A flow cytometric method for rapid selection of novel industrial yeast hybrids

Abstract

We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 x 10(6) cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.

FIG. 1

Mating of haploid strains of S. cerevisiae. (A) Strain SMC19-A stained with CT-Green BODIPY. (B) Strain PB1 stained with BR. (C) Preparation 5 min after two strains were mixed together under conditions suitable for mating. (D) After 16 h the gated region was used to sort mated cells. All values are expressed in arbitrary units.

FIG. 2

Gating region used for isolation of small clusters of mating cells. Linear gains were used for both SSC and FSC parameters to provide sufficient resolution for size discrimination. Values are expressed in arbitrary units.

FIG. 3

Rare mating of an industrial polyploid strain with a laboratory haploid strain of S. cerevisiae. Strain PB1 (haploid) stained with BR and strain N1 (polyploid) stained with CT-Green BODIPY were mixed under conditions suitable for mating. After 16 h cells from the gated region were sorted twice to enrich for rare mated hybrids. All values are expressed in arbitrary units.