Intraspecific Hybridization of a Methanol-utilizing Yeast,Candidasp. N-16, through Protoplast Fusion

Abstract

Auxotrophic mutants of a methanol-utilizing yeast, Candida sp. N-16, were hybridized through protoplast fusion using polyethylene glycol and Ca2+ ions. The complementation frequency of fusion was about 2 to 5 × 10 −4. Some of the prototrophic fusants obtained were very stable and distinguishable from the original prototroph. The fusants were uninuclear and contained 3.8 to 4.2 mg of DNA per 1011 cells as compared with about 1.5 mg of DNA in the parental strains. The cell volume of the fusants was greater than that of the wild and parental strains. The growth rate of the fusants was slightly lower than that of the wild strain. There was a difference between the fusants and the wild strain with respect to the levels of alcohol-dissimilating enzymes in cells grown on methanol, and one of the fusants possessed about 2.5 times higher activity of alcohol oxidase than that of the wild strain.