42 results about "Auxotrophic strain" patented technology

The invention discloses an endogenous tRNA (transfer ribonucleic acid) promoter. The nucleotide sequence of the endogenous tRNA promoter is shown as SEQ ID NO: 1; an expression cassette containing the endogenous tRNA promoter and a recombinant vector; the invention also relates to application of the endogenous tRNA promoter, the expression cassette or the recombinant vector in thraustochytriaceae microorganism gene editing, and a thraustochytriaceae microorganism gene editing method. The CRISPR / Cas9 gene editing system based on the endogenous tRNA promoter is successfully established, convenient, rapid and efficient fixed-point editing in thraustochytriaceae microorganisms is achieved, and the CRISPR / Cas9 gene editing system has high application value; a transformation system for screening the recombinant thraustochytriaceae microorganisms by using sodium acetate as an auxotroph is established, the transformation screening efficiency of the recombinant thraustochytriaceae microorganisms obtained based on an agrobacterium transformation method is remarkably improved, and rapid gene editing is assisted.

The invention provides a method for genetically modifying bordetella pertussis and application of the method. The method comprises the following steps: knocking down or knocking out a first nutrition screening gene of bordetella pertussis to generate a first auxotrophic cell; replacing the first genetic element with a second nutrition screening gene through recombination treatment to obtain a second auxotroph cell; replacing the second nutritional screening gene with the genetically modified second genetic element through recombination treatment to obtain a third nutritional deficient cell; and restoring the first nutritional screening gene to the genome of the third nutritional deficient cell through recombination treatment. In the modification process, use of antibiotics is not involved, no redundant fragments are introduced except for target modification sites, and the method has the advantages that the genetic background purity is high, and no biological safety risk exists in the modification process.

The invention provides an application of an AC2 gene and the like in improving the expression quantity of GFP (Green Fluorescent Protein) and / or inhibiting degradation of an mRNA (Messenger Ribonucleic Acid) attenuated substrate taking the GFP as a reporter gene. Researches find that AC2 has an mRNA decay inhibition function, can inhibit degradation of a nonsense-mediated mRNA decay substrate taking GFP as a reporter gene and degradation of an endogenous nonsense-mediated mRNA decay substrate of arabidopsis thaliana, can improve the expression quantity of mRNA of AT1G0106020, RPS6, SMG7, AT5G35490, AT5G64430, AT1G36730 and the like, can reduce the expression quantity of AT1G72450 and AT2G400, has interaction with Dcp1 and Dcp2, and can be used for inhibiting the expression quantity of mRNA of AT1G0106020, RPS6, SMG7, AT5G35490, AT5G6430, AT1G36730 and the like. The growth ability of the yeast in an auxotroph culture medium can be improved, the expression level of Dcp1 can be reduced, and a research result lays a foundation for clarification of AC2 functions.

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 invention provides an application of an AC2 gene, or an AC2 gene coding protein, or a recombinant vector or host bacteria containing an AC2 gene coding region in reducing the expression level of AtSGS3, and / or reducing the expression level of MeSGS3, and / or reducing an SGS3 / RDR6 compound. Researches find that SLCMV AC2 interacts with AtSGS3 and MeSGS3, the growth ability of yeast in an SD / -Ade / -His / -Leu / -Trp auxotroph culture medium and an SD / -Ade / -His / -Leu / -Trp auxotroph culture medium added with 3-AT can be improved, expression of AtSGS3 and MeSGS3 can be down-regulated, an SGS3 / RDR6 compound can be reduced, and a research result lays a foundation for clarification of a molecular mechanism of SLCMV AC2 regulation and control of host RNA silencing.

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

The invention provides auxotroph hansenula polymorpha as well as a construction method and application thereof. The method comprises the following steps: constructing a CRISPR gene editing plasmid of single-plasmid double-point editing yeast, introducing the plasmid into a hansenula polymorpha strain, and constructing the auxotroph hansenula polymorpha by replacing an sgRNA sequence in the plasmid, so as to obtain the auxotroph hansenula polymorpha. The auxotroph hansenula polymorpha keeps a high growth rate of an original strain, genetically engineered bacteria capable of efficiently producing exogenous target proteins can be rapidly screened out through auxotroph screening, expression products can be selectively accumulated in cells and can also be selectively secreted into fermentation liquor, and the yield of the heterotroph hansenula polymorpha is improved. The expressed recombinant protein has high biological safety and can be applied to multiple fields of medicine, medical beauty, instruments, food, animal husbandry and the like.

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 a multivalent expression sheep listeria vaccine for cervical cancer and a preparation method thereof, and the method comprises the following steps: on the basis of replacing sheep listeria LI [delta] ilo:: hly with hemolysin, constructing to obtain auxotrophic sheep listeria LI [delta] ilo:: hly [delta] dal [delta] dat; the fusion antigen is constructed by alternately arranging amino terminal and carboxyl terminal structural domains of four types of HPV6 / 11 / 16 / 18 E6 and E7 proteins and retaining an overlapping region of 16 amino acids at a connecting site; inserting the fusion antigen into a non-resistance plasmid pCW631 which carries a dal gene and replaces an Amp gene with an asd gene, so as to obtain a non-resistance plasmid pCW636; the resistance-free plasmid pCW636 is electrically transferred into the auxotroph sheep listeria monocytogenes LIdeltailo:: hly deltadal deltadat, and the sheep listeria monocytogenes cervical cancer vaccine is obtained. The cervical cancer vaccine provided by the invention can express the E6E7 protein in a multivalent manner, so that multiple protection on cervical cancer is realized.

The invention belongs to the technical field of biological catalysis and biological engineering, and relates to a lytic polysaccharide monooxygenase expression system and application thereof. The expression system comprises an expression vector containing a lysing polysaccharide monooxygenase gene expression cassette and a host, the host is a pyrG auxotroph type aspergillus niger strain. According to the present invention, the deltaglaA gene knockout pyrG auxotrophic aspergillus niger strain is adopted as the host, and the strong promoter GlaPr is introduced to drive the expression of the cellulose activity LPMO gene, such that the interference of the endogenous glucamylase is eliminated, and the LPMO secretion efficiency is enhanced; a dynamic fermentation process is adopted, and efficient production of the LPMO protein is achieved.

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 belongs to the technical field of genetic engineering, and particularly relates to application of turnip crinkling virus protein P38 to regulation and control of double-chain binding protein of arabidopsis thaliana. Research finds that turnip crinkling virus protein P38 interacts with Drb7.1 and Drb7.2, expression of Drb7.1 and Drb7.2 can be reduced, the growth capacity of yeast in an SD / -Leu / -Trp auxotroph culture medium and an SD / -Leu / -Trp / -His / -Ade auxotroph culture medium is improved, and a research result lays a foundation for clarification of a molecular mechanism of TCV P38 regulation and control of Drb7.1 and Drb7.2.

The present invention provides the use of genes such as AC2 for increasing GFP expression and / or inhibiting the degradation of mRNA decay substrates using GFP as a reporter gene. The present invention has discovered that AC2 has mRNA decay inhibition function, inhibiting the degradation of nonsense-mediated mRNA decay substrates using GFP as a reporter gene, as well as the degradation of endogenous nonsense-mediated mRNA decay substrates in Arabidopsis thaliana. It can increase the mRNA expression of genes such as AT1G0106020, RPS6, SMG7, AT5G35490, AT5G64430, and AT1G36730, while reducing the expression of AT1G72450 and AT2G400. It also interacts with Dcp1 and Dcp2, enhancing yeast growth in nutrient-deficient culture media and reducing Dcp1 expression levels. These findings will lay the foundation for elucidating the functions of AC2.

The invention belongs to the technical field of biological medicine, and discloses a method for preparing porcine parvovirus VP2 nanoparticles by auxotroph kluyveromyces marxianus. The dual-replicon expression vector applicable to the kluyveromyces marxianus is prepared by the applicant through screening and assembling, the vector realizes the effects of high conversion rate and high expression efficiency in the kluyveromyces marxianus, and the dual-replicon expression vector pGKD32 is shown as SEQ ID NO.2. The Kluyveromyces marxianus expression system provided by the invention is utilized by the applicant to successfully express the porcine parvovirus VP2 in a nanoparticle form, and a new thought is provided for research and development of a porcine parvovirus oral immunization strategy.

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.

The present invention discloses an endogenous tRNA promoter, whose nucleotide sequence is shown in SEQ ID NO: 1; an expression cassette and a recombinant vector containing the endogenous tRNA promoter; the application of the endogenous tRNA promoter, the expression cassette or the recombinant vector in the gene editing of Thraustochytriaceae microorganisms, and a method for gene editing of Thraustochytriaceae microorganisms. The present invention successfully establishes a CRISPR / Cas9 gene editing system based on the endogenous tRNA promoter, and realizes convenient, rapid and efficient site-specific editing in Thraustochytriaceae microorganisms, which has high application value; establishes a transformation system for screening recombinant Thraustochytriaceae microorganisms by using sodium acetate as an auxotrophic type, significantly improves the transformation screening efficiency of obtaining recombinant Thraustochytriaceae microorganisms based on Agrobacterium transformation method, and assists in realizing rapid gene editing.

The present invention provides the use of the AC4 gene, etc., for increasing GFP expression and / or inhibiting the degradation of mRNA decay substrates using GFP as a reporter gene. The present invention has discovered that AC4 has mRNA decay inhibition function, inhibiting the degradation of nonsense-mediated mRNA decay substrates using GFP as a reporter gene, as well as the degradation of endogenous nonsense-mediated mRNA decay substrates in Arabidopsis thaliana, without affecting ARE-dependent turnover. It can increase the mRNA expression of AtUPF1, AtXRN4, AT1G0106020, RPS6, and others, while reducing the expression of AT1G72450 and AT2G400. It can cause dwarfing and / or leaf curling in Arabidopsis seedlings, enhance the pathogenicity of PVX, interact with MeUpf1 or AtUpf1, and improve yeast growth ability in nutrient-deficient culture media. The research results will lay the foundation for clarifying the function of AC4.

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 a preparation method and application of a double-auxotroph engineering strain, and belongs to the technical field of gene engineering. The invention discloses a preparation method of a double-auxotroph engineering strain, which comprises the following steps: knocking out a leuA gene in a host cell, and constructing an auxotroph engineering strain EHA105leuA-; the method comprises the following steps: further knocking out a hisD gene from an EHA105uA-strain, and constructing a double-auxotroph type engineering strain EHA105hisD-uA-; the gene ID of the leuA gene is WP010972185.1, and the gene ID of the hisD gene is WP004432479.1. The gene ID of the leuA gene is WP010972185.1. Results of the embodiment of the invention show that survival cells of the double-auxotroph engineering strain EHA105hisD-leuA-in soil and plants are greatly reduced, the probability that agrobacterium escapes into the natural environment is reduced, and the biological safety in the plant genetic transformation process is improved.

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 invention relates to auxotrophic escherichia coli for producing N-acetylglucosamine as well as a construction method and application of the auxotrophic escherichia coli. NagB, nagA, manX, nagE, nagK and asd genes are deleted in the auxotrophic escherichia coli, the auxotrophic escherichia coli contains a recombinant expression vector, and the recombinant expression vector contains asd, glmS and gna1 genes. According to the invention, an auxotroph engineering bacterium is further constructed on the basis of constructing a high-yield N-acetylglucosamine strain, so that the efficient production of N-acetylglucosamine is realized, the use of antibiotics can be avoided, and the yield of fermented N-acetylglucosamine can reach 98g / L; a new method and a new idea are provided for efficient, low-cost and safe industrial production of the N-acetylglucosamine.

The invention discloses a multivalent expression listeria monocytogenes cervical cancer vaccine and a preparation method thereof. The method comprises the following steps: constructing attenuated listeria monocytogenes of which actA and plcB virulence genes are knocked out; on the basis of the attenuated listeria monocytogenes, constructing to obtain auxotroph listeria monocytogenes LM [delta] dal [delta] dat; the fusion antigen is constructed by alternately arranging amino terminal and carboxyl terminal structural domains of multiple types of E6 and E7 proteins of HPV (human papillomavirus); inserting the fusion antigen into a non-resistance plasmid pCW631 which carries a dal gene and replaces an Amp gene with an asd gene through PCR (Polymerase Chain Reaction) amplification, gel recovery and seamless cloning technologies to obtain a non-resistance plasmid pCW636; the non-resistance plasmid pCW636 is electrically transferred into the auxotroph listeria monocytogenes LM [delta] dal [delta] dat, and the listeria monocytogenes cervical cancer vaccine is obtained after PCR (polymerase chain reaction) screening, product verification and sequencing. The cervical cancer vaccine for multivalent expression of the E6E7 protein is constructed on the basis of the attenuated listeria monocytogenes, and multiple protection on cervical cancer can be achieved.

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 relates to a mutated aspergillus niger strain and application thereof. Specifically, the present invention relates to a mutated Aspergillus niger strain, which is an auxotrophic strain of orotidin-5 '-phosphate decarboxylase, and which has an increased production capacity of an endogenous enzyme and / or a foreign protein relative to a starting strain.

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 (Δalr1Δalr2); and an inactivated D-alanine aminotransferase (dat) gene. In another aspect, the disclosure features a method of making the recombinant Staphylococcus bacterium.

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.

The invention discloses an auxotrophic strain W12-1 [delta] murI of Escherichia coli and an application of the auxotrophic strain W12-1 [delta] murI. Belongs to the technical field of gene engineering. The D-glutamic acid gene deleted recombinant bacterium of the cow mastitis source Escherichia coli, provided by the invention, is a strain obtained by carrying out whole genome sequencing on genetically operable Escherichia coli separated from clinical cow mastitis and then deleting a D-glutamic acid synthesis related gene murI in the Escherichia coli. The toxicity of the auxotrophic strain in a mouse body and a mammary gland model is reduced, and a living imaging technology shows that the toxicity of the auxotrophic strain is weakened and the auxotrophic strain grows in a self-restrictive manner. The invention provides a design strategy of a strain of Escherichia coli capable of being used for genetic modification and a new viable vaccine for D-glutamic acid auxotrophy, and the design strategy can be widely applied to the development of live vaccines of various bacterial pathogens.

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.