7 results about "Auxotrophic strain" patented technology

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.

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 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 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 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 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.