites; and d) removing the second selection marker by the action of a recombinase which recognises the second pair of recombination sites. The first and second pairs of recombination sites have identical nucleic acid sequences within each pair, sharing 90-98% nucleic acid sequence identity. Also disclosed is a host cell genome polynucleotide comprising two recombinantly engineered regions, each adjacent to a single recombination site sharing 90-98% identity with each other and any additional recombination sites present in the host cell genome polynucleotide.

Provided are methods comprising expressing in a recombinant cell comprising a Cas gene a recombinant error-prone reverse transcriptase (RT) and a recombinant spacer RNA comprising a target sequence for mutagenesis of a DNA sequence in the Cas gene; making a mutagenized cDNA polynucleotide homologous to the DNA sequence in the recombinant cell; expressing a recombinant recombineering system in the recombinant cell; and recombining the mutagenized cDNA with the homologous DNA sequence of the Cas gene in the recombinant cell. Also provided are recombinant cells comprising recombinant coding sequences for a recombinant Cas protein, recombinant error-prone reverse transcriptase (RT), recombinant spacer RNA comprising the target sequence, and recombinant recombineering system.

The invention relates to the technical field of biology, in particular to a biological preparation method of a remegapam intermediate, which comprises the following steps: by taking remegapam intermediate precursor ketone with a certain concentration as a substrate, adding recombinant engineering bacteria, reacting in a reaction system with the pH value of 8.0-9.0 at the temperature of 30-40 DEG C, and obtaining a product after the reaction is completed, wherein the recombinant engineering bacterium is an engineering bacterium containing a ketoreductase mutant coding gene, and the amino acid sequence of the ketoreductase mutant is as shown in SEQ ID NO: 4, SEQ ID NO: 19 or SEQ ID NO: 39. According to the biological preparation method of the remegapam intermediate disclosed by the embodiment of the invention, the stereoselectivity of asymmetric reduction catalysis of the used ketoreductase mutant on precursor ketone of the remegapam intermediate is improved, the R-type remegapam intermediate with high chiral purity is obtained, and the synthesis efficiency of the remegapam intermediate is favorably improved.

The invention belongs to the field of microbial engineering, and provides a gentamicin C1a high-yield biological manufacturing method based on metabolic network modeling and reverse metabolic engineering renovation, which comprises the following steps: S1, constructing a genome scale metabolic model of a target production strain, predicting and screening a first target gene in positive correlation with gentamicin biosynthesis by using the model; s2, performing whole genome sequencing on the gentamicin high-yield mutant strain, and identifying a second target gene related to the high-yield character through comparative genomics analysis; s3, combining the first target gene predicted in the step S1 and the second target gene identified in the step S2, and constructing to obtain a recombinant engineering strain; s4, carrying out fermentation culture on the recombinant engineering strain, and supplementing materials in the fermentation process; by remarkably improving the production efficiency and strain performance of gentamicin C1a, important technical support and theoretical basis are provided for green biological manufacturing of antibiotics.

The application discloses a preparation method of CHO cell-expressed triple-helix structure type III recombinant human collagen and application thereof, and relates to the field of biotechnology. The preparation method comprises the following steps: obtaining the triple-helix structure type III recombinant human collagen through fermentation and purification of a recombinant engineering cell; and the construction method of the recombinant engineering cell comprises the following steps: transforming a host cell with a recombinant plasmid for expressing alpha 1 (III) and a recombinant plasmid for expressing hP4H alpha 1 and hP4HB to obtain the recombinant engineering cell. The preparation method can be used to prepare the type III recombinant human collagen with the triple-helix structure and a high proline hydroxylation rate, and the type III recombinant human collagen can be widely applied to the fields of cosmetics, medical cosmetology, medical devices, biomedical materials and the like, and has important market application value.

The invention belongs to the technical field of molecular biology and microbial engineering, and particularly relates to a fermentation medium and a fermentation process for preparing recombinant protein through high-density fermentation. According to the fermentation medium and the fermentation method provided by the invention, the thallus fermentation density of recombinant engineering bacteria can be remarkably improved, the OD600 fermentation density can reach 200 or more and even reach 240 or more, the thallus fermentation expression quantity can be remarkably improved, and the target protein expression quantity is 13 g / L or more.

The application discloses a fermentation method for producing thiamine pyrophosphokinase and belongs to the technical field of bioengineering. The BL21 recombinant engineering bacteria containing pET28a-TPK, pET28a-ProS2-TPK or pET28a-SUMO-TPK are constructed, the seed liquid is prepared, the isopropyl-beta-D-thiogalactoside is used for inducing expression, the bacteria are collected, the cells are broken and the supernatant is collected to obtain the TPK crude enzyme liquid. In the fermentation, the LB / TB culture medium containing 50 μg / mL kanamycin or 100 μg / mL ampicillin is used. The SUMO label is introduced to guide the correct folding of the TPK, so that the soluble expression ratio of the TPK is more than 85%, the ratio is improved by 2 times compared with the common system, the inclusion body ratio is greatly reduced, the process is stable and has good repeatability, and an effective scheme is provided for the industrialized large-scale preparation of the TPK.

A recombinant engineering bacterium for improving a yield of a recombinant human albumin is provided, which relates to the field of biological chemistry, particularly to the fields of gene engineering technologies and microorganisms. Targeted knockout is conducted on one or more genes of CCW14 gene, EMW1 gene, and PUN1 gene by utilizing a CRISPR-Cas9 gene editing technology, and / or one or more genes of EXG1 gene and SPR1 gene are over-expressed and integrated into a strain of the recombinant engineering bacterium, so as to improve the expression level of the recombinant human albumin.

This invention discloses a phospholipase D and its application in the preparation of ω-3 PUFAs-PA, belonging to the field of functional enzyme technology. The amino acid sequence of the phospholipase D is shown in SEQ ID NO. 9. The application of phospholipase D in the preparation of ω-3 PUFAs-PA is also disclosed. This invention also discloses a recombinant engineered bacterium expressing OUC-nPLD. The phospholipase D of this invention—OUC-nPLD—is a recombinant enzyme obtained by functional domain recombination based on the Non-HKD phospholipase D of *Streptomyces aquamarine*, and its PLD hydrolytic activity is 221.73 U / mg. This invention constructs a recombinant engineered bacterium expressing OUC-nPLD. The OUC-nPLD of this invention can be used for the efficient preparation of ω-3 PUFAs-PA, which is of great significance for the large-scale production of ω-3 PUFAs-PA.

The application discloses a recombinant engineering strain for biosynthesis of glabridin and a construction method and application thereof, and belongs to the technical field of synthetic biology and enzyme engineering. The recombinant engineering strain comprises a recombinant plasmid pRSFDuet-SiDR-SiTR-PmGDH; wherein the nucleotide sequence of SiTR is shown as SEQ ID NO:1, the nucleotide sequence of SiDR is shown as SEQ ID NO:2, and the nucleotide sequence of PmGDH is shown as SEQ ID NO:3. Through technical means such as optimization of gene sequences, construction of expression vectors and in-vivo directed evolution, the application successfully constructs the recombinant functional strain pTDG- E. coli BL21(DE3) and its evolution strain pTDGm258- E. coli BL21(DE3). The strains can efficiently catalyze the conversion of a substrate into glabridin, and have important industrial application value.

The invention provides a method for prokaryotically expressing cat allergen Fel d 1 recombinant protein, which comprises the following steps of: cloning a cat allergen Fel d 1 gene to multiple cloning sites of an expression vector pGEX-4T-1 to obtain a recombinant expression vector pGEX-4T-1-Fel d 1, transforming the recombinant expression vector into escherichia coli Rosetta (DE3) competent cells to construct recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), and carrying out prokaryotic expression on the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) to obtain the cat allergen Fel d 1 recombinant protein. IPTG (isopropyl-beta-d-thiogalactoside) is used for inducing protein expression, and a GST affinity chromatography medium is used for capturing high-purity soluble GST-Fel d 1 fusion protein. According to the invention, a pGEX-4T-1-GST label system is combined with Escherichia coli Rosetta (DE3), so that the problem of soluble expression of the cat allergen Fel d 1 protein is fundamentally solved.

This invention discloses a desmethylmenoprenone methyltransferase mutant, its construction method, and its applications, belonging to the field of biotechnology. Desmethylmenoprenone methyltransferase is a key enzyme in the MK-7 biosynthetic pathway, responsible for catalyzing the methylation reaction of desmethylmenoprenone to generate a series of methylnaphthoquinone (MK) products, ultimately forming MK-7. This invention, through metabolic network regulation and rational design, successfully obtained 19 single-point mutants, among which 5 mutants showed significantly higher catalytic efficiency than the wild-type enzyme. Experimental results show that the recombinant engineered bacteria constructed based on these mutants exhibit significant advantages in the whole-cell catalytic production of vitamin K2 (MK-7), with MK-7 yield increased by more than 20% compared to the wild-type strain. The excellent catalytic performance of the menG mutant provided by this invention provides strong technical support for the industrial production of vitamin K2, and has broad application prospects and market value.

The invention belongs to the technical field of biological medicine, and discloses an enteritis targeted probiotic delivery system and a preparation method and application thereof, the system comprises: i) a recombinant plasmid for coding an anti-inflammatory functional protein, the plasmid comprising an anti-inflammatory peptide fragment, a surface protein modification and an antibiotic switch; ii) Escherichia coli chassis bacteria which are subjected to genetic engineering modification and have the capability of high yield of outer membrane vesicles; and iii) introducing the recombinant plasmid into the escherichia coli chassis bacterium to obtain a recombinant engineering bacterium. The delivery system disclosed by the invention can target intestinal inflammation parts, deliver anti-inflammatory proteins through the outer membrane vesicles, and specifically release anti-inflammatory factors in an inflammation microenvironment, so that intestinal inflammation response is relieved; according to the present invention, the method can achieve the precise targeting on the intestinal inflammation, can effectively control the remote delivery and the continuous secretion of the anti-inflammatory protein in space and time, has advantages of strong targeting property, lasting effect, small side effect and the like, and provides the new strategy for the safe and efficient treatment of the intestinal inflammation diseases.

The present application relates to a kind of mediated zymolytic enzyme of brown algae and application of double transcription unit recombinant bacteria of oxyprotein and brown algae, belong to genetic engineering technical field, the recombinant engineering bacteria is composed of two transcription units, one transcription unit is GAP-alpha-Vgb-AOX1TT, another transcription unit is AOX-alpha-102C300C-AOX1TT, two transcription units are expressed in tandem in pPICZ alphaA plasmid.The present application also provides the preparation method and application of the recombinant bacteria.The recombinant bacteria of the present application can improve the secretion expression efficiency of brown algae zymolytic enzyme, especially can be expressed in low-oxygen environment.

The present application relates to a kind of chitosanase mutant WP-E80A, plasmid, recombinant bacteria and its application, belong to enzyme engineering technical field, the amino acid sequence of the chitosanase mutant as shown in SEQ ID NO:1, the amino acid sequence of wild-type chitosanase CsnWP as shown in SEQ ID NO:2.Compared with wild-type chitosanase CsnWP, the 80th amino acid is subjected to site-directed mutagenesis.The present application also provides plasmid and recombinant engineering bacteria comprising the chitosanase mutant WP-E80A gene, and the chitosanase mutant WP-E80A can be prepared to generate chitotriose with chitobiose as substrate.

The present application relates to the field of recombinant enzyme catalytic engineering technology, and provides a kind of intracellular expression lipase Ndbn and its related recombinant engineering bacteria.The lipase described in the present application has the characteristics of organic solvent resistance, can use aniline and ester as substrate in organic solvent, and enzyme synthesis amide compounds.Compared with existing lipases, lipase Ndbn shows high catalytic activity to weak nucleophile aniline, has a wide substrate spectrum, the aromatic amide catalyzed by it can be used as an intermediate of drug, avoids the shortcomings of high temperature and use of toxic reagent in the process of chemical synthesis of amide, has high atom economy, and meets the concept of green manufacturing.

The invention relates to the field of synthesis of natural dyes by genetic engineering and a biological method, in particular to a recombinant engineering bacterium for synthesizing N-acetyl blue as well as a construction method and application of the recombinant engineering bacterium. According to the invention, Escherichia coli BL21 (DE3) is used as an initial strain for modification; the method comprises the following steps: by using a genetic engineering bacterium as a template, expressing an ornamental blue synthetase encoding gene bpsA, a 4 '-phosphoric acid pantetheinyl transferase encoding gene entD, a glutamine synthetase encoding gene glnA, a glutamate dehydrogenase encoding gene gdhA, an acetyl-coenzyme A synthetase encoding gene acs and N-acetyltransferase encoding genes from different sources, and constructing to obtain a recombinant engineering bacterium. The recombinant engineering bacteria take sodium acetate as a substrate, and the sodium acetate not only meets the growth requirements of cells, but also can provide sufficient precursor substances. After the recombinant engineering bacterium constructed by the invention is subjected to shake flask fermentation culture for 72 hours, the purity of the N-acetyl blue can be up to 99% or above, and the recombinant engineering bacterium has the potential of industrial application.

The application belongs to the technical field of biotechnology, and particularly relates to a Clostridium perfringens bacteriophage lytic enzyme produced by Bacillus subtilis, a preparation method and application thereof, an optimized Clostridium perfringens bacteriophage lytic enzyme coding nucleic acid sequence according to the codon bias of Bacillus subtilis, and an optimized Clostridium perfringens bacteriophage lytic enzyme coding nucleic acid sequence as shown in SEQ ID NO. 1. The preparation method of the Clostridium perfringens bacteriophage lytic enzyme is as follows: firstly, a recombinant expression vector containing a Clostridium perfringens bacteriophage lytic enzyme gene is constructed, and then the vector is transferred into a Bacillus subtilis competent cell to obtain a Bacillus subtilis strain recombinant engineering bacterium. After the recombinant strain is induced to express, a fermentation liquor with Clostridium perfringens bacteriophage lytic enzyme activity can be obtained. The fermentation liquor can specifically inhibit the growth of Clostridium perfringens, and provides a safe, efficient and orally-taken preparation for treating animal diseases caused by Clostridium perfringens.

The invention discloses a high-osmotic-pressure-tolerant high-yield succinic acid recombinant engineering bacterium as well as a method and application thereof, aiming at the technical bottlenecks that in the existing process of producing succinic acid from escherichia coli, the growth of thalli is limited, the yield is reduced and the like due to high osmotic pressure of a fermentation environment after a product is neutralized by a neutralizing agent. The invention provides a genetically engineered bacterium for improving the yield of succinic acid. Wherein the engineering bacterium is constructed by overexpressing rpsR, cutA, gltI or queG genes from the engineering bacterium in escherichia coli ZX11. The genetic modification effectively enhances the osmotic pressure stress resistance of the strain, so that the viability and succinic acid production capacity of the strain under a high osmotic pressure fermentation condition are improved. Under strict anaerobic fermentation conditions, the highest succinic acid yield of shake-flask fermentation of the engineering bacteria reaches 30.92 g / L, and compared with an original strain, the succinic acid yield is remarkably improved.