Patent ID: 8357527

Claim:
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 genetically modified haploid yeast cell 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.