134 results about "Host bacterium" patented technology

The invention relates to a recombinant pichia pastoris strain capable of producing astaxanthin at high yield as well as a construction method and application of the recombinant pichia pastoris strain. The recombinant pichia pastoris strain is obtained by expressing ATP (adenosine triphosphate) citrate lyase (ACL) and acetyl-CoA synthase (ACS) or phosphoketolase (PK) and phosphotransacetylase (PTA), choline kinase (CK), inositol polyphosphate kinase (IPK) and vitreoscilla hemoglobin (VHB) in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified strain PP-LC2. The astaxanthin production performance of the recombinant strain is verified on the basis of comparison of precursor supply pathways, IUP pathway construction and improvement of the oxygen supply capacity of the engineering strain, and the astaxanthin production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, the synthesis of astaxanthin can be better promoted, the engineering strain can efficiently synthesize the astaxanthin by utilizing methanol through amplification fermentation of a 5L fermentation tank, and industrial production is facilitated.

The invention relates to a recombinant bacterium for overexpressing uranyl binding protein and application of the recombinant bacterium, and belongs to the technical field of microorganisms. Bacterial outer membrane protein and uranyl binding protein are expressed in host bacteria, and the uranyl binding protein is anchored on an outer membrane of recombinant bacteria through the bacterial outer membrane protein, so that the uranyl binding protein anchored on the bacterial outer membrane can adsorb uranyl ions. Meanwhile, the invention finds that the expression quantity of the uranyl binding protein can be increased by performing fusion expression on the uranyl binding protein by using LPP-OMPA, so that the adsorption effect of uranyl ions is improved. The recombinant bacterium is prepared into a freeze-dried capsule product, and the freeze-dried capsule product can penetrate through a stomach barrier and is accurately delivered to intestinal tracts so as to realize uranyl ion adsorption.

The invention discloses a bacteriophage host prediction method based on a weighted integrated convolutional neural network. The method comprises the following steps: obtaining a joint feature vector for an input bacteriophage protein sequence and a host receptor protein sequence; taking the bacteriophage-host pair as a positive sample, randomly selecting non-host bacteria for each bacteriophage to generate a negative sample, dividing the negative sample into M subsets, and combining each subset with all the positive samples to form M balance training subsets; for each balance training subset, training an independent one-dimensional convolutional neural network model; independently training a one-dimensional convolutional neural network sub-model for each balance training subset to obtain a plurality of sub-models with different performances; and distributing weights based on the performance indexes of the sub-models on the verification set, performing weighted fusion on the prediction probabilities of the M sub-models, and outputting a final host interaction probability. The method realizes accurate prediction of phage-host interaction on the premise of only needing basic sequence information, and has the advantages of light weight, high efficiency and wide applicability.

The invention provides an application of a TGBp1 gene, or a TGBp1 gene coding protein, or a recombinant vector or host bacteria containing a TGBp1 gene coding region in down-regulation of the MeGRXC3 expression level. Researches find that TGBp1 and MeGRXC3 interact with each other, so that the growth ability of yeast in an SD / -Leu / -Trp / -His / -Ade auxotroph culture medium can be improved, the expression of MeGRXC3 can be down-regulated, the active oxygen content of tobacco leaves for co-expression of TGBp1 and MeGRXC3 is improved, and the growth ability of the yeast in the SD / -Leu / -Trp / -His / -Ade auxotroph culture medium is improved. Research results lay a theoretical foundation for breeding of common cassava mosaic virus disease resistance.

The present disclosure provides engineered bacterial cells having one or more genetic modifications that result in increased production of expression products, such as increased production of plasmids with reduced occurrence of plasmid concatemers or multimers. Also disclosed herein are expression systems for product manufacturing in media having reduced antibiotic concentration, as well as methods of making cells for use in the expression systems. The cells and expression systems increase the growth rate and production yields of biomolecules produced within the host bacterial host cells without the use of antibiotics, and can produce the product with little to no endotoxins present.

The present disclosure provides engineered bacterial cells having one or more genetic modifications that result in increased production of expression products, such as increased production of plasmids with reduced occurrence of plasmid concatemers or multimers. Also disclosed herein are expression systems for product manufacturing in media having reduced antibiotic concentration, as well as methods of making cells for use in the expression systems. The cells and expression systems increase the growth rate and production yields of biomolecules produced within the host bacterial host cells without the use of antibiotics, and can produce the product with little to no endotoxins present.

Provided are a preparation method for a yeast-expressed type I recombinant human collagen with a tri-helical structure and use thereof. The preparation method comprises the steps of performing fermentation using an engineered bacterium and performing purification to obtain the type I recombinant human collagen with a tri-helical structure. A method for constructing the engineered bacterium comprises the step of transforming a host bacterium with plasmids pMChZ-α1(I), pMCrN-α2(I), pPIC9K-P4H(DP)-1, and pMCeH-[α1(I)] 2α2(I) to construct the engineered bacterium. The type I recombinant human collagen with a tri-helical structure prepared using the preparation method comprises hydroxyproline, and hydroxyproline accounts for 30% or more of total proline.

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 provides an anti-white spot syndrome virus peptide derived from litopenaeus vannamei, and belongs to the technical field of biology. The amino acid sequence of the anti-white spot syndrome virus peptide provided by the invention is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2. The invention further provides a recombinant expression vector and host bacteria containing the anti-white spot syndrome virus peptide gene, and application of the recombinant expression vector and host bacteria in anti-white spot syndrome virus drugs, vaccines, feeds and feed additives, and the recombinant expression vector and host bacteria have wide application prospects and economic benefits.

The invention discloses a method for improving the heterologous soluble expression efficiency of cytochrome P450, which is characterized in that plasmids containing coding genes of fungus-derived cytochrome P450 truncated protein and plasmids containing molecular chaperones are jointly transferred into host bacteria to obtain recombinant genetically engineered bacteria for improving the heterologous soluble expression efficiency of cytochrome P450. Through host screening, molecular chaperonin and cytochrome P450 gene modification, the soluble expression efficiency of cytochrome P450 in Escherichia coli is improved, and the problems of poor protein solubility and low expression efficiency when CYP450 is expressed in Escherichia coli at present are solved.

The invention discloses a method for efficiently expressing FAD-dependent glucose dehydrogenase in pichia pastoris. The method comprises the following steps: (1) constructing a recombinant plasmid containing a promoter, a signal peptide, a glucose dehydrogenase gene and a purification tag; the nucleotide sequence of the signal peptide is as shown in SEQ ID No.2; (2) transforming the recombinant plasmids into host bacteria pichia pastoris to obtain recombinant pichia pastoris engineering bacteria capable of expressing FAD-dependent glucose dehydrogenase; and (3) carrying out fermentation culture, separation and purification on the recombinant pichia pastoris engineering bacteria to obtain the FAD-dependent glucose dehydrogenase. According to the invention, the expression level and the final yield of the FAD-GDH are obviously improved.

The invention provides a preparation method of thermal-stability T7RNA polymerase and the thermal-stability T7RNA polymerase, belongs to the field of biology, and can solve the problems that an existing T7RNA polymerase preparation process is complex, the expression culture period is long, multi-step purification treatment is needed, liquid change treatment needs to be carried out independently before storage, and the production cost is high. The method comprises the following steps: transferring plasmids containing a T7RNA polymerase gene sequence into expression host bacteria M15, culturing, screening, cloning, adding glycerol for strain preservation, and establishing a strain three-level library; the method comprises the following steps: taking glycerol bacteria, carrying out streak resuscitation on a flat plate, selecting monoclone, inoculating the monoclone into an LB culture medium, culturing, carrying out enlarged culture, carrying out IPTG induced expression, centrifugally collecting thalli, cleaning, adding a lysis buffer solution, resuspending, carrying out ultrasonic cell disruption, centrifuging, and collecting supernate; purifying by nickel column affinity chromatography, removing impure protein by an impure washing buffer solution, eluting target protein, purifying by matching with anions and cations, and collecting the purified protein; and mixing the purified protein with a storage buffer solution, and subpackaging and storing. The method can be applied to rapid preparation of the thermal-stability T7RNA polymerase.

The invention belongs to the field of industrial application of microbial fermentation, and relates to a microorganism for efficiently producing ergothioneine as well as a preparation method and application of the microorganism. Specifically, the method comprises the following steps: firstly, artificially creating a promoter with a group response property by utilizing saturation mutation and screening of a fluorescence microscope; then, the promoter is used for mediating host bacterium heterologous histidine methyltransferase, sulfoxide synthase and PLP dependent C-S lyase to improve the yield of ergothioneine; on the basis, an endogenous promoter HisJ of host bacteria is replaced, and endogenous cysteine lyase yhaM is knocked out, so that the yield of the ergothioneine is increased again. According to combination of the above strategies, a heuristic and commercialized production thought is provided for efficient synthesis of ergothioneine, and a foundation is laid for reduction of the synthesis cost of ergothioneine.

The invention belongs to the technical field of biology, and particularly relates to perforin derived from staphylococcus warneri mild bacteriophage and application. Perforin has a nucleotide sequence as shown in SEQ ID NO: 1. The perforin is a key gene for bacteriophage splitting growth and plays an important role in the splitting process of host bacteria. Therefore, the research aims to verify whether the perforin of which the coding site is positioned on a staphylococcus warneri mild bacteriophage genome can be used as a cell lysis gene and can be used as a potential antibacterial substance or not, and a theoretical basis is provided for prevention and treatment of gram-negative pathogenic bacteria.

The present invention discloses a recombinant type III collagen with high thermal stability and high transdermal absorption and its application, belonging to the technical field of genetic engineering drugs. The core amino acid sequence of a secretion peptide is inserted upstream of the collagen active fragment gene, and a purification tag is added downstream of the collagen active fragment gene; then this gene sequence is subjected to protein expression and purification in a host bacterium; the amino acid sequence of the collagen active fragment is as shown in SEQ ID NO.3; the core amino acid sequence of the secretion peptide is as shown in SEQ ID NO.2. The amino acid sequence of the recombinant type III collagen with high thermal stability and high transdermal absorption is as shown in SEQ ID NO.1. By the above method, the recombinant type III collagen containing the secretion peptide of the present invention overcomes the poor absorbability of natural collagen, shows a higher transdermal absorption effect, and has high thermal stability.

The invention discloses a preparation method and application of a recombinant A-type botulinum toxin, and aims to industrially produce the recombinant A-type botulinum toxin by utilizing the preparation method of the recombinant A-type botulinum toxin provided by the invention. The method comprises the following steps: firstly, constructing genetically engineered bacteria capable of heterologously expressing a botulinum toxin A gene in host bacteria by virtue of a genetic engineering means, then accumulating recombinant botulinum toxin A in a strain by virtue of the genetically engineered bacteria through high-density fermentation, collecting fermented thalli, crushing and purifying to obtain the recombinant botulinum toxin A, and finally, preparing the recombinant botulinum toxin A by virtue of the recombinant botulinum toxin A. And adding an auxiliary poising agent and a protective agent to prepare a liquid agent or a freeze-drying agent to obtain the recombinant A-type botulinum toxin for injection. According to the technical scheme, the complete recombinant botulinum toxin A with the purity as high as 99.9% is obtained, the yield is as high as 7000000 U / L, and the complete recombinant botulinum toxin A has no obvious difference from natural botulinum toxin A in structure and function and can be applied to multiple fields such as medical treatment, medical beauty and military affairs.

The invention provides a method for improving the expression level of a meat toxin light chain. The method comprises the following steps: connecting a dissolution-promoting tag to an N end or a C end of meat toxin light chain protein to form soluble meat toxin light chain recombinant protein; the dissolution-promoting tag is a wild dissolution-promoting tag NT11 or a mutant protein mut7 of the wild dissolution-promoting tag NT11, enzyme digestion removal is not needed, the activity of a meat toxin light chain is hardly influenced and interfered, and the fusion protein can keep 93% of substrate enzyme digestion activity. The method further comprises overexpressing a molecular chaperone htpG in the genome of the host bacterium. According to the invention, a BoNT / A-LC high-efficiency expression system is constructed through multi-strategy optimization, so that the yield of BoNT / A-LC is increased by nearly 20% compared with that of wild type NT11-Lc, the yield reaches 647 mg / L through overexpression of a molecular chaperone htpG, and after combinatorial optimization, the yield is 8.5 times that of only expressed BoNT / A Lc, 1.79 times that of NT11-Lc and 1.43 times that of Mut7-Lc.

The application discloses a kind of exogenous bacteriophage inoculation methods for strengthening soil microbial arsenic methylation capacity, comprising the following steps: S1. preparation bacteriophage crude extract;S2. preparation concentrated solution;S3. preparation bacteriophage extract;S4. preparation inoculation soil sample;S5. in the 2, 4, 6th day of culture, bacteriophage extract is added to inoculation soil;S6. after three times inoculation, continue to flood culture 9 days.The application proposes a kind of inoculation method for retaining virulent bacteriophage, not retaining original host bacteria, inoculating after flooding two days, and inoculating three times, so that the proportion of lysogenization of bacteriophage in soil after inoculation is increased by 156%~347%, so that arsenic metabolism genes carried by bacteriophage genome can function, the arsenic methylation capacity of soil microorganism is strengthened, and the arsenic methylation capacity is increased by more than 300%, to efficiently methylate arsenic in soil.

The invention discloses an optimized recombinant chicken double-cell factor as well as a preparation method and application thereof. The chicken recombinant double-cell factor coding gene provided by the invention contains complete ChIL-2 and ChIFN-alpha structures. The preparation process comprises the following steps: selecting a pichia pastoris expression system; optimizing the recombinant chicken double-cell factor according to the codon preference of pichia pastoris; inserting the optimized gene sequence into an expression vector, and introducing host bacteria to construct a recombinant yeast engineering strain; carrying out inoculated culture by using the obtained recombinant yeast, and screening strains containing target protein based on expression level to obtain required culture bacteria; and breeding and culturing the obtained high-expression-level culture bacteria. According to the invention, ChIL-2 and ChIFN-alpha are connected through a modified flexible linker, and a nucleotide sequence is replaced by preferred codons of pichia pastoris, so that a chicken recombinant cell factor can be secreted to a supernatant, the defect of ChIL-2 expression quantity is overcome, and the problem that proteins of ChIL-2 and ChIFN-alpha mainly exist in bacteria and inclusion bodies in prokaryotic expression is solved.

The application discloses a biosynthesis method of blue copper peptide, comprising the following steps: synthesizing a recombinant protein according to an amino acid sequence shown in SEQ ID NO. 1, synthesizing a gene coding the recombinant protein, constructing a recombinant plasmid by using the gene, transferring the recombinant plasmid into a host bacterium, carrying out fermentation culture to induce protein expression, separating the protein, carrying out enzyme cutting and purification, and finally carrying out complexing with copper ions to obtain the blue copper peptide. Compared with the prior art, the biosynthesis method of the blue copper peptide has the advantages of low production cost, green environmental protection and high yield by optimizing the design of the fusion protein, and the yield of the final product can be significantly improved, and the fusion protein has better structure and isoelectric point characteristics.

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

A phage composition and an use thereof in strengthening soil carbon fixation, said phage composition including three phages, namely, a polyvalent phage φYSZKP that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa; a Bacillus cereus phage φYSZBA1; and a Bacillus cereus phage φYSZBA2. In the present invention, the phage composition is injected into the target soil, and an approach of strengthening soil carbon fixation by means of cooperation with the host bacteria through injection of AMGs from the phage is employed; at the same time, the phage has a function of regulating the soil microbial communities from bottom to top; a network of symbiosis with indigenous bacteria is further optimized, and the soil carbon fixation potential is improved. This technique is an environmental improvement technique that achieves a good carbon fixation effect, has a low price and is environment-friendly.

This invention discloses a method for the efficient synthesis of gastrodin using recombinant Corynebacterium glutamicum, belonging to the fields of genetic engineering, fermentation engineering, and synthetic biology. This invention uses Corynebacterium glutamicum as the host bacterium and knocks out… pobA The gene was expressed, and the engineered strain WN01 / pB6 was constructed using the expression plasmid pEC-XK99E to express cladoid-pyruvate lyase, carboxylic acid reductase, and glycosyltransferase, achieving heterologous synthesis of gastrodin. Further overexpression was then performed. aroG Genes, overexpression of protein-coding genes that activate carboxylic acid reductase sfp The engineered strain WN05 / pB6 was able to synthesize 7.37 g / L of gastrodin at the shake flask level, and the gastrodin yield reached 17.77 g / L at 72 h of batch fermentation in a 5-L fermenter, with a yield of 0.247 g / L / h. This provides a brand-new biomanufacturing platform for the industrial production of gastrodin.

This invention discloses an anti-phage system based on ESCRT-III and its applications, relating to the field of biotechnology. This invention utilizes the coding genes for two structural proteins of the Hoda_Snf7_1 and Hoda_Snf7_2 from the Hoda archaea ESCRT-III. Hoda_ snf7_1 and Hoda_snf7_2 This invention is applied to antiphage systems. Compared with existing technologies, the main advantages of the antiphage system of this invention are: (1) it consists of only two genes, making system construction simple and quick; (2) it is composed of structural proteins, making it less prone to losing antiphage activity due to gene mutations; (3) it has a broad-spectrum antiphage effect; and (4) it does not affect the normal growth of host bacteria. This system expands the understanding of antiphage systems and provides new ideas and technical means for preventing and controlling phage contamination in industrial microbial production processes.

The invention discloses a recombinant prokaryotic expression vector, an expression strain, soluble human-derived protein and application, the recombinant prokaryotic expression vector comprises a first nucleotide sequence for coding a target human-derived protein and a second nucleotide sequence for coding a cold-adapted molecular chaperone, and a rare codon corresponding to host bacteria is introduced into the first nucleotide sequence to obtain a recombinant prokaryotic expression vector. The translation rate of a key site is actively reduced, a cold-adapted molecular chaperone is introduced into a second nucleic acid sequence, and correct folding and soluble expression of the human-derived protein in a prokaryotic host are promoted in a low-temperature induction manner, so that the high-yield and high-solubility human-derived soluble protein is obtained. The invention provides a feasible way for large-scale preparation of human soluble protein, and has important industrial application value.

The present invention relates to a method for constructing a host bacterium with high expression of human serum albumin, which method comprises: S100, designing an optimized gene encoding human serum albumin; S200, constructing a recombinant expression vector; and S300, transforming the recombinant expression vector into a Pichia pastoris host bacterium. The optimization design of a full-length sequence of a target gene is performed at the molecular level to obtain a target gene with the most suitable expression in Pichia pastoris; at the cellular level, the latest international Pichia pastoris expression system host bacterium CBS7435 is introduced for the gene expression of human serum albumin; and Pichia pastoris transformants harboring high-copy-number exogenous genes are selected by means of using a G418 resistance gene for the construction of an engineering bacterium with high expression of recombinant human serum albumin.

This invention belongs to the field of biotechnology, specifically relating to a perforin derived from *Staphylococcus warwickiana* temperate bacteriophage and its applications. The perforin has the nucleotide sequence shown in SEQ ID NO:1. This perforin is a key gene for phage lysis and growth, playing a crucial role in the lysis of host bacteria. Therefore, this study aims to verify whether the perforin, whose coding site is located on the genome of *Staphylococcus warwickiana* temperate bacteriophage, as a cell lysis gene, can serve as a potential antibacterial substance, providing a theoretical basis for the prevention and control of Gram-negative pathogens.

This invention relates to the fields of environmental monitoring and biosafety technology, and in particular to a viral aerosol sampling patch for face masks, comprising: a capture layer for intercepting viral aerosol particles in the air; a culture layer disposed inside the capture layer, the culture layer comprising a culture unit supporting the virus to maintain activity and replicate in the presence of a host, a host bacterium sensitive to the virus, and a visual indicator indicating viral replication or host response; and a base layer disposed inside the culture layer; the sampling patch is fixed to the face mask through the base layer, and viral aerosol collection is achieved without external power through the capture layer, and countable detection results are generated after sampling through the culture layer; utilizing the airflow and humidity conditions naturally formed during wearing, effective interception, activity preservation, and visual detection of viral aerosols are achieved without complex equipment and additional operating steps, and the operation is simple and repeatable.

This invention belongs to the field of protein engineering and biomedicine, and particularly relates to a water-soluble membrane protein, a recombinant vector, a recombinant host bacterium, and their modification methods and applications. The method involves the following steps: First, an interface mutant is constructed based on the SQTY code, and its water solubility and ligand binding ability are evaluated. If the requirements are not met, multiple low-impact transmembrane regions are screened, and after mutation modification, the interface mutant is introduced to construct a single-transmembrane combined mutant, whose water solubility and ligand binding ability are evaluated. If the requirements are still not met, the multiple low-impact transmembrane regions are combined in pairs, and the interface mutant is introduced to construct various double-transmembrane combined mutants, whose water solubility and ligand binding ability are evaluated, and the optimal double-transmembrane combined mutant is selected. This method rationally mutates CXCR4 in stages to achieve water solubility, minimizing changes to the protein's structure and other physicochemical properties, thereby maintaining or even enhancing its binding ability to the ligand CXCL12.

The invention relates to a methanophilic genetically engineered bacterium for producing gamma-polyglutamic acid (gamma-PGA) as well as a construction method and application of the methanophilic genetically engineered bacterium, EQU2420670 gene is knocked out on a host bacterium genome, and meanwhile, an exogenous gamma-PGA synthetase encoding gene cluster pgsBCAE under the control of an inducible arabinose promoter (Pbad) is introduced at the site. The construction method comprises the following steps: obtaining a multi-cistron structure DNA fragment pgsBCAE for coding gamma-PGA synthetase through artificial gene synthesis in combination with a codon optimization strategy, and constructing the expression module Pbad-pgsBCAE under the control of a Pbad promoter. A suicide vector pK18mobsacB carrying a SacB sucrose reverse selection system is used for constructing a gene replacement vector, upstream and downstream homologous arms of an EQU2420670 gene on a genome of methanophilic bacteria M. buryatase 5GB1 are designed on two sides, an EQU2420670 site is replaced by a Pbad-pgsBCAE expression module, the methanophilic genetically engineered bacterium PGA is obtained, and the methanophilic genetically engineered bacterium PGA is applied to production of gamma-PGA and has important economic and social significance; the gamma-PGA produced by the engineering methanophilic bacteria by using methane as a carbon source and an energy source can be used as a cosmetic additive; the invention provides a novel low-carbon biosynthesis approach for commercial production and application of gamma-PGA, and has a good industrial application prospect.