METHOD FOR GENERATING A GENETICALLY MODIFIED MICROBE
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.
1 . 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.
2 . The genetically modified yeast cell of claim 1 that is heterothallic (ho).
3 . The genetically modified yeast cell of claim 1 that is diploid.
4 . The genetically modified yeast cell of claim 2 that is diploid.
5 . The genetically modified diploid yeast cell of claim 4 that is homozygous other than for its mating type allele.
6 . The genetically modified yeast cell of claim 1 that is haploid.
7 . The genetically modified yeast cell of claim 2 that is haploid.
8 . The genetically modified haploid yeast cell of claim 7 , further comprising a recombinant plasmid encoding a functional homothallism (HO) protein.
9 . The genetically modified haploid yeast cell of claim 8 , further comprising at least one recombinant plasmid encoding a functional copy of the at least one pheromone response gene that is functionally disrupted in said genetically modified haploid yeast cell.
10 . The genetically modified yeast cell of any one of claims 1 - 9 that is a Saccharomyces cerevisiae cell.
11 . The genetically modified yeast cell of claim 10 , wherein the Saccharomyces cerevisiae cell is of the Baker's yeast, Mauri, Santa Fe, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1 or AL-1 strain.
12 . The genetically modified yeast cell of claim 10 , wherein the Saccharomyces cerevisiae cell is of the PE-2 strain.
13 . The genetically modified yeast cell of claim 10 , wherein the Saccharomyces cerevisiae cell is of the CAT-1 strain.
14 . The genetically modified yeast cell of claim 1 , wherein said at least one sporulation gene is selected from the group consisting of IME1, IME2, NDT80, SPOIL SPO20, AMA1, HOP2, and SPO21.
15 . The genetically modified yeast cell of claim 1 , wherein said at least one pheromone response gene is selected from the group consisting of STE5, STE4, STE11, STE12, STE7, and STE11.
16 . The genetically modified yeast cell of claim 14 comprising a functional disruption of the IME1 gene.
17 . The genetically modified yeast cell of claim 15 comprising a functional disruption of the STE5 gene.
18 . The genetically modified yeast cell of claim 1 comprising a functional disruption of the IME1 gene and the STE5 gene.
19 . The genetically modified haploid yeast cell of claim 9 that comprises a functional disruption of the STE5 gene and a recombinant plasmid encoding a functional STE5 protein.
20 - 43 . (canceled)
44 . A MATα/a ste5/ste5 ime1/ime1 yeast cell.