4 results about "URA3" patented technology

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.

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 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.