81 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 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.

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.

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 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 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.

This invention discloses a gene CsANS1 encoding anthocyanin synthase and its applications, belonging to the field of biotechnology. The nucleotide sequence of the gene CsANS1 is shown in SEQ ID NO.1. The gene CsANS1 or related biological materials can be used to synthesize anthocyanins and quercetin in vitro. This invention verifies through recombinant engineered bacteria that the anthocyanin synthase CsANS1 not only has the activity of catalyzing the conversion of colorless anthocyanins to anthocyanins, but also has a strong catalytic activity of catalyzing the conversion of quercetin, effectively overcoming the limitations of existing technologies in the production of anthocyanins and quercetin. The bifunctional catalytic properties of the gene CsANS1 broaden the application prospects of ANS enzymes in metabolic engineering and help promote the development and utilization of natural active ingredients in the fields of food, medicine, and cosmetics.

This invention discloses the Clostridium difficile phage recombinant lyase Lys51, its preparation method, and its application, relating to the field of genetic engineering technology. The invention involves introducing the recombinant plasmid pET-28a-SUMO-Lys51 into Escherichia coli BL21(DE3) competent cells via heat shock transformation; screening to obtain the recombinant engineered bacterium BL21(DE3)-pET-28a-SUMO-Lys51; constructing the recombinant plasmid pET-28a-SUMO-Lys51 and the recombinant engineered bacterium BL21(DE3)-pET-28a-SUMO-Lys51, further inducing the expression of Clostridium difficile phage recombinant lyase Lys51 in the recombinant engineered bacterium BL21(DE3)-pET-28a-SUMO-Lys51, and then purifying it to achieve the complete process for preparing Clostridium difficile phage recombinant lyase Lys51.

The invention relates to a lipase mutant as well as a preparation method and application thereof, and the lipase mutant is obtained by performing single-site mutation or double-site mutation on 138-site arginine and 251-site tyrosine on the basis of lipase of which the nucleotide sequence is SEQ ID No.1 and the amino acid sequence is SEQ ID No.2. The lipase mutant or a recombinant expression vector or recombinant engineering bacteria thereof is used as an active enzyme, and a powerful biocatalyst is provided for synthesis of (S)-parachloro-beta-phenylalaninol. The invention provides a lipase mutant with remarkably improved catalytic activity, a nucleotide sequence of the lipase mutant, and a recombinant expression vector and recombinant engineering bacteria containing corresponding mutant genes, and the lipase mutant or recombinant cells containing corresponding mutant proteins are subjected to asymmetric reduction to obtain the lipase mutant. And the chiral (S)-p-chloro-beta-phenylalaninol with high optical purity can be prepared.

The present invention discloses 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;wherein the first pair of recombination sites have an identical nucleic acid sequence and the second pair of recombination sites have an identical nucleic acid sequence and the first and second pairs of recombination sites share 90-98% nucleic acid sequence identity.Also disclosed is a host cell genome polynucleotide comprising a first recombinantly engineered region and a second recombinantly engineered region, wherein a first single recombination site is adjacent to the first recombinantly engineered region, and a second single recombination site is adjacent to the second recombinantly engineered region, wherein the first and second recombination sites have nucleotide sequences which share 90-98% identity with each other and optionally with the nucleic acid sequence of any further recombination sites present in the host cell genome polynucleotide.

The invention discloses wide-temperature-range low-temperature beta-galactosidase OUC-JST derived from flavobacterium fusiformis as well as a coding gene and application of the wide-temperature-range low-temperature beta-galactosidase OUC-JST, and belongs to the technical field of functional enzymes. The beta-galactosidase OUC-JST has an amino acid sequence as shown in SEQ ID No.1, the optimum reaction temperature is 20 DEG C, the relative enzyme activity in a wide temperature range from 5 DEG C to 30 DEG C is kept at 50% or above, 20% of the enzyme activity is still kept at 0 DEG C, and the beta-galactosidase OUC-JST shows excellent wide-temperature-range low-temperature adaptability. The invention also provides a gene SEQ ID No.2 for coding the enzyme, a recombinant expression vector and recombinant engineering bacteria containing the gene, and a preparation method of the enzyme. The beta-galactosidase OUC-JST disclosed by the invention is suitable for hydrolyzing lactose at low temperature, and has a wide application prospect in the field of lactose-free dairy product production.

The invention discloses an isopentenyl transferase and an application of the isopentenyl transferase in synthesis of bakuchiol. Specifically, recombinant saccharomyces cerevisiae for expressing the isopentenyl transferase is constructed, the isopentenyl transferase CcPT6 from fructus psoraleae is obtained through screening, the isopentenyl transferase CcPT6 can catalyze an isopentenylation reaction of a coumaric acid substrate to generate bakuchiol, and the activity of the isopentenyl transferase CcPT6 is higher than that of a reported sequence BAK36. The CcPT7 is obtained by truncating 54 amino acids at the N end of the CcPT6, the catalytic activity is improved by 74%, and the activity of the CcPT7 is higher than that of a reported BAK36 mutant. In addition, the invention also provides a recombinant saccharomyces cerevisiae and yarrowia lipolytica engineering strain for integrally expressing the isopentenyl transferase CcPT6 or CcPT7, so that the artificial biosynthesis of the bakuchiol is realized. The discovery of the isopentenyl transferase and the construction of the recombinant engineering strain have important meanings for promoting the biosynthesis of the bakuchiol.

The present invention provides recombineering-editing systems using CRISPR and recombination enzymes as well as methods, vectors, nucleic acid compositions, and kits thereof. The methods and systems provide means for altering target DNA, including genomic DNA in a host cell.

This invention relates to the field of enzyme engineering technology, and in particular to a total synthetic ketone reductase and its applications. The amino acid sequence of the total synthetic ketone reductase is shown in SEQ ID NO:2 or SEQ ID NO:3. This total synthetic ketone reductase can be used to prepare (S)-3-hydroxytetrahydrofuran. Specifically, recombinant engineered bacteria are cultured, lysed, and added to a reaction system containing substrate compound II, NAD+, and isopropanol. The reaction yields compound I. The specific route is as follows: the recombinant engineered bacteria are *E. coli* BL21(DE3) as the host and pET-30a(+) as the vector, expressing the above-mentioned total synthetic ketone reductase. The ketone reductase of this invention can stably express heterologous functionality, and it exhibits highly efficient catalytic activity in the preparation of the chiral compound (S)-3-hydroxytetrahydrofuran, demonstrating significant potential for industrial application.

The invention provides recombinant III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. On the basis of human-derived III-type collagen COL3A1, an amino acid sequence suitable for efficient expression of pichia pastoris is obtained after optimization design and screening, a recombinant expression vector is provided on the basis, and recombinant engineering bacteria are constructed. The recombinant III-type micromolecular collagen is successfully expressed by adopting the constructed recombinant engineering bacteria, and the invention further provides a preparation method of the recombinant III-type micromolecular collagen. According to the present invention, the efficient expression of the recombinant III-type micromolecule collagen in the yeast is achieved, and the recombinant III-type micromolecule collagen has characteristics of high stability, and no significant degradation under different pH conditions, repeated freezing and thawing treatment, and different temperature and time duration treatment; the main band accounts for 90% or above in the fermentation stage, the purification difficulty is low, the sponge purity is 95% or above, and the cell adhesion activity is excellent while the stability is high.

The invention provides a combined module for enhancing symbiotic nitrogen fixation capacity of rhizobium and application, and belongs to the technical field of synthetic biology. The combined module for enhancing the symbiotic nitrogen fixation capacity of the rhizobium comprises an inducible promoter element Pi and a transcriptional regulatory factor coding gene tetR, wherein the inducible promoter element Pi and the transcriptional regulatory factor coding gene tetR are sequentially connected; the inducible promoter element Pi specifically responds to a symbiotic nitrogen fixation signal; according to the invention, the combined module is inserted into an initial vector to construct a recombinant expression vector, and the recombinant expression vector is transferred into different soybean rhizobium chassis through a triparental conjugation method to obtain two nitrogen fixation recombinant engineering strains; the recombinant engineering strain can recover the influence of glyphosate stress on the symbiotic nitrogen fixation capability of the rhizobium.