Patent ID: 11913016
Assignee: WISCONSIN ALUMNI RESEARCH FOUNDATION
Field: Biotechnology (Chemistry)
Classification: CPC C  Y | IPC C

Claim 17:
18. A method of making a synthetic polyploid yeast cell comprising:
i) introducing a first polynucleotide comprising a first promoter operably connected to an HO polynucleotide encoding a yeast Ho protein, a first selectable marker cassette, and a first yeast origin of replication into a first yeast cell from a first yeast species or hybrid to produce a first transformed yeast cell,
ii) introducing a second polynucleotide comprising a second promoter operably connected to an HO polynucleotide encoding a yeast Ho protein, a second selectable marker cassette, and a second yeast origin of replication into a second yeast cell from a second yeast species or hybrid to produce a second transformed yeast cell,
iii) co-culturing the first transformed yeast cell with the second transformed yeast cell to produce a mating mixture and allow for mating of the first transformed yeast cell and the second transformed yeast cell, and
iv) selecting at least a portion of the mating mixture for the first selectable marker cassette and the second selectable marker cassette to select for a first synthetic polyploid yeast cell produced by the mating of step iii,
v) selecting for loss of one of the first selectable marker cassette or the second selectable marker cassette to produce a single marker first synthetic polyploidy yeast cell,
vi) introducing a third polynucleotide comprising a third promoter operably connected to an HO polynucleotide encoding a yeast Ho protein, a third selectable marker cassette, and a third yeast origin of replication into a third yeast cell from a third yeast species or hybrid to produce a third transformed yeast cell, wherein the third selectable marker cassette is specific fora marker not found in the single marker first synthetic polyploidy yeast cell,
vii) co-culturing the single marker first synthetic polyploid yeast cell selected in step v with the third transformed yeast cell of step vi to produce a mating mixture and allow for mating of the single marker first synthetic polyploid yeast cell and the third transformed yeast cell, and
viii) selecting at least a portion of the mating mixture for the selectable marker cassette of the single marker first synthetic polyploidy yeast cell and the third selectable marker cassette to select for a second synthetic polyploid yeast cell produced by the mating of step vii,
wherein the first selectable marker cassette and the second selectable marker cassette encode different selectable markers, wherein the first yeast cell, the second yeast cell, and the third yeast cell are each from different species of the same genus, and wherein the second synthetic polyploid yeast comprises whole genomes from at least three different yeast species and has a ploidy of at least 6N.