11 results about "Yeast genome" patented technology

The invention belongs to the field of biosynthesis, and in particular relates to LBD (Levandanenediol Pyrophosphate) synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, the sclareol biosynthesis yield is generally low, LSLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained by performing mutation and optimization on LSLPPs pyrophosphate synthase, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 26.11 g / L and is greatly improved compared with the prior art.

The invention belongs to the field of biosynthesis, and particularly relates to sclareol synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, sclareol biosynthesis yield is generally low, sclareol synthase is mutated and optimized, an SsScs mutant containing at least one mutation of Y491F, G307L or N269F is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, and the yield of a 5L fermentation tank reaches 21.13 g / L and is greatly improved compared with the prior art.

This invention belongs to the field of biosynthesis, specifically relating to a variant of lysine pyrophosphate diol synthase and the constructed engineered yeast strain for perillaldehyde synthesis. To address the generally low yield of perillaldehyde biosynthesis in existing technologies, this invention mutates and optimizes the lysine pyrophosphate diol synthase to obtain an SsLPPs mutant containing the G379M mutation, which is then used for perillaldehyde synthesis. Simultaneously, to address the genetic instability issues associated with free plasmid expression in some Saccharomyces cerevisiae strains, this invention constructs a perillaldehyde synthesis pathway within the yeast genome. Furthermore, by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillaldehyde synthesis pathway, and by knocking out some transcriptional repressors, the substrate strain synthesizing perillaldehyde is modified, resulting in a significantly increased yield compared to existing technologies.

The invention belongs to the field of biosynthesis, and in particular relates to an improved variant of libdanenediol pyrophosphate synthase and engineered yeast for producing sclareol. In order to solve the problem that in the prior art, the sclareol biosynthesis yield is generally low, LsLPPs mutant containing N674L mutation is obtained by performing mutation and optimization on LsLPPs pyrophosphate synthase, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability caused by free plasmid expression of part of saccharomyces cerevisiae strains, a sclareol synthesis pathway is constructed in a yeast genome, and meanwhile, an acetyl coenzyme A synthesis pathway, an MVA pathway and a sclareol synthesis pathway are enhanced; and the chassis bacteria for synthesizing sclareol are modified in a manner of knocking out part of transcription inhibition factors, so that the yield is greatly improved compared with the prior art.

The invention relates to the technical field of saccharomyces cerevisiae genetic engineering transformation, in particular to a high-throughput saccharomyces cerevisiae transformation method. The method comprises the following steps: screening an IGR sequence on a saccharomyces cerevisiae genome through bioinformatics, respectively taking 45bp from upstream and downstream of the IGR sequence as genome homologous sequences for primer design, designing and subpackaging a PCR premixed solution and the designed primer by using an automatic platform script for PCR amplification, adding an obtained PCR product into competent cells, adding a conversion solution, and carrying out PCR amplification on the competent cells to obtain the saccharomyces cerevisiae gene. Performing heat shock; washing and resuspending the cells subjected to heat shock, and then coating the cells on a screened solid culture medium for culturing; the conversion process is carried out on an automatic platform island. According to the method, the transformation efficiency is improved, the operation time of high-throughput transformation is shortened, the intelligence and stability of each transformation are improved, and the method is suitable for large-scale yeast genome editing, functional gene screening and saccharomyces cerevisiae genome site screening.

The present application belongs to the field of biosynthesis, and particularly relates to a sclareol synthase and a yeast engineering bacterium for producing sclareol. In order to solve the problem of low yield of sclareol biosynthesis in the prior art, the sclareol synthase is mutated and optimized in the present application, a SsScs mutant containing at least one mutation of Y491F, G307L or N269F is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability existing in the expression of free plasmid in some Saccharomyces cerevisiae strains, the present application constructs a sclareol synthesis pathway in the yeast genome, strengthens the acetyl coenzyme A synthesis pathway, the MVA pathway and the sclareol synthesis pathway, and knocks out part of the transcriptional repressor to modify the chassis bacterium for synthesizing sclareol. The yield in a 5 L fermenter reaches 21.13 g / L g / L, which is greatly improved compared with the prior art.

The present application belongs to the field of biosynthesis, and particularly relates to pyrophosphate lyddane diol ester synthase and yeast engineering bacteria for producing sclareol. In order to solve the problem of generally low yield of sclareol biosynthesis in the prior art, the pyrophosphate lyddane diol ester synthase is mutated and optimized in the present application, and a SsLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability existing in the expression of free plasmid in part of saccharomyces cerevisiae strains, the present application constructs a sclareol synthesis pathway in the yeast genome, and at the same time, the acetyl coenzyme A synthesis pathway, the MVA pathway and the sclareol synthesis pathway are strengthened, and part of the transcriptional repressor is knocked out, so that the bottom bacteria for synthesizing sclareol are modified. The yield in a 5 L fermenter reaches 26.11 g / L, and the yield is greatly improved compared with the prior art.

This invention belongs to the field of biosynthesis, specifically relating to a variant of lysine pyrophosphate diol ester synthase and engineered bacteria for producing perillaldehyde. To address the generally low yield of perillaldehyde biosynthesis in existing technologies, this invention mutates and optimizes lysine pyrophosphate diol ester synthase, obtaining an SsLPPs mutant containing the D374L mutation, and uses this mutant for perillaldehyde synthesis. Simultaneously, to address the genetic instability issues associated with free plasmid expression in some Saccharomyces cerevisiae strains, this invention constructs a perillaldehyde synthesis pathway in the yeast genome. Furthermore, it modifies the substrate bacteria synthesizing perillaldehyde by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillaldehyde synthesis pathway, and by knocking out some transcriptional repressors, resulting in a significant increase in yield compared to existing technologies.

The invention discloses a method for constructing a vitamin E production strain by synchronously editing and recombining three sites of a yeast genome and application, and relates to the technical field of synthetic biology and metabolic engineering. According to the invention, a helper plasmid for expressing CRISPR-Cas9 protein and three targeted specific site guide RNA (sgRNA) is constructed, and the helper plasmid and three vitamin E pathway gene donor fragments which are assembled and cloned in escherichia coli in advance are co-transformed into saccharomyces cerevisiae, so that efficient editing and large-scale gene fragment integration can be simultaneously carried out on three chromosome sites of HO, GAL80 and ARO10. The three-site synchronous editing and recombination efficiency of the method exceeds 80%. By utilizing the method, an engineering strain of a complete vitamin E synthesis route can be rapidly constructed at one time, and fermentation verifies that the yield of the vitamin E can reach 5.9 g / L. The invention provides an efficient and universal tool for quickly constructing complex metabolic pathways in yeast.

The present application relates to the technical field of gene editing, and specifically provides a non-typical Pichia pastoris gene editing system and application thereof.The present application provides a non-typical Pichia pastoris gene editing plasmid, comprising a prokaryotic replication origin, a resistance screening marker gene, a Cas9 gene expression cassette, a gRNA gene expression cassette and an autonomous replication sequence; the Cas9 gene expression cassette comprises, from upstream to downstream, a Cas9 gene promoter, a Cas9 gene and a terminator; the gRNA gene expression cassette comprises, from upstream to downstream, an RNA pol III promoter, a tRNA Leu guide sequence, a scaffold RNA sequence and a polyT.The gene editing plasmid of the present application can edit the Pichia pastoris genome, has high editing efficiency and simple operation method, and provides a good gene editing system for Pichia pastoris gene modification research.

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.