HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette

Southern blot analysis of industrial yeasts showed that all top-fermenting yeasts, distiller's yeasts and a proportion of wine yeasts tested in the present study produced a hybridization signal (approximately 7 kb), corresponding to a Saccharomyces cerevisiae-type HO gene (Sc-HO). It also showed that bottom-fermenting yeasts gave rise to 7-kb and 4-kb hybridization signals, corresponding to the Sc-HO gene and the lager yeast HO gene (Lg-HO), respectively. Two wine yeasts produced a 4-kb hybridization signal, corresponding to Lg-HO; and one wine yeast produced 2.5-kb and 1.5-kb hybridization bands, corresponding to a S. uvarum-type HO gene (Uv-HO). Partial nucleotide sequences of HO genes amplified from these wine yeasts perfectly matched those of Lg-HO and Uv-HO, respectively. HO disruption vectors were constructed by inserting a dominant selective marker PGK1p-neo and the mating-type detection cassette MFα1p-PHO5 within the Lg-HO or Uv-HO gene. From transformants carrying a single-disrupted ho gene, mating-competent progenies were easily obtained through meiosis. Moreover, mating-competent derivatives appearing at very low frequency could be obtained from a double-disrupted ho transformant without meiosis (even from a wine yeast lacking sporulation ability), because the sensitive phosphatase-staining method allowed detection of the Pho+ mating-competent derivatives from confluent colonies by the random spore method. Our study describes a rapid and convenient method for isolating mating-competent clones from industrial yeasts.