2987 results about "Escherichia coli" patented technology

A recombinant humanized collagen and a preparation method therefor. The recombinant humanized type III collagen can be efficiently and stably expressed in three expression systems of yeast, Escherichia coli, and CHO cells, and has the biological activity superior to that of a commercially available collagen product. The recombinant humanized type III collagen has structural characteristics and functional activity similar to those of a natural collagen, and thus has wide application prospects in biomedical materials, tissue engineering products, cosmetics, foods, health care products or drugs, medical instruments, medical cosmetology and the like.

The present invention discloses a human serum albumin-targeting nanobody and its application. A variety of human serum albumin-targeting nanobodies that can be used to construct long-acting fusion protein drugs and their immune screening methods are disclosed. The antibody is composed only of the heavy chain variable region and can be fused and expressed with therapeutic proteins or polypeptide drugs such as interleukin, interferon, and tumor necrosis factor in an Escherichia coli expression system. The fusion protein has the activity of specifically binding to human serum albumin, has a relatively high affinity, can effectively extend the drug half-life, and does not affect the biological activity of the recombinant protein drug at the same time.

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

The invention belongs to the technical field of biochemical engineering, and provides a method for increasing L-carnosine yield of escherichia coli by increasing PRPP supply, and the yield and yield of L-carnosine are remarkably increased by up-regulating expression of a prs gene, increasing PRPP content in cells and promoting synthesis of L-histidine. Experiments show that the yield of the L-carnosine in different recombinant strains is increased by 55.56%-140%, and L-histidine and beta-alanine do not need to be added in the production process.

The invention relates to the field of microorganisms, in particular to a plant lactobacillus and application thereof in bacteriostasis. Specifically, the preservation number of the plant lactobacillus LP06 is CGMCC (China General Microbiological Culture Collection Center) No. 28412. The plant lactobacillus LP06 provided by the invention can be used for inhibiting the growth of debaryomyces hansenii, candida tropicalis and penicillium rochei, and also has an inhibiting effect on super bacteria acinetobacter baumannii, pseudomonas aeruginosa, klebsiella, escherichia coli, staphylococcus aureus and listeria monocytogenes. The plant lactobacillus LP06 provided by the invention has broad-spectrum bacteriostatic ability, and bacteriostatic powder prepared from the bacterium can be used as a biological preservative for corrosion prevention of dairy products such as yoghourt, cheese and the like and other foods.

The present invention relates to the construction of a high-yield indigo genetic engineering strain and a fermentation method. The present invention uses Escherichia coli as a starting strain, knocks out at least one of the yddG gene, trpR gene, tyrA gene or pheA gene in the host cell; integrates the tryptophanase gene tnaA and / or the flavin monooxygenase gene MaFMO on the genome; integrates one or more genes related to biofilm modification on the genome, and obtains the optimal engineering strain by comparing the fermentation yield; then transfers a plasmid into the strain to further strengthen the tryptophanase gene tnaA and the flavin monooxygenase gene MaFMO; finally, the yield is significantly increased by supplementing carbon sources and substrates and adding surfactants. The present invention provides an indigo-producing genetic engineering strain and a fermentation method with good stability and high synthesis efficiency, providing a reference for the subsequent development of more advantageous engineering strains.

The invention provides recombinant escherichia coli with high yield of ergothioneine and a method for preparing ergothioneine, the recombinant escherichia coli co-expresses ergothioneine anabolic enzymes Egt1 and Egt2, the methyltransferase structural domain of the Egt1 is derived from trichoderma reesei, mycobacterium smegmatis EgtD or rhizopus nigricans, and the methyltransferase structural domain of the Egt2 is derived from the trichoderma reesei, the mycobacterium smegmatis EgtD or the rhizopus nigricans. The histidine sulfoxide synthase structural domain is derived from trichoderma reesei, schizosaccharomyces pombe or rhizopus nigricans, and the Egt2 is derived from claviceps purpurea, neurospora crassa or aspergillus niger. After the recombinant escherichia coli obtained by screening is cultured for 68 hours in a 5L fermentation tank, the yield of ergothioneine can reach 5g / L or above; wherein after recombinant escherichia coli co-expressing Egt1 enzyme (the structural domain of methyltransferase is derived from mycobacterium smegmatis EgtD, and the structural domain of histidine sulfoxide synthase is derived from trichoderma reesei) and Egt2 enzyme (derived from neurospora crassa) are cultured in a 5L fermentation tank for 68 hours, the yield of ergothioneine can reach 8.7 g / L, and the production intensity reaches 127.9 mg / (L.h).

The invention provides a Lactobacillus paracasei, which has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.32951. The Lactobacillus paracasei has a preservation number of CGMCC No.32951. The Lactobacillus paracasei has a preservation number of CGMCC No.32951. The lactobacillus paracasei ID001 provided by the invention has an inhibition effect on enteropathogenic bacteria such as salmonella, escherichia coli, staphylococcus aureus and the like. The probiotic preparation is obtained through fermentation and the dendrobium officinale extract, and the probiotic preparation has the immunoregulation effect through performance verification and screening.

The invention discloses construction and application of an L-malic acid producing issatchenkia orientalis strain, and belongs to the technical field of microorganisms and the technical field of microbial fermentation and metabolic engineering. Through a metabolic engineering means, a cytoplasm metabolic pathway of an issatchenkia orientalis strain is transformed, and a pyruvate decarboxylase coding gene, glycerol dehydrogenase and lactic dehydrogenase are knocked out; a pyruvate carboxylase gene derived from aspergillus flavus, a malate dehydrogenase gene derived from corynebacterium glutamicum and phosphoenolpyruvate carboxylase derived from escherichia coli are screened and integrated, so that a cytoplasm rTCA cycle synthesis pathway is enhanced; inserting a dicarboxylic acid external transporter gene from schizosaccharomyces cerevisiae at a neutral site GD21, and knocking out endogenous dicarboxylic acid internal transporter; the L-malic acid yield of the modified yeast strain in the fermentation process is remarkably increased and reaches 140.16 g / L. The engineering strain provided by the invention has good industrial application potential in efficient L-malic acid production.

The invention provides recombinant escherichia coli for de novo synthesis of anisic acid. At least one of the following improvements is carried out on an escherichia coli starting strain: (1) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus, (2) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus, and (3) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus; the method comprises the following steps: carrying out overexpression on chorismate lyase UbiC derived from E.coli MG1655 or Providencia rustiana, and carrying out the overexpression on the chorismate lyase UbiC derived from E.coli MG1655 or Providencia rustiana; (2) carrying out overexpression on a 3-deoxy-D-arabic-heptanone acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroGD146N derived from E.coli MG1655 on the basis of the step (1); and (3) overexpressing synthase PpsA derived from phosphoenolpyruvate (PEP) on the basis of the step (2), and knocking out a pykF gene for coding pyruvate kinase I. Therefore, in the fermentation process of the recombinant escherichia coli, the accumulation of the anisic acid in the fermentation liquor can be realized, the maximum anisic acid shake flask yield of the recombinant escherichia coli can reach 284.9 mg / L, and a foundation is laid for the subsequent construction of high-yield anisic acid engineering bacteria.

The invention belongs to the field of bioengineering, and particularly discloses a tryptophan synthetase mutant with improved enzymatic activity and application of the tryptophan synthetase mutant in production of L-cysteine. Compared with wild tryptophan synthetase from escherichia coli, the amino acid sequence of the tryptophan synthetase mutant has the advantages that a beta catalytic subunit has T69A, S143A, R219E and F280H site combined mutation. Researches show that the enzyme activity of the mutant is increased by 2.4 times compared with that of a wild type through enzyme activity determination and analysis of inducible expression and purification. The beneficial tryptophan synthetase mutant provided by the invention can efficiently biocatalyze L-serine and sodium hydrosulfide to synthesize L-cysteine, and has a better industrial application prospect.

The invention discloses humanized III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. According to the method, firstly, on the basis of an original humanized III-type collagen gene, cell adhesion activity is taken as an evaluation index, a better cell adhesion sequence is screened out and spliced and integrated with the original collagen gene, and brand-new humanized III-type collagen is obtained. And constructing a recombinant expression vector of the humanized III type collagen containing the adhesion sequence by taking the probiotic EcN as a host cell, and introducing the recombinant expression vector into the EcN to obtain a strain of functional recombinant probiotic. Compared with a traditional escherichia coli engineering bacterium BL21 (DE3), the recombinant probiotics have good cell adhesion activity while efficiently expressing the humanized type III collagen, the bacterial endotoxin content of the recombinant humanized type III collagen obtained through purification meets the industrial standard, and the recombinant probiotics have good application prospects. The purification cost caused by industrial removal of endotoxin can be avoided, so that the production cost is reduced, and the method has a good application prospect.

The invention relates to a construction method of escherichia coli engineering bacteria with high yield of N-acetylglucosamine. According to the method, the metabolic network of escherichia coli is directionally modified through metabolic engineering, the problems of'product re-decomposition ', by-product accumulation, poor genetic stability and the like of natural strains are solved, and efficient industrial production of GlcNAc is realized.

The invention relates to the technical field of synthetic biology, in particular to recombinant XVII type collagen as well as a preparation method and application thereof.The amino acid sequence of the recombinant XVII type collagen comprises a sequence shown in any one of SEQ ID NO.1-SEQ ID NO.3. The recombinant XVII type collagen can be highly expressed in an escherichia coli and pichia pastoris system, the expression system is simple, the cost is low, and the recombinant XVII type collagen is suitable for industrial production. The yield is high; the biological activity is good. The protein can be compounded with other proteins to form raw materials of tissue engineering products, cosmetics, health care products or medicines, so that the protein can be widely applied to the fields of medicines, medical instruments, biological materials, tissue engineering, cosmetics and the like.

The invention provides an NMN efficient synthesis strain and a construction method and production application thereof, and belongs to the technical field of engineering bacteria. According to the invention, firstly, an NMN synthesis route taking NAM as a substrate is constructed in escherichia coli, and specifically, NadV and PrsD128A are introduced into the escherichia coli, so that the NMN synthesis flux is improved; pnuC is introduced to promote the external arrangement of the NMN; the method comprises the following steps: knocking out PncC, NadR, UshA, NudC and MazG; pfkA and TalA are knocked out to improve the carbon flux of the pentose phosphate pathway. GDH1 and GDH2 are heterologously expressed in engineering bacteria to establish a reducing power balance system, the reducing power balance system is used for converting NADPH excessively accumulated in an NMN synthesis route into NADH, and meanwhile, an esaR-esaI group response element is introduced to dynamically regulate and control the system. The engineering strain constructed by the method can accumulate 3.0 g / L of NMN after 24-hour shake flask fermentation.

The invention discloses SGNH family esterase SH2 screened in a Tibetan hot spring environment and application of the SGNH family esterase SH2, and belongs to the technical field of gene engineering. According to the invention, a new SGNH family esterase gene is screened from a Tibetan hot spring environment, and recombinant expression is realized in escherichia coli. The recombinant esterase prepared by the invention has the characteristics of thermal stability, salt resistance, organic solvent resistance and the like, can be safely applied to tooth whitening or skin exfoliating products, avoids damage of traditional chemical bleaching agents to enamel, and has important application value in the fields of daily chemicals, medicines, food processing and the like.

The embodiment of the invention discloses human-derived recombinant III-type collagen as well as a preparation method and application thereof. The amino acid sequence of the human-derived recombinant III-type collagen comprises a basic repetitive unit, and the amino acid sequence of the basic repetitive unit is as shown in SEQ ID NO.4. The amino acid sequence of the basic repetitive unit is as shown in SEQ ID NO.3. The amino acid sequence of the basic repetitive unit is as shown in SEQ ID NO.4. The amino acid sequence of the human recombinant III type collagen is shown as SEQ ID NO.3. The invention also discloses a preparation method of the human recombinant III type collagen. According to the invention, a human recombinant III-type collagen gene with the coding molecular weight of 60kD is designed, an escherichia coli expression strain is constructed and expressed, the yield of the human recombinant III-type collagen is increased through amplification culture on a fermentation tank, and the protein purity is improved by virtue of strategies such as ultrafiltration primary purification and affinity chromatography. The human-derived recombinant III-type collagen prepared by the invention avoids potential hidden dangers such as animal diseases or viruses and immunological rejection caused by a traditional extraction method, has excellent medical performance, can effectively control the production cost and the product quality by virtue of a fermentation preparation process, and is also easy to realize large-scale production.

The invention relates to the technical field of microorganisms, and particularly discloses a recombinant microorganism and application thereof in fermentation production of decarboxylated carnosine. Compared with an original strain, the recombinant microorganism disclosed by the invention has the advantages that the expression of the mdtK gene is enhanced; the starting strain is escherichia coli capable of producing decarboxylated carnosine. When the recombinant microorganism is used for fermentation production, the yield of the decarboxylated carnosine can be increased, L-histidine, histamine or beta-alanine and other precursors do not need to be added in the production process, and an effective novel method is provided for industrial production of the decarboxylated carnosine.

The invention belongs to the technical field of gene engineering, and particularly relates to an RNA polymerase beta subunit mutant and application thereof. The RpoB mutant is obtained through a gene editing technology, after the 560th amino acid of the RpoB is changed into leucine from proline, the number of living cells during strain fermentation is greatly increased, the tolerance of the RpoB mutant in the later environment of a fermentation tank is improved, the RpoB mutant is applied to escherichia coli for producing human milk oligosaccharide, and finally the yield of the human milk oligosaccharide is greatly increased.

The invention relates to Penicillium decumbens C01 and application of the Penicillium decumbens C01, and belongs to the technical field of microorganisms. The penicillium decumbens C01 disclosed by the invention is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the penicillium decumbens C01 is GDMCC NO.66456. The strain is obtained by separating, purifying and screening soil samples collected from botanical gardens in South China of Guangzhou; tests show that the bacteriostatic active substance generated by fermentation of the strain has a good bacteriostatic effect on staphylococcus epidermidis, staphylococcus aureus, escherichia coli, propionibacterium acnes, candida albicans and pseudomonas aeruginosa, and the bacteriostatic active substance has the characteristic of high temperature resistance and excellent preservative effect; the antibacterial preservative can be used as an antibacterial preservative raw material, is applied to the fields of food, health care products or daily chemical products and the like, and has wide application prospects and important conversion research values.

The invention belongs to the technical field of in-vitro cultivation of calculus bovis, and particularly relates to a method for in-vitro cultivation of calculus bovis. According to the invention, firstly, the escherichia coli is subjected to bile domestication and ray induced mutation, a high-activity oxgall-resistant bezoar production engineering bacterium E.coli-NH01 is obtained through screening, a method for in-vitro cultivation of bezoar by using the engineering bacterium E.coli-NH01 is provided, the prepared bezoar meets the pharmacopoeia standard, the production efficiency of in-vitro cultivation of bezoar is improved, and the production cost is reduced. And the production cost is reduced, the overall forming period is shortened, and the key that precious traditional Chinese medicinal materials are popular can be achieved.

The invention relates to the field of animal breeding, and discloses a pig beta defensin compound preparation which comprises pig beta defensin PBD1, PBD2 and PBD114, and the weight ratio of the PBD1 to the PBD2 to the PBD114 is 1: 1: 1. The porcine beta defensin PBD1, the porcine beta defensin PBD2 and the porcine beta defensin PBD114 are recombinant proteins, and are respectively expressed in escherichia coli BL21 (DE3) PLysS competent cells by a pET-32a expression vector. Through the synergistic effect of the defensins PBD1, PBD2 and PBD114, the minimum inhibitory concentration (MIC) of the EPEC is reduced to 18.75 mu g / mL from 75 mu g / mL of single defensins, and is reduced by 75%; within 24 hours, the bacteriostasis rate of the compound preparation reaches 90%, and the inhibition effect on EPEC is remarkably improved.

The invention discloses preparation of a human CD56 immune phage display antibody library, which comprises the following steps: S1, animal immunization: expressing recombinant hCD56 protein in escherichia coli, then immunizing a Balb / c mouse, and detecting immune titer; s2, splenocyte preparation: extracting RNA (Ribonucleic Acid) of immune mouse spleen cells, and performing reverse transcription to obtain cDNA (Complementary Deoxyribonucleic Acid); s3, preparation of an initial bacteriophage display library: amplifying a heavy chain variable region and a light chain variable region by using a PCR technology, and then assembling to form scFv; constructing scFv phagocytids, electrically transforming the scFv phagocytids into TG1 competent cells, and constructing a bacterial library; s4, constructing a phage single-chain library of the hCD56: infecting the bacterial library by using a helper phage M13K07, and constructing a phage single-chain antibody library; and S5, elutriation and screening of a phage library: specifically enriching a phage single-chain antibody library, screening positive clones from the phage single-chain antibody library, and determining a sequence. According to the invention, the human hCD56 antibody scFv sequence fragment can be screened out, and the research and development period is shortened.

The invention discloses a genetically engineered bacterium and an application of the genetically engineered bacterium in production of cis-3-hydroxy-L-proline. According to the genetically engineered bacterium, a proline dehydrogenase gene putA and a proline transporter gene putP in a starting bacterium are silenced, and meanwhile, a gamma-glutamyl kinase mutant gene proBD107N, a gamma-glutamyl phosphate reductase gene proA and a cis-3-hydroxy-L-proline hydroxylase gene P3H are subjected to overexpression. And by comparing a plurality of common escherichia coli, the cis-3-hydroxy-L-proline hydroxylase gene P3H and different gamma-glutamyl kinase mutant genes, the highest yield combination is preferably selected. The strain takes glucose as a raw material, so that the production cost is greatly saved, and the strain has a relatively good industrial utilization value.

The invention discloses an L-aspartic acid transaminase mutant and application thereof. The L-aspartic acid transaminase mutant is obtained by performing single mutation on the 104th amino acid of the amino acid sequence of L-aspartic acid transaminase. The L-aspartic acid transaminase mutant is used for synthesizing an L-phenylglycine derivative. The L-aspartic acid transaminase mutant is from escherichia coli, homologous sequence analysis and molecular docking simulation are carried out, site-specific mutagenesis verification is combined, and single-point mutation is carried out on the 104 amino acid to obtain the T104S mutant. The L-aspartic acid transaminase mutant disclosed by the invention can be used for efficiently catalyzing to generate an L-phenylglycine derivative, and compared with a wild type, the specific activity of the T104S mutant to o-chlorophenoxyacetic acid is 3.2 times that of the wild type.

The invention discloses construction and application of an escherichia coli L-tryptophan biosensor, and belongs to the technical field of bioengineering. The L-tryptophan biosensor provided by the invention comprises a mutated leader peptide fragment tnaC-tnaA, a promoter of the mutated leader peptide fragment tnaC-tnaA, a green fluorescent protein coding gene eGFP and a pTrc99a plasmid skeleton, a recombinant strain containing the L-tryptophan biosensor is cultured, the expression of green fluorescent protein in a culture solution is gradually enhanced along with the increase of the concentration of the L-tryptophan, and the fluorescence value of a unit cell shows better correlation when the concentration of the L-tryptophan is 0-1.5 g / L; therefore, when the biosensor is used for detecting the L-tryptophan in the fermentation liquor, the biosensor has the characteristics of simplicity in operation, sensitive response and high threshold value.

The invention discloses a recombinant human IV-type collagen as well as a preparation method and application thereof, and belongs to the field of bioengineering and protein engineering. According to the invention, three recombinant human IV type collagen new molecules are designed and screened on the basis of a part of sequences in a human IV type collagen alpha1 chain, soluble expression of a target protein is realized by using an escherichia coli system, and the recombinant collagen with the purity of more than 98% can be obtained through nickel column purification and acid regulation treatment. The recombinant collagen provided by the invention has good cell migration promotion activity and cell adhesion activity, and can be widely applied to the fields of tissue engineering, medical equipment, cosmetics, health care products and the like.

The invention discloses construction and application of a plasmid-free and auxotrophy-free L-tryptophan production strain, and belongs to the technical field of bioengineering. According to the invention, a plurality of key anti-feedback inhibition enzymes in a tryptophan synthesis route are optimized and combined, and the optimal combination is integrated into an escherichia coli genome by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats)-ASPR (clustered regularly interspaced short palindromic repeats)-trans-spansses (clustered regularly interspaced short palindromic repeats) system. According to the invention, the plasmid-free and nutritional deficiency-free engineering strain ZH-3 for producing the L-tryptophan is successfully constructed, and under the conditions of no use of antibiotics, use of inducers and no addition of additional substances, the total yield of the L-tryptophan reaches 43g / L and the sugar-acid conversion rate is increased to 0.180 g / g after the strain is cultured for 35h in a 3L fermentation tank. The strategy used by the invention is not only suitable for construction of tryptophan genome integration expression strains, but also provides reference for construction of other plasmid-free amino acid production strains.

The invention provides a genetically engineered bacterium for producing O-succinyl-L-homoserine as well as a construction method and application of the genetically engineered bacterium. In a chassis bacterium genome, the expression of a 2-ketoglutaric acid decarboxylase encoding gene sucA is enhanced, and the expression of a succinyl-coenzyme A synthetase encoding gene sucD is weakened, so that the supply of succinyl-coenzyme A is increased; the method comprises the following steps: increasing the NADPH (Nicotinamide Adenine Dinucleotide Phosphate) reducing capacity and ATP (Adenosine Triphosphate) energy supply of a chassis bacterium, increasing DNA (Deoxyribose Nucleic Acid) in combination with a transcription dual regulatory factor ompR to improve the stress resistance of escherichia coli under high osmotic pressure, and introducing an overexpression plasmid containing a homoserine transsuccinylase coding gene metA to construct the genetically engineered bacterium for producing O-succinyl-L-homoserine. The engineering strain obtained through a systematic metabolic engineering modification strategy can realize effective accumulation of OSH, the shake flask yield of OSH reaches 19.8 g / L, the fed-batch fermentation yield of a 5L fermentation tank reaches 110.5 g / L, the sugar-acid conversion rate reaches 52.6%, and a foundation is laid for subsequent construction of high-yield OSH engineering bacteria.

The invention discloses a functional probiotic composition and application thereof, and relates to the technical field of microbial engineering. Bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis are combined to serve as a microbial agent, the microbial agent and active substances of the tea leaves form a composition, then the composition is combined with prebiotics, it is ensured that viable bacteria directly reach the intestinal tract through a microencapsulation technology, and the effect of improving the immune function is achieved; tea polyphenol in the active substances of the tea leaves can serve as a carbon source of prebiotics to stimulate proliferation of the bifidobacterium longum and the lactobacillus acidophilus, so that the acid resistance and intestinal colonization capacity of the microbial inoculum are enhanced, and meanwhile, theanine in the active substances of the tea leaves, the bifidobacterium longum and the lactobacillus acidophilus synergistically promote secretion of gamma-aminobutyric acid, so that the effect of improving the intestinal colonization is achieved. And the tea polyphenol can inhibit the large intestine rod from adhering to the intestinal mucosa, and the probiotic agent secretes antibacterial peptide, so that the integrity of the intestinal barrier is maintained together.