158 results about "Recombineering" patented technology

The present invention relates to a recombinant human type IV collagen and a preparation method therefor, which belong to the technical field of bioengineering. A recombinant human type IV collagen is designed on the basis of a partial sequence in an α2 chain of human type-IV collagen, and DNA encoding of the protein is ligated into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into competent Escherichia coli cells, and a recombinant expression plasmid is extracted; after the plasmid is linearized, the linearized plasmid is transformed into an engineered strain HCPB-PPKEX2 to obtain a recombinant engineered strain; and the engineered strain is cultured, the expression of a target protein is induced, and the target protein is purified and identified. It is further demonstrated experimentally that the collagen can be completely cleaved by Kex2, Ste13 and CPB enzymes and efficiently secreted and expressed extracellularly, and the obtained sequence is completely consistent with a theoretical sequence. It is also verified experimentally that the recombinant collagen has a cell adhesion activity and a cell migration-promoting activity. The method avoids the risk of residual exogenous proteins, shortens the time and reduces the cost of the subsequent purification process, and thus has a relatively high safety and application value.

Provided is a recombinant human serum albumin-collagen binding domain (CBD) fusion protein for tumor-specific targeting matrix and its use in specific targeted treatment of a solid tumor. HSA and the CBD generate gene fusion in the form of different molecular structures, and can be expressed in a constructed recombinant engineered host. A pharmaceutical recombinant fusion protein is obtained through large-scale fermentation. The recombinant fusion protein shows tumor-specific targeting of solid tumors and can be used as a starting material of a broad-spectrum matrix carrier. The recombinant fusion protein is coupled with any tumor treatment drug, small-molecule compound, or cytotoxic protein to form an innovative pharmaceutical composition. The pharmaceutical composition of the recombinant fusion protein is also long-acting, can prolong a half-life of the conjugate drug in vivo, and has a significantly better clinical curative efficacy on anti-tumor than that of a monomer drug.

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

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 provides a recombinant engineering bacterium for preparing recombinant equine albumin and application of the recombinant engineering bacterium, and relates to the technical field of genetic engineering. According to the invention, one or more of genes SED1 and TIP1 related to cell wall composition is / are knocked out in a targeted manner while the recombinant equine albumin is expressed, and / or one or more of genes PAC10 and YDJ1 related to protein folding promotion is / are overexpressed and integrated into a strain, so that the expression quantity of the recombinant equine albumin is improved. According to the recombinant engineering bacterium disclosed by the invention, the yield of the recombinant equine albumin can be obviously improved, the highest yield can reach 51.27 g / L, and compared with an unmodified recombinant engineering bacterium, the yield is improved by 5.38 times, and the recombinant engineering bacterium has a wide 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 discloses an application of silkworm fatty acid dehydrogenase Bmdesat13 in enzymatic catalytic generation of trans-palmitoleic acid and a method thereof, the silkworm fatty acid dehydrogenase Bmdesat13 gene ORF sequence is cloned from silkworms, and a brewer's yeast eukaryotic expression system is used for heterologous expression of Bmdesat13, so that the trans-palmitoleic acid is obtained. An enzyme activity product which can be synthesized by the gene in a yeast positive recombinant engineering strain is identified to be trans-9-hexadecenoic acid (trans-palmitoleic acid) through GC-MS (gas chromatography-mass spectrometry), so that theoretical and technical supports are provided for mass production of trans-palmitoleic acid.

The invention provides a mutant of xylose reductase and application of the mutant in synthesis of D-tagatose. According to the present invention, the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification on the basis of the sequence and structure-oriented semi-rational modification strategy so as to obtain the high activity xylose reductase mutant, and the xylose reductase mutant has the following characteristics that: the xylose reductase (XR) derived from Saccharomyces cerevisiae is subjected to molecular modification so as to obtain the xylose reductase mutant, according to the invention, the mutant of the xylose reductase is integrated into a redox pathway for synthesizing the D-tagatose by virtue of a genetic engineering means, and is expressed in bacillus subtilis 168, so that a recombinant engineering bacterium for synthesizing the D-tagatose by fermenting D-galactose is successfully constructed, the efficient synthesis of the D-tagatose is realized, and the yield of the D-tagatose is improved. And the method has obvious practical application value and economic benefit.

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 a nucleic acid molecule for preparing indigoidine, an expression vector and a recombinant engineering strain. The nucleic acid molecule comprises a sequence as shown in SEQ ID NO.2 or a nucleotide sequence which has at least 98% sequence identity with the SEQ ID NO.2. According to the present invention, the Wlpl protein encoded by the nucleic acid molecule is derived from Williamsiapylosphaerae bacteria, the amino acid sequence of the Wlpl protein is represented by SEQ ID NO.1, the protein has excellent 4 '-phosphoric acid pantetheine transferase catalytic activity, and when the Wlpl protein is used for catalyzing the synthesis process of glutamine to synthesize the indigoidine, the yield of the indigoidine can achieve 14.26 g / L, such that the indigoidine yield is substantially improved, and the good industrialization prospect is provided;

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 present invention provides a fusion protein KP-Ag1 used as a Klebsiella pneumoniae vaccine antigen, which is composed of KP6 protein, KP21 protein and KP24 protein connected by a flexible amino acid Linker; wherein, the amino acid sequence of the KP6 is SEQ ID NO:4, the amino acid sequence of the KP21 is SEQ ID NO:5, and the amino acid sequence of the KP24 is SEQ ID NO:6. The present invention also provides its expression vector and recombinant engineering diagram, as well as its application as a Klebsiella pneumoniae antigen. The antigen protein prepared by the method described in the present invention can effectively stimulate the body to produce a higher humoral immune response, and can provide obvious protection against lethal-dose Klebsiella pneumoniae infection, and can be used as a candidate antigen for Klebsiella pneumoniae vaccine.

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.

The present invention provides a recombinant engineered bacterium for producing recombinant canine albumin and its application, relating to the field of biochemistry, specifically, genetic engineering technology. By integrating overexpression of one or more of the protein-folding-related genes PBN1, KAR2, and ERV2 into the strain while simultaneously expressing recombinant canine albumin, the present invention significantly improves canine albumin expression, with relative protein expression reaching up to 491% and specific yields reaching up to 32.83 g / L, demonstrating broad application prospects.

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 invention relates to the technical field of gene engineering, and discloses a recombinant Escherichia coli engineering strain for producing phenethyl alcohol by using L-phenylalanine as a substrate, the recombinant Escherichia coli engineering strain comprises a recombinant Escherichia coli engineering strain for producing phenethyl alcohol by using L-phenylalanine as a substrate, and the recombinant Escherichia coli contains a key gene capable of synthesizing phenethyl alcohol. Proteins coded by the key gene are a transaminase gene, a phenylpyruvate decarboxylase gene and an ethanol dehydrogenase gene. According to the invention, a recombinant plasmid containing a phenethyl alcohol biosynthetic pathway is constructed, the recombinant plasmid is transformed into Escherichia coli, and a recombinant engineering strain for improving the yield of phenethyl alcohol is screened out. The recombinant escherichia coli engineering strain contains transaminase KmARO8 derived from kluyveromyces marxianus, phenylpyruvate decarboxylase VvPDC1 derived from wine grapes and ethanol dehydrogenase AtADH1 derived from arabidopsis thaliana, the yield of phenethyl alcohol can be 7.29 g / L after fermentation culture of the recombinant strain, and the recombinant escherichia coli engineering strain has a good industrial prospect.

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.