16 results about "Auxotrophic strain" patented technology

The present disclosure provides recombinant Staphylococcus bacterium (e.g. S. epidermidis) that are dependent on D-alanine for growth. In one aspect, the disclosure features a recombinant Staphylococcus bacterium comprising two inactivated alanine racemase genes (Δalr1 Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In another aspect, the disclosure features a method of making the recombinant Staphylococcus bacterium. In another aspect, the disclosure features a method of treating or preventing a rash in a subject, comprising administering to the subject a population of the recombinant Staphylococcus bacterium of any one of the aspects or embodiments described herein, in an effective amount to treat or prevent the rash in the subject.

The invention discloses heat-resistant saccharomyces cerevisiae of a heterozygous human DNA methylation system as well as a construction method and application of the heat-resistant saccharomyces cerevisiae. The construction method comprises the following steps: determining a to-be-knocked-in saccharomyces cerevisiae target gene locus CAN1, and carrying out annular PCR amplification by taking a target sgRNA sequence as a homologous arm and an original gRNA plasmid as a template to obtain a gRNA plasmid; constructing a to-be-knocked-in gene segment, and transferring the constructed gRNA plasmid, the segment 1 and the segment 2 into BY4741-Cas9 saccharomycetes through a saccharomyces cerevisiae lithium acetate conversion method; the CRISPR plasmid is lost by adopting an iteration method to obtain a saccharomyces cerevisiae strain which is integrated into a genome and has DNA methylation modification, and the saccharomyces cerevisiae strain is named as yHL006. The invention constructs a saccharomyces cerevisiae chassis strain capable of stably expressing a DNA methylation system. Any auxotrophic selection marker is not occupied, and the complete amino acid synthesis capability and corresponding gene loci of the yeast are completely reserved. The heat stress resistance phenotype of the DNA methylation strain is verified, and the heat resistance of the yeast can be improved by the introduced DNA methylation system.

The invention relates to a construction method of a metallothionein yeast surface display engineering strain capable of efficiently enriching copper ions. According to the method, the metallothionein yeast surface display engineering strain is constructed based on a saccharomyces cerevisiae cell surface display technology. According to the method, a metallothionein gene CUP1 after codon optimization is connected to a saccharomyces cerevisiae expression vector pYD1 through the steps of double digestion, connection, transformation and the like, and a recombinant display plasmid pYD1-CUP1 is successfully constructed. A lithium acetate method is adopted to convert the recombinant display plasmid pYD1-CUP1 into tryptophan auxotroph saccharomyces cerevisiae EBY100, a plurality of recombinant transformants are obtained through MD plate screening, and screening and verification are carried out. The engineering yeast strain constructed by the method can improve the display efficiency of metallothionein on the surface of saccharomyces cerevisiae cells and enhance the enrichment capacity of the engineering strain on copper ions, so that inorganic copper is replaced by copper-enriched yeast, the side effect of copper is reduced, and the absorption and utilization rate of livestock and poultry on copper is improved.

Provided is a mutated lysophospholipase. Further provided are a gene encoding the lysophospholipase, and a vector and a host cell comprising the gene. In addition, further provided is a use of the lysophospholipase. The present invention also provides a mutated Aspergillus niger strain and a use thereof. Specifically, the present invention provides a mutated Aspergillus niger strain, which is an orotate phosphoribosyltransferase auxotrophic strain, and has improved production capacity of endogenous enzymes, preferably lysophospholipase, with respect to an unmutated strain. The present invention further provides uses of the strain. For example, the strain can be used for expressing the mutated lysophospholipase of the present invention.

This invention discloses a genetically engineered strain of *Saccharomyces cerevisiae* and its application in cholesterol synthesis. The strain was obtained through multiple genetic engineering steps, starting with *Saccharomyces cerevisiae* SquP1: DHCR24 gene was integrated at the YPL062W site to achieve 7-dehydrocholesterol synthesis; the ERG6 promoter was replaced with the ERG7 promoter and the DWF5 gene was integrated to achieve cholesterol synthesis; the TGL3, TGL4, and ROX1 genes were knocked out to enhance cholesterol accumulation; the FLX1 and ZWF1 genes were integrated to strengthen cofactor supply; the HXK1 promoter was replaced and the ERG11 gene was integrated to optimize the synthesis pathway; and auxotrophic genes were reintroduced to obtain the CC37N strain. This strain achieved a maximum yield of 6.23 g / L in a 5L bioreactor fermentation, providing an excellent strain for efficient cholesterol biosynthesis.

The invention discloses an engineering strain with high yield of echinocandin B as well as a construction method and application of the engineering strain, and belongs to the technical field of gene engineering. According to the invention, wild aspergillus nidulans BGCm5 is modified by a CRISPR / nCas9-CBE (clustered regularly interspaced short palindromic repeats / nCRas9-CBE (clustered regularly interspaced short palindromic repeats / nCRas9-CBE) technology, so that nutritional deficient strains of NHEJ (NkuA mutation), pyridoxine (PyroA mutation) and riboflavin (RiboB mutation) are obtained. And then, introducing an orthogonal Lox site by utilizing CRISPR / Cas9, and constructing a chassis cell. Through a Cre / LoxP and Cre / Lox2272 recombination system, a combined overexpression cassette of aniF, aniF 2, aniG, aniH, aniI, aniK and odeA genes is integrated into a genome, a series of engineering strains are obtained, and the production efficiency is remarkably improved.

The application provides application of a TGBp1 gene, or a protein coded by the TGBp1 gene, or a recombinant carrier or host bacteria containing a coding region of the TGBp1 gene in down-regulating expression level of MeGRXC3. The application research finds that there is interaction between TGBp1 and MeGRXC3, the growth ability of yeast in SD / -Leu / -Trp / -His / -Ade auxotrophic medium can be improved, the expression of MeGRXC3 can be down-regulated, the active oxygen content of tobacco leaves co-expressing TGBp1 and MeGRXC3 is improved, and the growth ability of yeast in SD / -Leu / -Trp / -His / -Ade auxotrophic medium is improved. The research result lays a theoretical foundation for cassava common mosaic virus disease-resistant breeding.

The application relates to a method for improving the yield of attenuated Listeria monocytogenes membrane vesicles and application, wherein, starting from attenuated Listeria monocytogenes LIDeltaAplcB, an LIDeltaAplcBdaldat attenuated strain with dal and dat genes knocked out is constructed, a back-supplement plasmid pCW633 carrying the dal gene is constructed, and the plasmid is electroporated into the attenuated auxotrophic strain LIDeltaAplcBdaldat to construct a stable recombinant strain LIDeltaAplcBdaldat::pCW633. Compared with the wild strain LI, the yield of MVs secreted by the recombinant strain LIDeltaAplcBdaldat::pCW633 can be increased by 1.84 times, and the shape structure, particle size and protein component of the MVs secreted by the recombinant strain are not obviously different from those of the MVs secreted by the wild strain.

The invention discloses a construction method of a saccharomyces cerevisiae surface display engineering strain for efficiently expressing aflatoxin oxidase, which is characterized in that an aflatoxin oxidase gene Afo after codon optimization is connected to a saccharomyces cerevisiae expression vector pYD1 through the steps of double enzyme digestion, connection, transformation and the like, and a recombinant display plasmid pYD1-Afo is successfully constructed. A lithium acetate method is adopted to convert the recombinant display plasmid pYD1-Afo into tryptophan auxotroph saccharomyces cerevisiae EBY100, a plurality of recombinant transformants are obtained through MD plate screening, and screening and verification are carried out. And determining the whole-cell enzyme activity of the recombinant strain with the aflatoxin oxidase displayed on the surface by using enzyme-linked immunosorbent assay, and screening out the strain with high enzyme activity. The engineering yeast strain constructed by the method not only can obviously improve the detoxification efficiency, but also can reduce the cost and the pollution to the environment.

This invention belongs to the field of biomedical technology and discloses a method for preparing porcine parvovirus VP2 nanoparticles from the auxotrophic Kluyveromyces martensii. The applicant, through screening, assembly, and preparation, developed a double-replicant expression vector suitable for Kluyveromyces martensii. This vector achieved high conversion rate and high expression efficiency in Kluyveromyces martensii. The double-replicant expression vector pGKD32 is shown in SEQ ID NO.2. Using the Kluyveromyces martensii expression system provided by this invention, the applicant successfully expressed porcine parvovirus VP2 in nanoparticle form, providing a new approach for the development of oral immunization strategies for porcine parvovirus.

The invention discloses a construction method and application of squalene-producing saccharomyces cerevisiae, and belongs to the technical field of biology. According to the invention, an auxotroph strain of S.cerevisiae BY4742 is taken as an original strain, and by optimizing an intracellular mevalonic acid pathway, the expression level of a key enzyme is enhanced and the metabolic bottleneck is relieved, so that the flux of intermediate metabolites is remarkably improved; by introducing an exogenous NADH dependent HMGR and strengthening an Acetyl-CoA supply approach, supply and demand of cofactors are effectively balanced, the availability of a precursor is improved, the yield of the constructed engineering strain AG15 fermented for 240 hours in fermentation at a 5-L fermentation tank level reaches 20.9 g / L, the production efficiency of squalene and the applicability of a carbon source are greatly improved, and the method has important industrial application potential and economic advantages.

The invention belongs to the field of gene engineering, and particularly relates to a non-interval loop iteration yeast genome editing system. The system is composed of Cas and gRNA double plasmids, wherein the gRNA plasmids comprise auxotrophy gRNA-URA3, gRNA-HIS3 and gRNA-LEU2, and the gRNA plasmids comprise gRNA-URA3, gRNA-HIS3 and The resistance is gRNA-BleoR, gRNA-HygR, gRNA-NrsR, gRNA-BleoR, gRNA-HygR and gRNA-NrsR; through coordinated use with a Cas selection marker, the method realizes interval-free, cyclic and iterative efficient editing operation on a yeast genome, and can be used as a practical tool for multi-gene continuous iterative editing of saccharomyces cerevisiae and other fungi. The method has extremely high editing efficiency, an experimental terminal eliminates Cas and gRNA plasmids to realize traceless operation, and the method is low in off-target rate, universal, accurate and easy to operate and can be applied to large-scale gene knockout, knockin, site-specific mutagenesis and the like.

The invention provides a resistance marker-free auxotrophic bacillus subtilis and a construction method and application thereof, the newly constructed strain takes bacillus subtilis 168 as a starting strain, indole-3-glycerophosphate synthase gene trpC of the bacillus subtilis 168 is knocked out, synthesis of tryptophan in the growth and metabolism process of the strain 168 is eliminated, and the resistance marker-free auxotrophic bacillus subtilis is obtained. The shuttle vector YH46 is constructed by taking pMD-19T as a framework, so that the shuttle vector YH46 can normally grow only in the presence of exogenous tryptophan, and meanwhile, a universal auxotroph shuttle vector YH46 is constructed by taking pMD-19T as the framework, so that convenience is provided for high-efficiency expression of genes in bacillus subtilis; the tryptophan auxotrophic strain BS-TR is constructed through double exchange by using a homologous recombination principle, the construction process is simple and easy to implement, and the strain BS-TR does not contain a resistance gene selection marker, can be used as a food-grade engineering bacterium, and can be used for intracellular expression or extracellular secretion of target protein for production.

The invention discloses a construction method and application of pyruvate-producing Kluyveromyces marxianus, and belongs to the technical field of biology. According to the invention, an auxotroph strain of K. marxianus NBRC1777 is used as an original strain, and a kluyveromyces marxianus engineering strain K. marxianus PA6 for efficiently producing pyruvic acid is obtained through metabolic engineering transformation. The strain can produce 83.6 g / L pyruvic acid in the fermentation period of 60 hours in the fermentation at the level of a 5 L fermentation tank. The strain effectively blocks a by-product pathway and enhances key enzyme activity, realizes high concentration of carbon flow to pyruvic acid, obviously improves the yield and final production concentration in unit time, reduces pollution risk and saves cooling cost through high-temperature fermentation, improves the final production concentration and fermentation stability of pyruvic acid through acid resistance, and improves the yield of pyruvic acid. The industrial application potential of pyruvic acid production is realized.

The application discloses a preparation method and application of a double auxotrophic engineering strain, and belongs to the technical field of genetic engineering. The preparation method of the double auxotrophic engineering strain disclosed by the application knocks out a leuA gene in a host cell, constructs an auxotrophic engineering strain EHA105leuA-, further knocks out a hisD gene in the EHA105leuA- strain, and constructs a double auxotrophic engineering strain EHA105hisD-leuA-. The gene IDs of the leuA gene and the hisD gene are WP_010972185.1 and WP_004432479.1 respectively. The results of examples of the application show that the double auxotrophic engineering strain EHA105hisD-leuA- has a large reduction in survival cells in soil and plants, reduces the probability of agrobacterium escaping into a natural environment, and improves the biosafety in a plant genetic transformation process.

The invention relates to the technical field of molecular biology, in particular to a biocontrol agent containing a helicospora pink genetic transformation system, a construction method of the biocontrol agent and application of the biocontrol agent in biological control of plant diseases. The spiropolyspora pink uracil auxotroph strain is constructed for the first time, a genetic transformation system with pyrF as a marker gene is established by taking the strain as a recipient bacterium, and compared with a conventional transformation mode, a transformant obtained by the system does not contain an antibiotic resistance tag, and the yield of the transformant is improved. And engineering strains obtained by screening are high in transformation efficiency, biocontrol strains with excellent characters are easier to obtain, and a foundation is laid for genetic modification, popularization and application of biocontrol fungi.