Method for generating a genetically modified microbe
AssigneeAmyris, Inc.
InventorJeffrey A. Ubersax
Life of this patent
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Abstract
Provided herein are methods of generating genetically modified microorganisms, e.g., genetically modified yeast strains, which comprise functional disruptions in one or more pheromone response genes and one or more sporulation genes, and genetically modified yeast cells, e.g., genetically modified diploid and haploid yeast cells, that lack sporulation capability and endogenous mating capability, produced thereby.
Claims44
A genetically modified yeast cell comprising:
- (a) a functional disruption in at least one sporulation gene;
- (b) a functional disruption in at least one pheromone response gene;
- and
- (c) a chromosomally integrated heterologous nucleotide sequence encoding a protein of interest;
- wherein said genetically modified yeast cell is sporulation impaired and endogenous mating impaired.
A method of generating a genetically modified diploid yeast cell that is sporulation and endogenous mating impaired, the method comprising:
- (a) transforming each of a first and a second genetically modified haploid yeast cell with at least one plasmid encoding a protein capable of complementing an endogenous mating impairment of said first and second genetically modified haploid yeast cells,
- wherein said first genetically modified haploid yeast cell is sporulation and endogenous mating impaired and comprises a chromosomally integrated heterologous nucleotide sequence encoding a protein of interest, and wherein said second genetically modified haploid yeast cell is sporulation and endogenous mating impaired, is of the opposite mating type as the first genetically modified haploid yeast cell, and comprises a chromosomally integrated heterologous nucleotide sequence encoding said protein of interest;
- (b) mating the first genetically modified haploid yeast cell with the second genetically modified haploid yeast cell, thereby forming a genetically modified diploid yeast cell;
- and,
- (c) eliminating the one or more plasmids from the genetically modified diploid yeast cell,
- wherein the resulting genetically modified diploid yeast cell is sporulation and endogenous mating impaired and comprises two copies of a chromosomally integrated heterologous nucleotide sequence encoding said protein of interest.
A method for generating a sporulation and endogenous mating impaired genetically modified heterothallic (ho) diploid yeast cell, the method comprising:
- (a) transforming a population of a first genetically modified heterothallic haploid yeast cell with a plasmid encoding a functional homothallism (HO) protein to yield a first genetically modified haploid yeast cell, wherein expression of the HO protein is capable of inducing a mating-type switch of the first genetically modified haploid yeast cell, whereby a second genetically modified haploid yeast cell of the opposite mating type as the first genetically modified haploid yeast cell is obtained,
- wherein the first genetically modified heterothallic haploid yeast cell comprises a chromosomally integrated heterologous nucleotide sequence encoding a protein of interest and functional disruptions in the STE5 gene and the IME1 gene;
- (b) transforming each of the first and the second genetically modified haploid yeast cells with a plasmid encoding a STE5 protein, whereby said transforming results in mating of the first genetically modified haploid yeast cell with the second genetically modified haploid yeast cell, thereby forming a genetically modified diploid yeast cell;
- and,
- (c) eliminating any plasmids from the genetically modified diploid yeast cell to yield a genetically modified heterothallic diploid yeast cell,
- wherein the resulting genetically modified heterothallic diploid yeast cell is sporulation and endogenous mating impaired and comprises two copies of a chromosomally integrated heterologous nucleotide sequence encoding said protein of interest.
A MATα/a step5/ste5 ime1/ime1 yeast cell.
Dependencies are inferred from the claim text.