Hiroshi Fukuhara Institut Curie, Section de Recherche, UMR2027, Centre Universitaire Paris XI, Orsay, France. Tel.: 133 1 69 86 3063; fax: 133 1 69 86 9429; e-mail: hiroshi.fukuhara@curie.u-psud.fr Received 27 April 2005; accepted 31 May 2005. First published online 28 November 2005. doi:10.1111/j.1567-1364.2005.00012.x Editor: Lex Scheffers Keywords Kluyveromyces lactis; model yeast; nonconventional yeast.
The use of Kluyveromyces lactis for research started in
early 1960s. In contrast to most cases of yeast research, the study of this particular species was initially motivated by a purely academic question, that is, possible adaptive regulation of sugar metabolism in a lower eukaryote. Biotechnological interest in K. lactis came much later. Until about 1980, K. lactis research was barely visible in the shadow of the formidable development of the Saccharomyces cerevisiae system. The early 1960s were the era of lactose regulation in Escherichia coli, which led to the birth of molecular biology. Following the achievements of the investigators of the E. coli system, a number of laboratories were trying to find an inducible enzyme system in a eukaryotic organism in order to evaluate the general significance of the operon concept. Harlyn O. Halvorson at Madison, Wisconsin, was one of those people. He contacted the great taxonomist L. J. Wickerham (USDA, Peoria) who knew how different yeast species assimilated various sugars. Apparently, it was he who suggested the use of K. lactis, a species that assimilated bglucosides in an adaptive mode. Halvorson and his colleagues have thus started to work on this yeast (then called Saccharomyces lactis), using two isolates obtained from Peoria, NRRL Y-1140 (CBS 2359 [Mat a]) and Y-1118 (CBS 6315 [Mat a]). A mating system for genetic analysis was elaborated. The b-glucosidase system of K. lactis turned out to be complicated by the fact that it was paraconstitutive (half-inducible, half-constitutive). After this pioneer work of the Madison group, only a few laboratories continued to use K. lactis, essentially in the field of mitochondrial genetics and biogenesis, in comparison with the S. cerevisiae system. After all, yeast species available for formal genetic analysis were rare and are still few even now: apart from the fission yeast Schizosaccharomyces pombe and the nonfermenting Yarrowia lipolytica, we have practically only K. lactis. In the early 1980s, two lines of study, specific to K. lactis, revived interest in this yeast. One was the series of works on FEMS Yeast Res 6 (2006) 323324
the regulation of lactose metabolism, and the other was the
discovery of the new killer system involving DNA plasmids. The studies on the lactose regulon in K. lactis illustrated how the regulatory system of this unicellular eukaryote differed from the bacterial lactose operon. The lactose regulon, a close variation of the galactose regulon of S. cerevisiae, was shown to involve many genetically unlinked positive and negative regulatory genes. In parallel, the works of the killer system involving the linear DNA plasmids pGKL1 and 2 had also played an important role in the development of K. lactis biology, because at that time the only plasmids known in yeast were the 2 mm circular DNA and the double-stranded killer RNA of S. cerevisiae. In addition to their new killing mechanism and their linear mode of replication, pGKL plasmids were interesting in many aspects. The fact that K. lactis secreted a high-molecular weight killer toxin retained the attention of a few people who were looking for an efficient system to produce recombinant proteins in a secreted form. Indeed the 1980s were years that were full of enthusiasm for gene engineering for biotechnology. Thus, the milk-coagulating enzyme chymosin was produced industrially from K. lactis. Such achievements by a few industrial companies encouraged research on this particular yeast system. The successive biotechnology programs funded by the European Commission were a timely support, and it was through these programs that the first network of K. lactis research was set up in 1988. Since then the workshop, Biology of Kluyveromyces, continues to be an important instrument of communication and collaboration of the research community. Collaboration between K. lactis workers has been facilitated by two circumstances. First, the researchers have used, from the beginning, only a very small number of K. lactis isolates, including those used by the Madison group, thus a relatively homogeneous genetic system has rapidly emerged and has been shared by most laboratories, allowing the efficient exchange of strains. Second, after a deliberate 2005 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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search, a 2 mm type circular plasmid pKD1 was discovered in
the species Kluyveromyces drosophilarum (hence the name of the plasmid), which was capable of replicating in K. lactis, offering a replicating vector system equivalent to the 2 mm vectors. Kluyveromyces drosophilarum was later incorporated into K. lactis. The yeast species that assimilate lactose aerobically are widespread, but those that ferment lactose are rather rare. Beside K. lactis, Kluyveromyces fragilis is one of such lactosefermenting yeasts, and is well known in industry. This species is now incorporated into K. marxianus (E. C. Hansen) van der Walt (1971), distinct from K. lactis. At present the taxonomists recognize two varieties in K. lactis. One is K. lactis (Dombrowski) van der Walt var. lactis (1986), the other is K. lactis (Dombrowski) van der Walt var. drosophilarum (1986). Whereas the former is heterothallic and ferments lactose, the latter is homothallic and does not assimilate lactose (Lachance, 1998). Nearly all the published works on K. lactis concern the variety lactis. If S. cerevisiae stands in a very exceptional position among yeasts because of its fermentation-oriented Crabtree-positive physiology, K. lactis appears to be a good model of the large number of more aerobic species that are used in todays yeast biotechnology. At the opposite extreme, the fermentation-less Yarrrowia lipolytica may be a model for highly aerobic species, with its well-established genetic system.
2005 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
H. Fukuhara
While continuously stimulated by industrial interests, the
present research in K. lactis mostly focuses on fundamental aspects of physiology and gene regulation. The themes cover a wide range of topics, and the articles gathered in this issue do not represent the diversity of the ongoing research in this area. The total DNA sequence of the K. lactis genome has been established in 2004 through the Ge nolevures project. Thus, K. lactis has become, after S. cerevisiae, a most useful instrument of yeast study, combining both genetic and genomic information. In view of the distinctive physiologies of the two species, and their relatively recent common origin suggested by genomic sequence analyses, the comparison of these two species will be particularly useful to unveil details of the process of evolution of gene regulation and genome organisation. Such a comparative approach is a basic practice of K. lactis workers, as most of them are active in S. cerevisiae research and also exploring the biology of other nonconventional species.
Reference Lachance MA (1998) The Yeasts, A Taxonomic Study. 4th edn (Kurtzman CP & Fell JW, eds), pp. 227247. Elsevier Science B. V., Amsterdam.