12 results about "URA3" patented technology

The invention discloses a saccharomyces cerevisiae recombinant strain based on a uracil metabolism gene URA3 as well as a construction method and application of the saccharomyces cerevisiae recombinant strain. The construction method comprises the following steps: taking saccharomyces cerevisiae rich in farnesyl pyrophosphate as an original strain, introducing a uracil metabolism gene URA3, synchronously overexpressing a ribosome biosynthetic gene UTP10 and an iron metabolism gene FIT3, and utilizing the uracil metabolism gene URA3 to express up-regulation correlation with the ribosome biosynthetic gene UTP10 and the iron metabolism gene FIT3, so as to obtain the farnesyl pyrophosphate-rich saccharomyces cerevisiae. The metabolic flux of the terpenoids is enhanced, and the saccharomyces cerevisiae recombinant strain for efficiently producing the terpenoids is constructed. According to the method, a metabolic network of yeast is systematically optimized, and combined regulation and control on precursor flux distribution, protein translation capability and metal ion steady state are realized, so that the microbial synthesis efficiency of sesquiterpenoids such as patchouli alcohol and beta-elemene is remarkably improved.

The invention discloses a composition for gene editing and application thereof in gene editing, and belongs to the technical field of genetic engineering. The invention provides an efficient traceless gene editing method for pichia pastoris. According to the technical scheme provided by the invention, traceless knockin or knockout of the pichia pastoris gene can be realized only by constructing the donor fragment, and Ura3 can be repeatedly used as a selection marker, so that knockin or knockout of a plurality of genes is realized, and the pain point that no selection marker is available in multiple rounds of gene editing in pichia pastoris is solved.

The application discloses a plant source campesterol Yarrowia lipolytica production strain and a construction method. 12 The application is constructed as follows: (1) taking Yarrowia lipolytica strain AHH 7 as a chassis, introducing Δ 24 \-sterol-C5(6)-desaturase gene 28 _DWF7, 7-dehydrocholesterol reductase gene At _DWF5 and Δ At \-sterol reductase gene Ar _DWF1 from Arabidopsis thaliana, and obtaining a recombinant strain Yl001; (2) recovering a URA3 nutritional marker in the strain Yl001, integrating the genes ARE1 and the URA3 nutritional marker into an ERG4 site of the Yl001, and knocking out the gene ERG4, to obtain a plant source campesterol Yarrowia lipolytica production strain Yl006; the yield of the plant source campesterol of the strain Yl006 is increased by 21.83%, reaching 343.34 mg / L.

The invention discloses a yarrowia lipolytica hypermutation system strain based on double-chain specific cytidine deaminase as well as a construction method and application of the yarrowia lipolytica hypermutation system strain. The construction method comprises the following steps: integrating a Ura3 gene into a yarrowia lipolytica genome to obtain a stable expression strain HM00; the method comprises the following steps: connecting a PTEFIN regulation and control element, a DddAtox gene, a linker sequence and a MutH * gene to a pWLH plasmid by virtue of a seamless cloning technology, and constructing recombinant plasmids pWLH-DddAtox-linker-MutH * and pWLH-DddAtox-linker-MutH; the method comprises the following steps: transforming recombinant plasmids pWLH-DddAtox-linker-MutH * and pWLH-DddAtox-linker-MutH into a strain HM00, so as to respectively obtain a strain HM22 of a hypermutation system and a strain HM21 of a hypermutation system.

The invention relates to the field of yeast double hybrid in molecular biology, in particular to a three-plasmid yeast double hybrid strain as well as a construction method and application thereof. According to the invention, an AH109 strain is taken as a chassis strain, an endogenous URA3 gene is knocked out, a URA3 screening site is released, and a NatMX gene resistance marker is introduced, so that a three-plasmid yeast two-hybrid strain is obtained. The strain can be compatible with three independent plasmids TRP1, LEU2 and URA3 at the same time, the limitation that only binary interaction can be researched in traditional yeast two-hybridization is broken through on the basis that the normal screening function is not affected, the cost and difficulty of binary interaction research are close to those of binary interaction research, more complex ternary interaction research is achieved, and the application prospect is broad. The method greatly widens the research field, improves the research efficiency and success rate, saves the time cost for researching the three-factor interaction regulation, is stable in plasmid expression level and good in experiment repeatability, and provides a powerful, efficient and low-cost new technical platform for researching the complex life process.

The invention discloses a method for constructing a low-beauvericin cordyceps sobolifera strain based on a non-resistance marker technology and application of the low-beauvericin cordyceps sobolifera strain, and belongs to the technical field of fungus molecular biology. In order to obtain a low-beauveria-bassiana cordyceps sobolifera strain (Cordyceps canhua KIVI with a preservation number of CCTCC NO: M2025637) without a resistance marker, the preparation method comprises the following steps: firstly, carrying out double enzyme digestion on a plasmid pPK2-bar to remove a herbicide-resistant gene bar, and inserting an upstream region and a downstream region of a coding region of cordyceps sobolifera orotic acid nucleotide decarboxylase URA3 into the plasmid to obtain a knockout plasmid pPK2-URA3 of the URA3. And then obtaining the URA3 gene deleted cordyceps sobolifera strain through a homologous recombination technology and the screening effect of 5-fluoroorotic acid (5-FOA) on the uridine / uracil auxotroph strain, so as to obtain the URA3 gene deleted cordyceps sobolifera strain. The content of the beauveria bassiana in the obtained cordyceps sobolifera strain meets the requirement (the content of the beauveria bassiana is less than or equal to 3mg / kg) of the'announcement of 15 kinds of'three-new foods' of national health commission about cordyceps sobolifera sporocarp (artificially cultivated) and the like '(No.9 in 2020).

The invention discloses an engineering strain for enhancing methanol metabolism capability of yarrowia lipolytica as well as a construction method and application of the engineering strain. The construction method comprises the following steps: by taking a pWLH plasmid as a carrier, integrating a non-homologous end connection (NHEJ) repair gene DL4, PAXX and an XRCC4 expression cassette, constructing a recombinant plasmid pWLH-DL4-PAXX-XRCC4, and transforming yarrowia lipolytica Po1f, so as to obtain an NHEJ enhanced strain PO1f / pWLH-DL4-PAXX-XRCC4; the method comprises the following steps: respectively constructing recombinant plasmids pWLH-Aox-Das-Dak and pWLH-Mdh-Hps-Ph i by using methanol metabolism key genes Aox, Das and Dak as well as Mdh, Hps and Ph i; the Xump and Rump path fragments of the plasmid are connected with a truncated Ura3 tag (Ura41), and a multi-copy methanol utilization gene integration library is constructed through five rounds of iterative transformation and SC-Ura screening; the methanol consumption is quantified through the cell accumulation amount and high performance liquid chromatography (HPLC), and the efficient methanol utilization strain is obtained through screening. According to the constructed engineering strain, by enhancing NHEJ-mediated multi-copy gene integration, the methanol metabolism efficiency of yarrowia lipolytica is remarkably improved, and a new strategy is provided for development of non-natural methylotrophic chassis cells.

The invention discloses a yarrowia lipolytica hypermutation system strain based on cytidine deaminase as well as a construction method and application of the yarrowia lipolytica hypermutation system strain. The construction method comprises the following steps: integrating a Ura3 gene into a yarrowia lipolytica genome to obtain a stable expression strain HM00; the preparation method comprises the following steps: connecting a Trex2 gene and a PTEFIN regulatory element to a pWLH plasmid through a seamless cloning technology, and constructing a recombinant plasmid pWLH-Trex2; a PmCDA1 gene and a MutH * gene are assembled to the recombinant plasmid in series through a linker sequence and a PTEFIN fragment respectively, and a composite expression recombinant plasmid pWLH-Trex2-PmCDA1-linker-MutH * is constructed; and transforming the recombinant plasmid yeast into an HM00 strain to obtain the hyper-mutation system strain HM07. The strain forms a'nicking-melting-deamination 'cascade reaction mechanism, so that the mutation frequency of the yarrowia lipolytica is increased to 3.3 * 10 < 7 > times that of the original strain. The hypermutation system established by the invention is simple and convenient to operate, and the mutation frequency of the yarrowia lipolytica can be obviously improved.

This invention provides a genome editing method for *Yersinia lipolytica* based on homologous recombination, belonging to the fields of microbial genetic engineering and synthetic biology. This invention constructs the tool plasmid pUrloxP3 to achieve efficient knockout and combined knockout of ylAS and ylPDH, completing site-specific integration and functional verification of exogenous genes RgTAL, 4CL, and CHS. The Cre / loxP system is used to recover the URA3 marker and eliminate the pYLXP'-Cre helper plasmid. This invention features high editing efficiency, standardized procedures, cyclical selection markers, and stable integration of exogenous genes, ultimately yielding a stable engineered strain without markers or helper plasmids. It is suitable for multi-round metabolic engineering and cell factory construction of *Yersinia lipolytica*.

The application relates to the field of biotechnology, and particularly discloses a Saccharomyces cerevisiae engineering strain LSc274 with high yield of sirenol and a construction method and application thereof, aiming to solve the problems of low content of sirenol in a natural host, difficulty in chemical synthesis, and low yield of biosynthesis. ROX1 、 EXG1 、 DPP1 、 TRP1 , the GAL80 gene is knocked out in a Saccharomyces cerevisiae host, and meanwhile, sirenol synthase coding genes, tHMG1, EGR20, TRP1, URA3, LEU2 and HIS3 genes are highly expressed. The yield of the constructed engineering strain reaches 905.83+33.41 mg / L in a flask fermentation, and the yield reaches 16.29+0.12 g / L in a 5 L fermenter, which is the highest yield reported at present, and the engineering strain has the application prospect of large-scale production of sirenol.

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 application belongs to the technical field of biotechnology, and particularly relates to a high-efficiency expression of a Kluyveromyces marxianus strain and application thereof. The Kluyveromyces marxianus strain is obtained by using CRISPR / Cas9 gene editing technology to knock out genes in a protein synthesis metabolic pathway, a secretion and transport pathway, a cytoskeleton synthesis or an intercellular information transmission pathway in a genome of the Kluyveromyces marxianus, and taking the Kluyveromyces marxianus FIM-1 in which a key gene URA3 of a uracil synthesis pathway is knocked out as a starting strain. A FIM-1 ura3Delta mnn11Delta is screened and obtained, and the alpha-galactosidase secretion expression level can be increased by more than 3 times. The Kluyveromyces marxianus strain can be applied to high-efficiency preparation of biological enzymes such as alpha-galactosidase, ferulic acid esterase, chitosanase, saccharifying enzyme, xylanase, mannanase, cellulase and beta-amylase, and has high industrial application value.