Nonconventional Yeasts in Biotechnology

Yeasts are of benefit to mankind because they are widely used for production of foods, wine, beer, and a variety of biochemicals. Yeasts also cause spoilage of foods and beverages, and are of medical importance. At present, approximately 700 yeast species are recognized, but only a few are commonly known. Relatively few natural habitats have been thoroughly investigated for yeast species; consequently, we can assume that many more species await discovery. Because yeasts are widely used in traditional and modern biotechnology, the exploration for new species should lead to additional novel technologies.

This chapter deals with the possible use of protoplast fusion for hybridization and genetic analysis of yeasts. For a more general overview the reader is referred to a review by Ferenczy (1981) on microbial protoplast fusion.

Electrophoretic karyotyping means the separation of intact chromosomal DNA according to its size on an agarose gel. Depending on the number and size of the chromosomes present in a strain, a specific banding pattern will be obtained. In order to reach this goal, two demands must be met. First, it is important to prepare the DNA without degradation by mechanical stress or by DNAses. Second, a method for the electrophoretic separation of the extremely large molecules must be developed. Conventional DNA electrophoresis is able to separate molecules of up to 50 kilobases. Yeast chromosomes range from several hundred to several thousand kilobases.

The ascomycetous yeast Schwanniomyces occidentalis (Schw. occidentalis) was first described by Kloecker in 1909, who isolated it from soil of the island of St. Thomas in the West Indies, hence the species name. Subsequently, a variety of other species, namely Schw. castellii, Schw. alluvius, an Schw. persoonii were accepted under the same genus name (Phaff 1970). These were distinguished from Schw. occidentalis by their different fermentation properties. On the basis of molecular analysis involving DNA reassociation experiments, Price et al. (1978) placed all four species under the same species name, Schw. occidentalis. The former species Schw. occidentalis, Schw. castellii, and Schw. alluvius showed more than 97% sequence homology and were therefore renamed Schw. occidentalis var. occidentalis. The former species Schw. persoonii, however, had only about 80% sequence complementarity with Schw. occidentalis and for that reason was named Schw. occidentalis var. persoonii. Recent analysis of the long-chain fatty acid compositions (Cottrell et al. 1986) and of the nucleic acid sequences of the cytochrome c gene, as well as of ribosomal RNAs, supports this view, the latter placing Schwanniomyces in close proximity to Candida and especially to Debaryomyces (Amegadzie et al. 1990; Kurtzmann and Robnett 1991). The high degree of identity of the rRNA sequences prompted Kurtzman and Robnett to suggest the transfer of Schw. occidentalis to the genus Debaryomyces (see Chap. 1, this Vol.).

Genetic studies of Kluyveromyces lactis (Dombrowski) van der Walt began in the early 1960s. Saccharomyces lactis was the name of the yeast at that time. Since then, the genus Kluyveromyces has been the object of intensive taxonomical studies. The position of Kluyveromyces lactis with respect to other members of the genus has been discussed in detail by a number of authors (van der Walt 1970; Johannsen and van der Walt 1978; Sidenberg and Lachance 1986; Lachance 1989; Fuson et al. 1987; Vaughan-Martini and Martini 1987; see also Kurtzman and Phaff 1989, for molecular taxonomy of yeasts). K. lactis had been thought to be closely related to the so-called K. fragilis (K marxianus), another lactose-assimilating yeast well known in industry. On the basis of cellular hybridization studies, Johannsen (1980) and van der Walt and Johannsen (1979) considered K. lactis as a variety of K marxianus (Hansen) van der Walt, but later taxonomic studies cited above showed that they are distinct species, as judged by various molecular criteria. DNA complementarity is less than 15–20% according to Fuson et al. (1987) and Vaughan-Martini and Martini (1987); the electrophoretic karyotype and the mitochondrial DNA restriction pattern are also completely different between the two yeasts (see below). At present, K. lactis is considered as a separate species from K. marxianus. K. lactis now incorporates K. drosophilarum and K. vanudenii. To our knowledge, the latest review on Kluyveromyces systematics is that of Lachance (1993).

Interest in the study of nonconventional yeasts (yeasts other than Saccharomyces cerevisiae and Schizosaccharomyces pombe) has increased dramatically in the past few years (Reiser et al. 1990). One such category is methylotrophic yeasts (Wegner and Harder 1986; Harder et al. 1986), e.g., Pichia pastoris, Hansanula polymorpha, Candida boidinii, etc. Methylotrophic yeasts have the ability to use methanol as a sole source of carbon and energy. Adaptation to growth on methanol is associated with induction of methanol oxidase, MOX (also referred to as alcohol oxidase, AOX), dihydroxy acetone synthase DAS, and several other enzymes involved in methanol metabolism. The most spectacular increase, however, is seen with alcohol oxidase, which is virtually absent in glucose-grown cells, but can account for over 30% of the cell protein in methanol-grown cells. Extensive proliferation of peroxisomes, accounting for over 80% of the cell volume, is also observed in methanol-grown cells (Veenhuis et al. 1983). Due to these characteristics, methylotrophic yeasts have gained the attention of biochemists, molecular biologists, cell biologists, biotechnologists, microbiologists, and chemists in academics and industry.

Pichia guilliermondii Wickerham represents a collection of sporogenous strains which formerly belonged to the asporogenous species Candida guilliermondii (Cast.) Langeron a. Guerra (Wickerham and Burton 1954; Wickerham 1966; Kreger van Rij 1970). This means that each strain of C. guilliermondii which is able to hybridize with any strain of P. guilliermondii must be transferred to the latter species. For example, even the strain type C. guilliermondii ATCC 9058 must now be considered as P. guilliermondii (Sibirny et al. 1977b).

Pichia methanolica was first described by Kato and coworkers (Kato et al. 1974). The yeast strain used in the laboratory of Dr. I.I. Tolstorukov (Moscow) for thorough genetic study was isolated in the Institute of Biotechnology, Leipzig, Germany, and was formerly identified by German scientists as Pichia pinus, strain MH4 (Tolstorukov 1994; see below).

A small and closely related group of yeasts is capable of using methanol as sole source of carbon and energy. Among these methylotrophic organisms, Hansenula polymorpha in particular has gained increasing attention in recent years, in both basic and applied sciences. This is mainly due to two physiological features that are an integral part of the methanol-utilizing machinery of this yeast. One is the expression of huge amounts of the key methanol-metabolizing enzymes, which can, in the case of the methanol oxidase, amount to up to one third of the total cellular protein. The expression is driven by very strong promoters that form the basis for a highly competitive system to produce foreign proteins at industrial scale (Gellissen et al. 1994). The other prominent characteristic of H. polymorpha is that the expression is accompanied by a dramatic growth and proliferation of microbodies, i.e., peroxisomes (Veenhuis and Harder 1987). H. polymorpha thus served and, in addition to the recent introduction of other systems such as Saccharomyces cerevisiae, still continues to serve as a valuable model organism to study the biogenesis of these organelles. Although we will briefly discuss the contributions of H. polymorpha to the field of peroxisome research, our main focus within this chapter belongs to the practical aspects that are of importance in working with this yeast.

Interest in Candida lipolytica (Harrison) Diddens et Lodder 1942 initially arose from its rather uncommon physiological characteristics. Strains of this species were more often isolated from lipid- or protein-containing substrates like cheese or sausage than from sugar-containing substrates. Indeed, strains of Candida lipolytica used few sugars (mainly glucose) as carbon source, but did readily assimilate various polyalcohols, organic acids, or normal paraffins. They were noted in the late 1940s by dairy technologists (Peters and Nelson 1948a,b) for their high extracellular protease and lipase activities, although these purified enzymes were never put to work industrially.

More than 30 years have passed since the first description of the yeast species Candida (C.) maltosa by Komagata et al. (1964a,b). Since then, C. maltosa has become of considerable academic and commercial interest. Now, together with some related Candida species and Yarrowia (Y.) lipolytica (cf. Barth and Gaillardin, Chap. 10, this Vol.), it is best known for its ability to grow on a wide variety of substrates including n-alkanes, fatty acids, or carbohydrates, and is therefore intensively investigated in its physiology, biochemistry, and molecular genetics. More recent investigations also use these yeast species for the study of fundamental cellular processes such as protein targeting, organelle biosynthesis, and drug resistance.

The yeast Trichosporon cutaneum belongs to the genus Trichosporon Behrend, which was described as early as 1890 (Behrend 1890). This genus includes yeasts which are characterized by budding cells of various shapes, a more or less developed pseudomycelium, or a true mycelium and arthrospores (Fig. 1). Trichosporon yeasts may form asexual endospores, but sexual reproduction has not been demonstrated so far (Do Carmo-Sousa 1970). Biochemical characteristics such as hydrolysis of urea, utilization of mono-, di-, tri-, or polysaccharides, etc., as well as studies concerning DNA base composition and DNA relatedness, led Guého et al. (1984) to propose that Trichosporon yeasts should be classified into two separate groups. The first group, which appears to be related to the Ascomycetes, includes 13 species with a G+C content lower than 50% (34.7–48.8%) and lacks urease, with T. margaritiferum being an exception. The second group appears to be related to the Basidiomycetes and contains 15 species with a G+C content higher than 50% (57–64%) including T. cutaneum, T. beigelii, and T. pullulans, and has the ability to hydrolyze urea. The basidiomycetous nature of some of the Trichosporon yeasts is demonstrated by the lamellar structure of the cell walls (Kreger-Van Rij and Veenhuis 1971) and the presence of xylose (Weijman 1979). Furthermore, the diazonium blue B test (van der Walt and Hopsu-Havu 1976) has been applied to a number of T. beigelii strains (Kemker et al. 1991). The positive test results are in agreement with the described basidiomycetous affinity of these strains.