Genetics and Molecular Physiology of the Yeast Kluyveromyces Lactis

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Fungal Genetics and Biology 30, 173–190 (2000) doi:10.1006/fgbi.2000.1221, available online at http://www.idealibrary.com on

REVIEW Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

Raffael Schaffrath1 and Karin D. Breunig

Institut fu¨ r Genetik, Martin-Luther-Universita¨ t Halle-Wittenberg, D-06099 Halle (Saale), Germany

Accepted for publication July 21, 2000

Schaffrath, R., and Breunig, K. D. 2000. Genetics and nable to genetic manipulation almost like Saccharomyces molecular physiology of the yeast Kluyveromyces lac- cerevisiae have been developed, (ii) its evolutionary dis- tis. Fungal Genetics and Biology 30, 173–190. With tance to S. cerevisiae often allows delineation of functional the recent development of powerful molecular genetic domains from sequence comparison of homologous genes, tools, Kluyveromyces lactis has become an excellent (iii) the regulation of carbon and energy metabolism re- alternative yeast model organism for studying the reflects its adaption to aerobic conditions and contrasts with lationships between genetics and physiology. In par- the well-studied but exceptional physiology of S. cerevi- ticular, comparative yeast research has been providing siae, and (iv) it has specific properties such as the “DNA- insights into the strikingly different physiological strat- killer system” that provide insight into new mechanisms of egies that are reflected by dominance of respiration microbial competition. This review puts particular empha- over fermentation in K. lactis versus Saccharomyces sis on the latter two aspects and points out the important cerevisiae. Other than S. cerevisiae, whose physiology insights that have been gained from studies of K. lactis. is exceptionally affected by the so-called glucose ef- Like the classical S. cerevisiae, K. lactis is an ascomyce- fect, K. lactis is adapted to aerobiosis and its respira- tous budding yeast that belongs to the endoascomycetales. tory system does not underlie glucose repression. As Formerly (mis)classified as S. lactis and K. fragilis var. a consequence, K. lactis has been successfully estab- lactis, K. lactis has been the subject of intensive taxonom- lished in biomass-directed industrial applications and ical studies; using molecular typing techniques such as large-scale expression of biotechnically relevant gene mtDNA-RFLP, rDNA alignments, and PFGE karyotyp- products. In addition, K. lactis maintains species-spe- ing (Herman and Halvorson, 1963; Fuson et al., 1987; cific phenomena such as the “DNA-killer system,” Vaughan-Martini and Martini, 1987; Cai et al., 1996; analyses of which are promising to extend our knowl- Kurtzman and Robnett, 1998), today K. lactis is reconsid- edge about microbial competition and the fundamen- ered to be a unique species. tals of plasmid biology. © 2000 Academic Press Its property of growing on lactose as a sole carbon source distinguishes K. lactis from most other yeasts and The yeast Kluyveromyces lactis is attracting increasing can be used to select isolates from dairy industry products attention from molecular biologists working in widely dif- (Valderrama et al., 1999), hence its name dairy or milk ferent fields. Several aspects have contributed to this de- yeast. In fact, it can use a wide range of substrates by velopment: (i) various molecular tools that make it ame- respiration. K. lactis is a so-called “petite-negative” yeast in which maintenance of mitochondrial DNA is essential. It 1 To whom correspondence should be addressed. E-mail: schaffrath@ has a respiratory–fermentative metabolism and most genetik.uni-halle.de. strains can grow on fermentable carbon sources when

1087-1845/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 173 174 Schaffrath and Breunig respiration is blocked. Nevertheless, K. lactis appears to be Despite organization similar to that of other yeast CEN- an obligate aerobe that is not able to grow under strict DNAs, the K. lactis centromeres have been shown to anaerobic conditions. function species specifically (Heus et al., 1990, 1994). Deletion of the K. lactis gene coding for the CDEI- binding protein, Cpf1p, is lethal (Mulder et al., 1994), whereas in S. cerevisiae a cbf1⌬ strain is only partially MENDELIAN GENETICS AND impaired for centromere function and is viable (Mellor et CHROMOSOMAL DNA al., 1990). A CEN-binding factor CBF5 homologue was isolated from K. lactis and implicated in chromosome separation (Winkler et al., 1998). Telomere structure– In contrast to that of S. cerevisiae, mitotic growth of K. function analysis has shown that, other than in S. cerevi- lactis is predominantly restricted to the haplophase, caus- siae, K. lactis maintains a perfect 25-bp telomere repeat ing diploid cells to spontaneously enter meiosis and sporu- present, on average, as 15 tandem copies, suggesting high ␣ lation. Most laboratory strains maintain stable and a fidelity of telomerase activity (McEachern and Blackburn, ␣ mating types encoded by the MAT or MATa genes but K. 1994). Telomere addition by telomerase uses a 30-nucle- lactis also carries cryptic mating-type alleles that are si- otide template region of its endogenous 1.2-kb TER1- lenced in a Sir4p-dependent manner and, as in S. cerevi- 1 RNA component that is complementary to 1 ⁄5 telomeric siae, mating-type switching can occur (Astrom and Rine, repeats (Fulton and Blackburn, 1998). Telomere growth is 1998). deregulated in ter1 mutants and dependent on interaction The chromosomal DNA has a GC content of 40%, of the most distal repeat unit with Rap1p, a dsDNA- comparable to that of S. cerevisiae. Karyotyping of K. lactis binding protein of the telomeric cap (Krauskopf and has revealed the presence of only six chromosomes, rang- Blackburn, 1996, 1998). Uncapping or cap complex dis- ing in size from 1 to 3 Mb (Sor and Fukuhara, 1989). The ruption ultimately causes cells to die (Smith and Black- estimated genome size of 12 Mb may not be significantly burn, 1999). The severity of this phenotype can be rescued smaller than that of S. cerevisiae but the larger size and on stabilizing deregulated telomeres by recapping, sug- smaller number of chromosomes are typical of non-Sac- gesting that maintenance of the terminal telomeric cap charomyces yeasts. Codon adaptation indices, studied in complex is essential for telomere function and cellular 47 chromosomal K. lactis genes by Lloyd and Sharp integrity (Smith and Blackburn, 1999). Consistently, lack (1993), follow the S. cerevisiae rules with a notable excep- of functional telomerase due to TER1 deletion results in tion for the unique CYC1 cytochrome c gene, which gradual telomere loss and progressive cellular senescence reflects the importance of respiratory metabolism in K. (Roy et al., 1998; Smith and Blackburn, 1999). Surpris- lactis. Despite the strikingly different karyotypes of K. ingly, cells surviving this catastrophic situation emerge at lactis and S. cerevisiae, the extent to which the local high frequencies. Life without telomerase, however, fully arrangement of gene order along the chromosomes has depends on RAD52, suggesting that recombination acti- been conserved amounts to 75% for 31 adjacent gene vated by telomere uncapping can restore telomere main- neighbors studied (Keogh et al., 1998). Consistent with the tenance and cell viability (McEachern and Blackburn, recent interpretation of gene arrangement data by K. 1996). Wolfe and co-workers that S. cerevisiae must have had its whole genome duplicated (Wolfe and Shields, 1997; Seoighe and Wolfe, 1998), gene redundancy in K. lactis is generally lower. The genetic map of K. lactis is poor, with NON-MENDELIAN GENETICS AND about only 30 linkage groups established and physically PLASMID BIOLOGY mapped to individual chromosomes (Wesolowski-Louvel et al., 1996). However, large-scale sequencing of random Mitochondrial DNA tags has identified 292 new K. lactis genes corresponding to about 5% of its estimated gene number (Ozier-Kalog- The mitochondrial DNA of K. lactis is a 39-kb circular eropoulos et al., 1998). Also, genetic/physical genome molecule and maintained, on average, as 30 copies per mapping is in progress and recent efforts are being made haploid genome, which roughly equals 10% of the total to initiate a separate K. lactis genome project (M. Bolotin- DNA content (Wesolowski-Louvel et al., 1996). Despite Fukuhara, pers. communication). strain-specific mtDNA polymorphism, partial DNA se-

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Genetics and Molecular Physiology of K. lactis 175 quence analysis has established a physical map that accom- been repeatedly used for gene cloning and identification. modates the 15S and 21S rRNA genes, 22 tRNA loci, and The Kluyveromyces host range of pKD1 is broader than ORFs encoding apocytochrome B (CYTB), as well as cy- that of the 2-␮m plasmid, allowing maintenance of pKD1 tochrome oxidase (COX1, COX2, and COX3) and mito- vectors in K. marxianus, K. wickerhami, K. thermotoler- chondrial ATPase (ATP6, ATP8, and ATP9) subunits. Con- ans, and K. waltii (Chen et al., 1989). sistent with evidence from mtDNA transcription in other yeast species, the mitochondrial RNA polymerase of K. Cytoplasmic Episomal DNA lactis recognizes a conserved nonanucleotide promoter A ( /TTATAAGTA), and all dairy yeast mtORFs are tran- Certain K. lactis strains maintain “killer DNA,” an en- scribed unidirectionally (Osinga et al., 1982). dogenous, cytoplasmic plasmid pair (k1 and k2; Fig. 1) Unlike the high frequency of spontaneously arising S. which encodes an anti-yeast toxin complex known as zy- cerevisiae petite (␳0) mutants, K. lactis is petite-negative. mocin (Gunge et al., 1981; Fig. 1). k1 and k2 have been Usually, loss of mtDNA is lethal for dairy yeast; however, thoroughly studied and represent a model system for an- if mutated at any of three nuclear loci called MGI (mito- alyzing the fundamentals of linear plasmid replication and chondrial genome integrity), K. lactis can be artificially gene expression (for reviews see Stark et al., 1990; Gunge, converted into a ␳0 yeast by continued growth in the 1986, 1995; Fukuhara, 1995; Meinhardt et al., 1997; Schaf- presence of DNA-targeting drugs (Chen and Clark- frath et al., 1999). Genetic analysis has shown that k1 Walker, 1993). Interestingly, disruption of the MGI genes encodes the three zymocin subunits (␣ and ␤: ORF2; ␥: which code for the mitochondrial F1-ATPase subunits ␣, ORF4; see below) and an immunity determinant (ORF3). ␤, and ␥ does not lead to the production of mitochondrial In contrast, the helper plasmid k2 provides vital k1/k2- genome deletion mutants and petite colonies, indicating maintenance functions (Schaffrath and Meacock, 1995), that an assembled F1 complex is needed for the “gain-of- including factors for replication (ORF2, ORF5, and function” of the mgi phenotype (Chen and Clark-Walker, ORF10), mRNA capping (ORF3) (Larsen et al., 1998), 1995, 1996). Inactivation of the F1-ATPase ␣ and ␤ sub- DNA unwinding (ORF4), and a plasmid-specific RNA unit genes in S. cerevisiae converts this petite-positive polymerase (RNP) (ORF6 and ORF7) (Wilson and Mea- yeast into a petite-negative yeast (Chen and Clark-Walker, cock, 1988; Schaffrath et al., 1995a, b, 1997a). k1 and k2 2000). Thus, comparative studies on nuclear genes affect- genes are activated from plasmid-specific promoter motifs ing mtDNA maintenance promise to yield important in- (UCSs) shown to be incompatible with the K. lactis host- sights into nuclear–mitochondrial interactions. encoded RNPs (Romanos and Boyd, 1988). A body of evidence, including terminal proteins attached to k1 and Nuclear Episomal DNA k2 and genes on both k1 (ORF1) and k2 (ORF2) that code for DNA polymerases, suggests a viruslike protein-primed The nuclear 2-␮m-type episome, pKD1, originally iso- mode of plasmid replication with replicases that function lated from K. drosophilarum, can be stably maintained in plasmid specifically (Kitada and Gunge, 1988; Salas, 1991; ϩ K. lactis to create cir hosts (Chen et al., 1986; Falcone et Schaffrath et al., 1995b). k2 encodes two other likely al., 1986). In analogy to the 2-␮m plasmid of S. cerevisiae, maintenance factors: TRF1 (ORF10), a plasmid-specific pKD1 is phenotypically cryptic and exists as two isomers replication initiator (McNeel and Tamanoi, 1991), and that can be interconverted by the gene A product, an Orf5p (ORF5) (Schaffrath and Meacock, 1995), which FLP-like site-specific recombinase. Genes B and C prob- binds to single-stranded DNA in vitro and probably pro- ably code for pKD1 partitioning factors, and autonomous tects replicative intermediates from exonucleolytic degra- replication requires a cis-acting ori sequence (Bianchi, dation in vivo (Schaffrath, 1995; R. Schaffrath and P. A. 1992; Bianchi et al., 1991). pKD1 can be used to transform Meacock, submitted). Summing up, in the fundamental S. cerevisiae cir0 strains but plasmid maintenance is very processes of replication and transcription, these plasmids low; vice versa, 2-␮m-based vectors get lost readily in K. function remarkably independent of their host cell. lactis without maintaining constant selective pressure. Mi- Successes in genetically manipulating k1 and k2 by totic stability, however, is increased in both species by allelic replacement have provided powerful tools for introducing the 2-␮m ori into pKD1-based vectors and studying these unusual plasmids at the molecular level ϩ using cir K. lactis or S. cerevisiae hosts carrying resident (Ka¨mper et al., 1989, 1991; Tanguy-Rougeau, 1990; Schaf- pKD1 or 2-␮m plasmids. This type of vector allows gene frath et al., 1992). To do so, the transcriptional barrier and library shuttles between both yeast species and has between host- and plasmid-specific genes was overcome

FIG. 1. The K. lactis killer system. (A) Genetic organizations of killer plasmids, k1 and k2. Functionally related genes are identically color–coded according to Schaffrath et al. (1999): zymocin biogenesis/immunity: blue; DNA replication: grey; transcription and mRNA modiﬁcation: orange; unassigned orphans: red. k1- and k2-speciﬁc terminal proteins (TPs) and terminal inverted repeats (TIRs) are illustrated as white/black circles/triangles,

␣ ␤ ␥ by introducing suitable markers fused to UCSs and ﬂanked termed , , and in decreasing order of their Mr (98, 30, by target sequences to direct k1/k2-speciﬁc integration. and 28 kDa, respectively), are encoded by the killer plas- This knockout technique has allowed dispensable and es- mid k1 (see above). The amino-terminal residues of the ␣ ␤ ␥ sential genes to be distinguished (Schaffrath et al., 1999). mature , , and polypeptides are A30 (ORF2), G895

In addition, development of a cytoplasmic gene shuffle (ORF2), and A19 (ORF4), respectively. Hence the two system has facilitated the swapping of genes between k1 larger subunits are the processed products of a single and k2 to reintroduce site specifically mutagenized or gene, k1ORF2 (Stark and Boyd, 1986; see above), and modified alleles (Schaffrath et al., 1996, 1999, 2000). Thus, result from proteolysis of an ␣␤ precursor at about two- Orf5p, a protein essential for maintenance of plasmid thirds along the primary ␣␤ translation product; the ␥ integrity (Schaffrath and Meacock, 1995), has been shown subunit, however, is the processed product encoded by to cause severe plasmid instability in vivo when carboxy- k1ORF4 (Stark and Boyd, 1986; see above). As for the terminally mutated and identified by way of epitope tag- latter, its mature NH2 terminus (A19) colocalizes within the ging (Schaffrath and Meacock, 1996; R. Schaffrath and first of three potential signal peptidase recognition sites P. A. Meacock, submitted). Functionally, the tagged vari- 2 2 2 (C16AA A A ). In contrast, despite the existence of such ant is indistinguishable from the wild-type protein and is a recognition site within the ORF2 leader region as abundant as TRF1 (see above), implying a structural 2 ␣ (V19QG ), the amino-terminus of the subunit (A30 of function (Schaffrath and Meacock, 1996). In addition, the ORF2)inthe␣␤ precursor does not appear to result from major transcriptase gene (ORF6) has been swapped and signal peptidase cleavage, but rather from proteolytic pro- ⌬ shown to complement an orf6 knockout created on plas- cession by another endopeptidase, presumably the K. lac- mid k2 when ectopically shuffled onto k1 (Schaffrath et al., tis Kex2p homologue: Kex1p (Stark et al., 1990; see be- 2000). In conclusion, by use of the gene shuffle technique, low). elucidation of k1/k2 gene function, regulation of k1/k2 Zymocin biosynthesis can be prevented in the presence gene expression, and UCS promoter analysis has been of tunicamycin, suggesting that the complex is glycosylated experimentally addressable (Cong et al., 1994; Schaffrath (Sugisaki et al., 1985). As judged from endo-␤-N-acetyl- et al., 1996, 2000; R. Schaffrath and P. A. Meacock, sub- glucosaminidase H (endo H) digestion of purified zymocin mitted). As a consequence, k1 and k2 have acquired con- preparations, the ␣ subunit shows a decrease of about 3.4 siderable interest as an alternative vector system (see be- kDa in its M (Stark and Boyd, 1986). Together with the low; Meinhardt et al., 1997; Schaffrath et al., 1999). r observation that mannose-rich moieties are detected only for the ␣ subunit by use of a concanavalin A conjugated- horseradish peroxidase-based chromogenic assay (Gunge, THE K. lactis ZYMOCIN 1995; Schaffrath et al., 1997b), this indicates a single, N-linked oligosaccharide chain associated with the ␣ Structure and Biogenesis polypeptide. In contrast, none of the other subunits appears to be N- nor O-glycosylated and both are endo H The holo-zymocin secreted by killer strains of K. lactis is resistant (Stark and Boyd, 1986). Structural integrity of the a heterotrimeric glyco–protein complex. Using micro-se- ␣␤␥ glyco–protein complex is maintained by internal cys- quence analysis, it became evident that all three subunits, teine bonds within the ␣ subunit and an intermolecular

respectively. DNP, DNA polymerase; RNP, RNA polymerase; SSB, single-stranded DNA-binding protein; TRF1, terminal recognition factor 1; for explanations see text. (B) Killer phenotype. K. lactis killer (K) and nonkiller (NK) strains were grown on a lawn of sensitive S. cerevisiae cells; growth inhibition is visualized by halo formation (C) Zymocin-mediated cell cycle arrest. Sensitive S. cerevisiae cells were treated with zymocin and analyzed by FACS. Cell fractions with 1n and 2n DNA contents are indicated by arrows. FIG. 2. The LAC/GAL Regulon of K. lactis. (A) Structural genes of the LAC/GAL regulon are shown as arrows (not drawn to scale) with KlGal4p binding sites as green triangles. Regulatory genes are colored. On top, the function of the gene products in the lactose–galactose metabolic pathway is shown. The gray bow indicates the plasma membrane with integrated sugar permeases. Galactose can enter the cell via Lac12p and independently of Lac12p. The Kht1p/Rag1p hexose transporter is a candidate. (B) Interactions regulating Gal4p transactivator activity. The Gal80p protein blocks Gal4p function by interacting with the activation domain. Gal1p relieves the inhibition by binding Gal80p in a galactose- and ATP-dependent way. In S. cerevisiae the formation of a Gal4p–Gal80p–Gal3p trimeric complex could be demonstrated (Platt and Reece, 1998).

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 178 Schaffrath and Breunig disulfide bridge between the ␤ and the ␥ polypeptides. that still contain k2 are phenotypically sensitive toward Hence, treatment of purified zymocin with ϪSH reagents zymocin and indistinguishable from plasmid-free nonkiller causes subunit disintegration and completely abolishes strains (Niwa et al., 1981). In contrast, nonkiller strains biological activity (Stark et al., 1990). carrying the k1-based orf2⌬ knockout plasmids k1-NK2, Zymocin secretion is blocked in a K. lactis kex1 mutant pGKL1S, or pGKL1D retain immunity but fail to produce and can be rescued by single-copy complementation with zymocin (due to being deleted in the ␣␤ subunit structural the KEX2 gene from S. cerevisae. Vice versa, the K. lactis gene, ORF2) (Gunge, 1995). Since strains maintaining the KEX1 gene is able to functionally complement a kex2⌬ k1 orf2-4⌬ deletion plasmids F1 and F2 fail to confer both gene knockout in baker’s yeast (Wesolowski-Louvel et al., autoimmunity and zymocin expression (Kikuchi et al., 1988; Tanguy-Rougeau et al., 1988). Kex2p localizes to the 1985), the k1ORF3 gene was considered to code for im- Golgi and is essentially involved in biogenesis of secreted munity. In fact, on cloning ORF3 into a K. lactis ARS proteins such as prepro-␣-pheromone and the dsRNA- vector and retransformation into a k1-free host strain, encoded K1 killer toxin precursor. Not only do recombi- immunity toward zymocin was partially restored as long as nant k1/k2-carrying S. cerevisiae strains process and se- the cell comaintained k2 (Tokunaga et al., 1987). There- crete authentic zymocin but the kex2⌬ mutant of baker’s fore, k2 was postulated by Stark et al. (1990) to be essen- yeast is also unable to produce secretable zymocin (Toku- tially needed for ORF3 gene expression on the ARS vec- naga et al., 1990; Stark et al., 1990). Thus, zymocin pro- tor; alternatively, it might encode another immunity factor duction appears to be very similar in both yeast species (in addition to k1ORF3). Although there is sequence ho- and processing is obligatorily coupled with secretion dur- mology between the k1ORF3 and the k2ORF1 genes ing its biogenesis and/or assembly into its holo-form. Con- (Stark et al., 1990), a role of the latter in immunity has sistently, in the absence of ␣␤ secretion the ␥ subunit fails been all but ruled out by targeted gene disruption: strains to be secreted but accumulates intracellularly (Tokunaga carrying a k2-based orf1⌬ϻLEU2 null mutation are indis- et al., 1989). A zymocin-resistant S. cerevisiae strain har- tinguishable from wild-type killer strains in expression of boring a conditional galactose-regulatable ORF4 secretion both killer and immunity phenotypes (Schaffrath et al., vector does not allow for ␥ subunit export under inducing 1992). Immunity is also seen in S. cerevisae strains toward conditions; instead, despite its functional secretion signal, expression of intracellular ␥ toxin subunit (see above; which has been shown to efficiently direct the secretion of Tokunaga et al., 1989), evidence which may implicate that heterologous proteins, the ␥ subunit also accumulates in- immunity does not work simply by exclusion of the zymo- tracellularly (Tokunaga et al., 1988, 1989). However, its cin from the cell. Instead, it suggests that immunity func- secretion can be effectively restored by genetically engi- tions by interaction of the Orf3p determinant with the ␥ neering a heterologous pro-sequence from S. cerevisiae subunit itself or with its putative intracellular target(s) in-frame between the ␥ leader peptide and its mature (Stark et al., 1990). coding region (Tokunaga et al., 1989, 1990). If this foreign ␣␤ pro-sequence can circumvent the requirement for the Mode of Action precursor to promote ␥ subunit export, why is ␣␤ needed during zymocin biogenesis? Explanations may include a The K. lactis zymocin is active against the growth of a role in assisting the correct folding of the ␥ polypeptide or variety of sensitive yeast genera, including Saccharomyces, a requirement for the ␣␤ precursor to assemble the ␥ Kluyveromyces, and Candida but not Schizosaccharomy- subunit into a secretable holo-zymocin either by facilitat- ces (Gunge et al., 1981; Gunge and Sakaguchi, 1981; Fig. ing the formation of the intermolecular ␤–␥ disulfide 1). Unlike the killer toxin K1 of S. cerevisiae, the K. lactis bridge (see above) or by providing the unglycosylated ␥ zymocin does not cause sensitive cells to hyperactivate subunit with a glycosylated entity typical for yeast exo- Tok1p and elicit rapid ion effluxes (Ahmed et al., 1999) but proteins. rather inhibits cell division (Butler et al., 1991a). Zymocin- treated S. cerevisiae strains accumulate as unbudded cells, Immunity implying that a G1 cell cycle-specific event may be affected (Butler et al., 1991a; Fig. 1). Further evidence that Not surprisingly, a K. lactis killer strain also expresses the zymocin acts specifically in G1 is provided by the immunity toward its own secreted exo-zymocin. Immunity observation that cells that have been chemically arrested is strictly dependent on the maintenance of plasmid k1: as in S phase by hydroxyurea prior to zymocin treatment are shown by curing experiments, k1-free isolates of K. lactis able to complete one round of cell division and get ar-

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Genetics and Molecular Physiology of K. lactis 179 rested in the new unbudded G1 cell cycle stage once they losamidin (Butler et al., 1991c) and the hydrophobicity have been released from the chemical S block in the predicted for the ␤ subunit, this suggests that ␣␤ may presence of zymocin (Butler et al., 1991a). Indeed, FACS associate with the plasma membrane. It is therefore analysis demonstrates a prereplicative DNA content (1n) tempting to speculate that ␣␤ may be required for native suggestive for a G1 cell cycle block prior to START (Butler zymocin to act from the cell’s exterior by docking to cell et al., 1991a; L. Fichtner and R. Schaffrath, unpublished wall-associated chitin and mediating ␥ toxin translocation. data; Fig. 1). Consistently, as shown by incorporation of Indeed, depletion of chitin by way of deleting, inactivating, radiolabeled precursors into RNA and protein, treated or mistrafficking major yeast chitin–synthase III activity cells are still metabolically active, allowing for transient renders cells refractory to the inhibitory effects of exo- macromolecular biosynthesis to be permitted (Butler et zymocin. Thus, chitin appears to be the zyomocin receptor al., 1991a; M. J. R. Stark, pers. communication). Also, they required for the initial interaction with sensitive cells (D. increase in volume by some 30–50%, similar to the bona Jablonowski and R. Schaffrath, unpublished data; see be- fide pre-START G1 arrests induced by the mating phero- low). mone cascade or growth of cdc28ts strains at the nonper- Based on their ability to grow in the presence of the missive temperature (Leberer et al., 1997). holo form, zymocin-resistant S. cerevisiae mutants Earlier reports that the zymocin functions on S. cerevi- (termed skt (sensitivity to K. lactis toxin), iki (insensitive to siae by inhibiting the CDC35 gene product, adenylate killer), and kti (K. lactis toxin insensitive), respectively) cyclase, and hence abolishing the roles of cAMP essential have been isolated independently (Kawamoto et al., 1992; for mitotic growth and cell division (Sugisaki et al., 1983) Butler et al., 1994; Kishida et al., 1996). Sensitivity of these have been disproven by knocking out BCY1, which en- mutants toward intracellular, conditional expression of the codes the regulatory subunit of cAMP-dependent protein ␥ toxin from inducible promoters (see above) can distin- kinase; the bcy1⌬ mutant which no longer requires cAMP guish zymocin binding/uptake (class I) from ␥ toxin target to undergo cellular division does grow in the absence of a site mutants (class II). Consistent with the role that the ␣ functional CDC35 gene product but behaves as sensitive subunit may play in assisting zymocin docking to the cell to and G1 arrestable by the exo-zymocin as its wild-type surface by virtue of its exo-chitinase activity (see above) parent strain (White et al., 1989). Thus, it is highly unlikely are studies on chs (chitin synthesis-deficient) S. cerevisiae that the zymocin exerts its toxicity by inhibiting Cdc35p; mutants (Takita and Castilho-Valavicius, 1993). The ma- yet, studying its mode of action may prove a useful tool for jority of these behave as class I mutants; i.e., they are dissecting stage-specific events in G1. resistant to exo-zymocin but sensitive toward endogenous Interestingly, toxicity of the zymocin complex appears to expression of the ␥ toxin. Moreover, KTI2 is allelic with reside solely within the smallest subunit, the so-called ␥ CHS3, and KTI10 may very well correspond to CHS6 toxin: conditional expression of its structural gene from (M. J. R. Stark, pers. communication); CHS3 codes for the tightly regulated yeast promoters (UASGAL or UASMET) catalytic subunit of chitin synthase III (CSIII), the in vivo mimics exogenous treatment of the holo-zymocin and is activity of which is lacking in chs6 mutants and usually fully lethal to sensitive strains of S. cerevisiae under in- amounts to 90% of a wild-type cell’s chitin synthesis (Cos ducing conditions (Tokunaga et al., 1989; Butler et al., et al., 1998; Ziman et al., 1998). In addition, knocking out 1991b; Schaffrath et al., 1997b; F. Frohloff and R. Schaf- SKT5 (isoallelic with CHS4), which encodes a posttrans- frath, unpublished data). Endogenous expression of ma- lational activator of CSIII, renders cells resistant to exo- ture ␥ toxin (i.e., without its native secretion leader) re- zymocin (Kawamoto et al., 1993; Trilla et al., 1997). Al- sults in biologically active ␥ toxin, whereas, exogenously though the class I gene KTI6 is nonallelic with CHS3, applied, the ␥ polypeptide is not able to inhibit cell growth CHS4, and CHS6, its mutation obviously affects zymocin (Tokunaga et al., 1989). As for the roles played by the ␣ binding or ␥ toxin uptake (M. J. R. Stark, pers. commu- and ␤ subunits within the secreted exo-zymocin, this nication). So it is possible that it corresponds to either strongly implies that the holo form must assist the ␥ toxin CHS5 or CHS7, two genes that have been recently shown subunit to be taken up by the cell. As a precondition for ␥ to function in targeting and trafficking of Chs3p from the subunit entry and action, the holo-zymocin is therefore ER to chitosomes (Ziman et al., 1996; Santos and Snyder, expected to first of all bind to the cell surface to be able to 1997; Trilla et al., 1999). Summing up, we propose that the signal its toxicity (Stark et al., 1990). Together with the primary interaction of the holo-zymocin with its sensitive finding that the ␣ subunit has an essential exo-chitinase cell is facilitated by docking to cell wall chitin. Thus, chitin activity in vitro that can be specifically inhibited by ␤-al- may serve as a receptor molecule and all scenarios that

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 180 Schaffrath and Breunig lead to a reduction of chitin synthesis due to abolishing, progression into S phase (Di Como and Arndt, 1996; Luke reducing, and/or mistrafficking CSIII activity render S. et al., 1996). Sit4p is required for execution of START and cerevisiae cells resistant toward K. lactis zymocin (D. Jab- sit4ts strains arrest late in G1 prior to START, in part due lonowski and R. Schaffrath, unpublished data). to the role of Sit4p in expression of the G1 cyclin genes The presence of as many as 10 distinct target site class CLN1 and CLN2. Interestingly, sit4⌬ null mutants which II mutants (see above) suggests that a complex pathway are viable only in certain SSD1-␯ backgrounds are fully transduces the zymocin’s inhibitory effect (Butler et al., resistant to partially purified zymocin (Stark, 1996). Thus, 1994). Whereas, some of them may be involved in the though it is tempting to speculate that zymocin might expression of targets inhibited by the ␥ toxin, a number of antagonize G1 cyclin function, we can only conclude that proteins could also participate in the process that is Sit4p is required for cells to respond to it. Also, neither a blocked by it. These might act as a biochemical pathway hyperactive dominant CLN3-1 allele nor overexpression of or, alternatively, form a complex containing several com- CLN2 can significantly reduce zymocin sensitivity (Butler ponents. To identify ␥ toxin target site (tot) mutant(s) we et al., 1994; Schaffrath et al., 1997b). Therefore, the mode are exploiting genetic screens involving two-hybrid inter- of zymocin action still remains unclear. However, we an- action trapping and gene knockout transposon tagging ticipate that molecular genetic analyses of zymocin-resis- (Schaffrath et al., 1997b). As for the latter, a pool of yeast tant mutants (see above) rather than biochemical ap- transformants which carry a Tn3ϻlaczϻLEU2 minitrans- proaches will provide more direct routes to identifying and poson randomly inserted into the genome are screened for characterizing the ␥ toxin target site, TOT. viability and ␥ toxin resistance, the tot phenotype, by conditionally switching on ␥ toxin expression (see above). Candidate clones are then subjected to plasmid rescue in TOOLS FOR GENETIC MANIPULATION Escherichia coli and characterized by DNA sequencing. In this way we and others have identified several resistant yeast disruptants. So far seven genes (IKI1/TOT5, IKI3/ The use of K. lactis as a model nonconventional yeast ELP1/TOT1, ELP2/TOT2, ELP3/TOT3, KTI12/TOT4, has been significantly advanced as a result of several tech- SIT4, and SAP155) have been implicated in affecting in- nical developments. Standard prototrophic yeast markers tracellular ␥ toxin action. Iki1p/Tot5p is part of an insol- isolated from S. cerevisiae (URA3, LEU2, TRP1, etc.) were uble fraction in cell extracts and Iki3p/Tot1p is predicted shown to function in the appropriate K. lactis auxotrophic to possess a membrane-spanning region, raising the pos- backgrounds and vice versa; the K. lactis homologues sibility that an insoluble Iki1p/Iki3p-containing compart- could be identified in S. cerevisiae by means of interge- ment is involved in toxin action (Yajima et al., 1997). The neric complementation (Shuster et al., 1987; Stark and recent reidentification of Iki3p as Elp1p, a complex com- Milner, 1989; Zhang et al., 1992; Bai et al., 1999). There ponent of elongating RNA polymerase II holo-enzyme, are also dominant markers conferring resistance to gene- which may associate with two other elongator constituents, ticin (G418R), bleomycin (BLER), and aureobasidin A Elp2p/Tot2p and Elp3p/Tot3p, provides substantial evi- (AUR1) or enabling acetamide utilization (amdS) dence for this hypothesis (Otero et al., 1999; Wittschieben (Sreekrishna et al., 1984; Chen and Fukuhara, 1988; et al., 1999; Fellows et al., 2000; F. Frohloff and R. Hashida-Okado et al., 1998; Castrillo et al., 1999). Trans- Schaffrath, unpublished data). Depletion or overproduc- formation techniques include PEG-mediated DNA trans- tion of Kti12p/Tot4p confers ␥ toxin resistance; so Kti12p fer into protoplasts, chemical lithium acetate protocols, may be a potential ␥ toxin target (Butler et al., 1994). If and electroporation (Das and Hollenberg, 1982; Klebe et absent (kti12⌬) or mutated (as in the kti12 background), al., 1983; Meilhoc et al., 1990). The latter yields high the ␥ toxin cannot bind to Kti12p, whereas high KTI12 transformation efficiencies and is generally preferred gene dosage might lead to excess, unbound Kti12p which when using genetic screens based on single and/or multi- competes for a downstream effector molecule, thereby copy genomic libraries. diluting the toxic signal of the ␥ subunit (Schaffrath et al., Currently, there are several plasmid vector systems 1997a). Consistent with this we found that Tot4p interacts available to transform K. lactis cells. These are based on with Elp1-3p/Tot1-3p and Iki1p/Tot5p (L. Fichtner, F. either the multicopy episomal pKD1 plasmid (see above), Frohloff, and R. Schaffrath, unpublished data). Sap155p the K. lactis autonomously replicating sequences (KARSs) associates in a cell cycle-dependent manner with the TOR (Das and Hollenberg, 1982; Thompson and Oliver, 1986), pathway phosphatase Sit4p, which functions late in G1 for or the K. lactis centromeric sequences (K1CENs, see

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Genetics and Molecular Physiology of K. lactis 181 above) (Heus et al., 1990). Thus, depending on the type of extracellular acid phosphatase, ␣ pheromone, invertase, genetic manipulation, one can choose from a variety of glucoamylase, and human serumalbumin (Bui et al., 1996; vectors, including low and multicopy episomal plasmids, Gellissen and Hollenberg, 1997; Ferminan and integrative vectors (Chen, 1996), intergeneric plasmids Dominguez, 1998). and libraries to shuttle DNA between S. cerevisiae, K. High-level gene expression can be approached from lactis, and E. coli (Wesolowski-Louvel et al., 1996), or even either integrative or episomal strategies allowing mitotic shuffle-recombinant DNA constructed in vitro between stability or multicopy dosage effects of the foreign gene. the linear plasmids k1 and k2 in vivo (Schaffrath and Both approaches have been successfully exploited with Meacock, 1996; Schaffrath et al., 1999, 2000). Regulation expression yields on the order of several grams for bovine of gene expression can be studied by standard reporter prochymosin and human serumalbumin per liter fed-batch assays (Chen and Fukuhara, 1988); to utilize the E. coli fermenter culture (van den Berg et al., 1990; Fleer et al., lacZ gene, one should work on a lac4 mutant lacking 1991). Strong transcriptional activation is obtained by both endogenous dairy yeast ␤-galactosidase. constitutive and conditional promoter systems (van den In contrast to the high fidelity of gene replacement in S. Berg et al., 1990; Bui et al., 1996; Ferminan and cerevisiae, knocking out genes by homologous recombina- Dominguez, 1998; Saliola et al., 1999; Mustilli et al., 1999; tion in vivo is less efficient in K. lactis (Wesolowski-Louvel Hsieh and Da Silva, 2000). As far as posttranslational et al., 1996). Nonetheless, targeted gene replacements are modifications are concerned, recombinant ␤-lactoglobulin possible by means of one- or two-step disruption tech- secreted from K. lactis has been shown to be virtually niques and, recently, Bundock et al. (1999) have described indistinguishable from the native protein based on PAGE a modified protocol that avoids illegitimate recombination analysis, reactivity to antibodies, CD, fluorescence spec- and results in a 10-fold increase of homologous targeting troscopy, and N-terminal sequencing (Rocha et al., 1996). studied at the K. lactis TRP1 locus. Also, other than in S. Recently, the zymocin system itself has acquired agri- cerevisiae, knocking out genes essential for viability in a cultural interest for use as anti-yeast additive during model diploid background requires constant selective pressure to silage fermentations. Aerobic spoilage by lactic acid-utiliz- suppress the transitory nature of the diplophase in K. ing yeast genera (Pichia and Candida) was shown to be lactis. significantly reduced in laboratory-scale experiments using lactose and K. lactis killer strains that were defective for growth on lactic acid due to deletion of the K1PCK1 gene BIOENGINEERING WITH K. lactis (coding for PEPCK, a key enzyme for gluconeogenesis; Kitamaoto et al., 1998, 1999). Whether the k1/k2 plasmid pair can be exploited as an alternative vector system awaits K. lactis has received increasing attention from bioin- further analysis. Gene dosage effects (Ն50 molecules per dustries. Attractive properties include its GRAS (generally haploid genome), mitotic stability, and a yeast host spec- regarded as safe) status (Bonekamp and Osterom, 1994), trum potentially broader than that of pKD1 are in favor of molecular genetic accessibility using both integrative and k1 and k2, and pilot expression has been achieved with highly stabilized episomal vector systems (Wesolowski- bacterial and viral genes (Meinhardt et al., 1994; Louvel et al., 1996; Hsieh and Da Silva, 1998), and excel- Schru¨ nder et al., 1996; Schickel et al., 1996). However, lent fermentative characteristics allowing for large-scale gene activation appears to be poor compared to nuclear industrial applications (Fleer, 1992; Swinkels et al., 1993). transcription (Schaffrath et al., 1996, 1999, 2000) and a Moreover, its capability of exporting the zymocin complex comparative study between a k1- and a pKD1-based ex- suggests that K. lactis is suitable for secretion of large pression approach has shown that, despite similar mRNA heterologous proteins. Thus, both the ␣␤ and the ␥ zymo- levels, the latter is superior with regards to total protein cin subunit secretion leader signals have been reproduc- yields (Reliene and Sasnauskas, 1997). Therefore, in ad- ibly used to export heterologous proteins such as human dition to increase in gene activation, another ascet to be interleukin 1␤ and serum albumin, mouse ␣-amylase, hep- optimized is translational initiation. Experimentally, this atitis C virus envelope glycoprotein E2, as well as bacte- should be approachable by gene shuffle-mediated UCS rial/fungal ␤-lactamase and xylanases to name a few (Fleer optimization and introduction of strong ribosome-binding et al., 1991; Tokunaga et al., 1992, 1997; Walsh et al., 1998; sites (Schaffrat et al., 1999, 2000; see above). Saliola et al., 1999; Mustilli et al., 1999; Durand et al., In a number of studies in which identical proteins were 1999). Other efficient secretion signals derive from yeast expressed in S. cerevisiae and K. lactis or other noncon-

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 182 Schaffrath and Breunig ventional yeasts, the latter have proven superior both in phenotype) has been used to select for mutants affecting Ϫ product yield and in secretion efficiency (Bui et al., 1996; fermentative metabolism (Goffrini et al., 1989). Rag mu- Gellissen and Hollenberg, 1997; Fleer et al., 1991; Van tants depend on oxidative phosphorylation and are unable den Berg et al., 1990). These empirical findings suggest to grow on glucose when respiration is blocked. Most RAG that nonconventional yeasts have particular properties that genes were shown to be involved in controlling the glyco- are different from those of S. cerevisiae, making them lytic flux. They encode glycolytic enzymes, a low-affinity attractive hosts for recombinant protein production. glucose transporter, or regulators of glycolysis (reviewed in Breunig et al., 2000). In the absence of respiration inhib- itors many of the rag mutants grow normally. Thus, im- pairment of glycolysis does not have a general negative MOLECULAR PHYSIOLOGY AND influence on cell physiology in K. lactis, in contrast to the PRIMARY CARBON METABOLISM situation in S. cerevisiae. Several metabolic activities have been pointed out that circumvent the necessity for a high The Crabtree Effect glycolytic flux in K. lactis. First, respiratory enzymes are not subject to strong glucose repression (Mulder et al., One of the properties of K. lactis favorable for recom- 1995). Second, the pentose phosphate cycle can efficiently binant protein expression is its so-called Crabtree-negative bypass a block in glycolysis (Jacoby et al., 1993). Third, a phenotype (De Deken, 1966). Crabtree-positive yeasts high biosynthetic capacity seems to allow for efficient such as S. cerevisiae form ethanol under aerobic condi- biomass accumulation even at a very low glycolytic flux tions, thus reducing ATP and biomass yield compared to (Breunig et al., 2000). Crabtree-negative yeasts (Verduyn, 1991). Ethanol formation can be avoided by sugar-limited cultivation but this Glucose Uptake results in low specific growth and protein synthesis rates. It was therefore important to understand the molecular Apparently, K. lactis is able to restrict the glycolytic flux basis of the Crabtree effect. by tightly regulating glucose uptake. Only one constitu- Insight was provided from studies of pyruvate metabo- tively expressed glucose transporter encoded by the HGT1 lism in K. lactis. A mutant in which pyruvate dehydroge- gene has been reported (Billard et al., 1996) but this nase was blocked by a mutation in the PDA1 gene (en- high-affinity transporter is insufficient to support fermen- coding its largest subunit) was phenotypically reversed tative growth and thus seems to have a low capacity. The into Crabtree positivity. This result indicated that K. lactis other known transporters are regulated at the transcrip- is able to produce ethanol under aerobiosis. Obviously, in tional level. The first one described encodes a low-affinity the wild type the capacity of Pdh allows channeling of all permease, Rag1p. Deletion of the RAG1 gene leads to a Ϫ glycolytic pyruvate into the TCA cycle by converting it into Rag phenotype and natural isolates have been shown to acetyl-CoA, and only when this step is down-regulated or contain a mutant rag1 allele (Goffrini et al., 1990). RAG1 blocked does the alternative enzyme pyruvate decarboxyl- is induced by several hexoses but induction is rather slow ase convert pyruvate into acetaldehyde and ethanol (Zee- (Chen et al., 1992). Possibly, oxygen limitation in high- man et al., 1998; Kiers et al., 1998). In addition, any density batch cultures contribute to full induction. ethanol eventually produced will be reassimilated effi- The other two known glucose transporters are Kht1p ciently due to the presence of an ethanol-inducible etha- and Kht2p. Kht1p is almost identical to Rag1p except for nol dehydrogenase in K. lactis (Mazzoni et al., 1992). the last two amino acids (Weirich et al., 1997) and is The difference between Crabtree-positive and similarly regulated (C. Milkowski and K. D. Breunig, sub- Crabtree-negative yeasts may thus be due to a difference mitted). In fact, the genes KHT1 and KHT2 replace the in Pdh and/or TCA cycle enzyme activity or to a difference RAG1 gene in some strains. Sequence analysis indicated in regulation of the glycolytic flux or both. that the tandemly arranged KHT genes had undergone a recombination event to give rise to the chimeric RAG1 RAG Genes and Glycolysis gene which is found in most laboratory strains in use (Weirich et al., 1997). Kht2p, like Rag1p/Kht1p, is able to Even in media containing high concentrations of glu- support fermentative growth and is expressed at high cose the glycolytic flux seems to be tightly controlled in K. levels in the absence of glucose. However, in high-glucose lactis. Resistance against antimycin A on glucose (Rag medium the KHT2 gene is repressed, indicating that

Kht1p is the major uptake systems under these conditions permease, respectively, are specific for lactose metabolism (C. Milkowski et al., submitted). Although strains lacking and these genes are sufficient to convert S. cerevisiae into ϩ all known transporters can still grow on glucose, none of a Lac yeast (Sreekrishna and Dickson, 1985). Four the remaining uptake systems that remain to be identified Gal4p-binding sites upstream of LAC4 and LAC12 control is able to support fermentative growth, indicating that the both genes in a coordinate fashion (Go¨decke et al., 1991) regulation of Rag1p (or Kht1p–Kht2p) is of major impor- (Fig. 2). In common with S. cerevisiae is the genetic tance in controlling glycolytic flux. organization and topology of the K. lactis GAL gene cluster, indicating evolutionary pressure on this particular ar- Glucose Repression rangement (Webster and Dickson, 1988). Whereas galactose induction does not depend on galac- Several lines of evidence suggest that a restriction of the tose metabolism, lactose induction requires lactose per- glycolytic flux may also contribute to the low sensitivity of mease and ␤-galactosidase activity (Fig. 2). In fact, intra- K. lactis to glucose repression. First, repression is stronger cellular galactose is evidently the activation signal for in strains containing the KHT1–KHT2 gene pair than in Gal4p (Zenke et al., 1996; Cardinali et al., 1997). Partly RAG1 strains. Second, overexpression of KHT1–KHT2 due to comparative studies in S. cerevisiae and K. lactis, further supports repression. Third, mutation of RAG1 or transduction of the galactose signal to the transcription KHT1–KHT2 abolishes glucose repression (Weirich et al., machinery is one of the best-understood examples of nu- 1997; Breunig, 1989). However, the sensitivity to glucose trient signaling in eukaryotes. A crucial finding was the repression is highly strain dependent and seems to be bifunctional nature of the K1GAL1 gene product, which influenced by multiple genetic loci. has galactokinase and an additional regulatory activity Gluconeogenic genes and genes involved in the utiliza- (Meyer et al., 1991). Both functions are separable by tion of alternative carbon sources have been shown to be mutation and only the regulatory function is essential for expressed at a very low level in high-glucose media, at least KlGal4p activation (Vollenbroich et al., 1999; Zenke et al., in the KHT1–KHT2 strain JA6, which has been used for 1996, 1999). In S. cerevisiae, the regulatory function is most studies on glucose repression (Breunig, 1989; Za- mainly conferred by the GAL3 product, which is highly chariae et al., 1993; Kuzhandaivelu et al., 1992; Weirich et similar to ScGal1p and KlGal1p but lacks enzymatic activ- al., 1997; Georis et al., 1999, 2000). However, except for ity (Bhat et al., 1992; Bajwa et al., 1988). the GAL1 gene (Dong et al., 1998), in none of these cases Gene shuffling between S. cerevisiae and K. lactis was does repression seem to be mediated by the K. lactis instrumental in elucidating the regulatory function of Kl- homologue of Mig1p (Georis et al., 1999, 2000), in con- GAL1 and ScGAL3 and provided genetic evidence for trast to S. cerevisiae. Thus, the regulators conferring glu- species-specific interaction between Gal1/Gal3p and cose repression in K. lactis remain to be identified. By Gal80p (Zenke et al., 1996). Gal80p is highly conserved screening for mutants that affect the utilization of glu- between S. cerevisiae and K. lactis (Zenke et al., 1993) and coneogenic carbon sources, two genes, FOG1 and FOG2, negatively regulates Gal4p in the absence of galactose by that affect the derepression of glucose-repressed genes, interacting with its activation domain. KlGal80p–KlGal1p were characterized. FOG1 turned out to encode the K. interaction was confirmed by in vitro studies (Zenke et al., lactis homologue of GAL83, and FOG2 is the K. lactis 1996) and two-hybrid experiments (Vollenbroich et al., SNF1 homologue (Goffrini et al., 1996). 1999). It depends on the regulatory, not the enzymatic activity, of Gal1p and requires galactose and ATP, sug- Lactose and Galactose Metabolism gesting that substrate binding to galactokinase leads to a conformational change that allows interaction with Gal80p The utilization of lactose is a characteristic trait for K. followed by activation of Gal4p (Fig. 2). Galactose phos- lactis and has been studied extensively. Lactose metabo- phorylation is not required for induction; instead, it leads lism is inducible and coregulated with galactose metabo- to inducer consumption, thus providing a direct link be- lism (Sheetz and Dickson, 1980; Wray et al., 1987; Web- tween metabolism and gene regulation (Zachariae, 1994). ster and Dickson, 1988) by a transcriptional activator, Whereas the basics of the “galactose switch” appear to be Lac9p or KlGal4p, homologous to Gal4p of S. cerevisiae essentially the same in S. cerevisiae and K. lactis (Platt et (Salmeron and Johnston, 1986; Ruzzi et al., 1987; Breunig al., 1998; Blank et al., 1997; Yano et al., 1997; Zenke et al., et al., 1987; Zachariae and Breunig, 1993). Two genes, 1996), there may be differences in the way the inhibitory LAC4 and LAC12, encoding ␤-galactosidase and lactose influence of Gal80p on Gal4p is relieved upon Gal80p–

Gal1p/Gal3p interaction. ScGal4p is subject to carbon mechanisms of microbial competition, and vector devel- source-dependent phosphorylation (Hirst et al., 1999; My- opment. Supported by the developments of powerful mo- lin et al., 1989; Sadowski et al., 1996, 1991), whereas in K. lecular genetic tools, K. lactis has been successfully estab- lactis KlGal80p is phosphorylated (Zenke et al., 1999). A lished as an alternative system for biomass-directed indus- de- or hypophosphorylated form of the latter accumulates trial applications and large-scale expression of biotechnically upon induction and the KlGal1p regulatory function is relevant gene products. required for this shift, suggesting a functional significance of regulated phosphorylation. Possibly, this difference between S. cerevisiae and K. lactis contributes to the fact that KlGal80p-mediated inhibition of ScGal4p or KlGal4p can- ACKNOWLEDGMENTS not be relieved by ScGal3p (Zenke et al., 1996). Regulation of lactose metabolism has also served as a We thank Lars Fichtner (University of Halle, Germany) who kindly model system for glucose repression. In this case glucose contributed in part to data illustrated in Fig. 1. The work was supported repression is primarily due to an inhibition of induction by grants from the EC Framework IV “Cell Factory” (BIO4-CT96-0003) (Zachariae et al., 1993; Kuzhandaivelu et al., 1992; Zenke to K.D.B. and the Deutsche Forschungsgemeinschaft (DFG) to R.S. et al., 1993; Zachariae, 1994). A low level of LAC12 gene (Scha750) and K.D.B. (Br921). In addition, R.S. acknowledges receipt of a Feodor-Lynen-Fellowship (FLF-DEU/1037031) from the Alexander expression and a Gal4p-inducible KlGAL80 gene is a pre- von Humboldt-Foundation, Germany. requisite for the glucose effect (Zachariae, 1994; Zenke et al., 1993). 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