Genetic manipulation of non-conventional yeasts by conventional and non-conventional methods.

Abstract

In recent years, yeasts other than those belonging to the species Saccharomyces cerevisiae and Schizosaccharomyces pombe have become increasingly important in industrial processes. Species such as Pichia stipitis, Hansenula polymorpha, Zygosaccharomyces rouxii, Saccharomyces exiguus, Torulaspora delbrueckii, Yarrowia lipolytica and others whose perfect stage is known, can be manipulated genetically by classical methods, but those belonging to the genera Candida (C. utilis, C. tropicalis, C. bombicola, C. zeylanoides, C. boidinii, etc.), Brettanomyces, Cryptococcus, Rhodotorula, and others of the different form genera, cannot be treated in this way. Some, such as Schwanniomyces and Debaryomyces spp., which have a perfect stage, are still difficult to manipulate by conventional means. Genetic manipulation of these yeasts can be approached from two points of view; the first involving improvement of strains by cross-breeding within one species, and the second, the introduction of desirable genes from unrelated species and even from plants or animals. Two techniques are available for construction of industrially-useful strains from these yeasts: protoplast fusion and transformation with chimaeric plasmids containing the gene(s) it is desired to introduce into the recipient strain. The methods for the latter procedure are well known but can be laborious and time-consuming, especially if it is desired to introduce genes from plant or animal sources for production of enzymes, hormones, vaccines and similar products. Protoplast fusion is a simple technique which can be utilized in most laboratories and used for construction of improved yeast strains for brewing, baking, ethanol production and wine-making, either by the fusion of desirable strains of the same species which do not sporulate, or by introduction of genes from non-Saccharomyces species. Methods for fusion of species from different genera and isolation of the desired hybrids have been improved considerably in recent years. We have developed a method for isolation of strains carrying the desired genes by fusing a non-Saccharomyces species with an auxotrophic strain of Saccharomyces cerevisiae and selecting hybrids having the desired characteristics on appropriate media, after which the genes are transferred to the industrial strain by rare-mating, repeated protoplast fusion, or classical mating as required. The advantages and limitations of the method are under investigation.