Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

Schaffrath, Raffael; Breunig, Karin D.

doi:10.1006/fgbi.2000.1221

Cited by 144 publications

(104 citation statements)

References 180 publications

(192 reference statements)

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“…The S. cerevisiae GAL regulon has become a paradigm for transcriptional control in lower eukaryotes and a model system for gene regulation (1)(2)(3). Although its importance is uncontested, the present results illustrate that this regulatory network may be restricted to yeasts and not representative for other ascomycetous fungi in general.…”

Section: Discussionmentioning

confidence: 73%

“…Blockage of the latter in a ⌬xyl1 also strongly impairs cellulase induction on lactose, and the effect is not additive with that in a ⌬gal1, as seen in a ⌬gal1⌬xyl1 strain. 2 In other words, D-galactose 1-phosphate, as well as galactitol or catabolites thereof, is needed for high induction of cellulase formation. Whether they both cooperate in the induction themselves, or first need to be converted to another metabolite, which then acts as an inducer, is currently investigated.…”

Section: Discussionmentioning

confidence: 99%

“…This allows the transcriptional activator ScGal4 to recruit the RNA polymerase II to each of the GAL genes (reviewed in Refs. [1][2][3].…”

mentioning

confidence: 99%

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Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

Hartl

Kubicek

Schink

2007

Journal of Biological Chemistry

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The Saccharomyces cerevisiae galactokinase ScGal1, a key enzyme for D-galactose metabolism, catalyzes the conversion of D-galactose to D-galactose 1-phosphate, whereas its catalytically inactive paralogue, ScGal3, activates the transcription of the GAL pathway genes. In Kluyveromyces lactis the transcriptional inducer function and the galactokinase activity are encoded by a single bifunctional KlGal1. Here, we investigated the cellular function of the single galactokinase GAL1 in the multicellular ascomycete Hypocrea jecorina ‫؍(‬ Trichoderma reesei) in the induction of the gal genes and of the galactokinase-dependent induction of the cellulase genes by lactose (1,4-O-␤-D-galactopyranosyl-D-glucose). A comparison of the transcriptional response of a strain deleted in the gal1 gene (no putative transcriptional inducer and no galactokinase activity), a strain expressing a catalytically inactive GAL1 version (no galactokinase activity but a putative inducer function), and a strain expressing the Escherichia coli galK (no putative transcriptional inducer but galactokinase activity) showed that, in contrast to the two yeasts, both the GAL1 protein and the galactokinase activity are fully dispensable for induction of the Leloir pathway gene gal7 by D-galactose and that only the galactokinase activity is required for cellulase induction by lactose. The data document a fundamental difference in the mechanisms by which yeasts and multicellular fungi respond to the presence of D-galactose, showing that the Gal1/Gal3-Gal4 -Gal80-dependent regulatory circuit does not operate in multicellular fungi.The enzymes of the Leloir pathway are responsible for the conversion of D-galactose to D-glucose 1-phosphate and have been identified in all biological kingdoms. In the model organism Saccharomyces cerevisiae, the genes encoding the three main Leloir pathway enzymes, GAL1 (galactose kinase), GAL7 (UDP-galactose uridylyltransferase), and GAL10 (UDP-galactose epimerase and aldose 1-epimerase), are clustered and coordinately regulated at the level of transcription. The S. cerevisiae GAL genes are repressed by D-glucose, expressed only at a very low basal level on other respiratory carbon sources, and highly induced (up to 1000-fold) when the cells are switched to a medium containing D-galactose. This transcriptional activation is mediated by the interplay of three proteins; in the presence of D-galactose and ATP, the transcriptional inducer ScGal3 associates with the ScGal80 repressor thereby alleviating its repressing effects. This allows the transcriptional activator ScGal4 to recruit the RNA polymerase II to each of the GAL genes (reviewed in Refs.1-3).The S. cerevisiae galactokinase ScGal1 displays a 73% amino acid identity to the ScGal3, but galactokinase activity is absent in ScGal3. However ScGal3 can be converted into a catalytically active galactokinase through the insertion of the two amino acids, Ser and Ala, into its sequence (4). Overexpression of ScGal1, or a phenomenon termed long-term adaptation, can recover a gal3 ph...

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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

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Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

Hartl

Kubicek

Schink

2007

Journal of Biological Chemistry

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“…In contrast to S. cerevisiae and K. lactis, which are widely used for molecular biology and industrial applications (Mattiazzi et al, 2012;Schaffrath and Breunig, 2000;Schwimmer et al, 2006;Smith and Snyder, 2006;van Leeuwen et al, 2012), K. marxianus has received relatively little attention, although there are an increasing number of reports describing the potential application of K. marxianus for the production of ethanol, biomass, endogenous enzymes and aromatic compounds (Bansal et al, 2008;Fonseca et al, 2008;Gao and Daugulis, 2009;Limtong et al, 2007;Nonklang et al, 2008Nonklang et al, , 2009Rodrussamee et al, 2011;Wittmann et al, 2002) and for protein structural analysis (Watanabe et al, 2012;Yamaguchi et al, 2012aYamaguchi et al, , 2012b. Thus, in the present study, we generated and characterized 79 auxotrophic mutants and identified 35 complementing genes using a novel integrative transformation approach for the development of genetic tools in K. marxianus.…”

Section: Discussionmentioning

confidence: 99%

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yarimizu

Nonklang

Nakamura

et al. 2013

Yeast

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The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

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“…The best characterized of these episomes is the cytoplasmic k1 and k2 killer plasmid system from the dairy yeast Kluyveromyces lactis (Schaffrath et al, 1999;Schaffrath and Breunig, 2000). Gene functional analysis has shown that k1 is instrumental in the expression of a zymotoxin (Stark and Boyd, 1986), whereas k2 provides vital maintenance functions for both plasmids (Schaffrath and Meacock, 1995;Schaffrath et al, 1995a).…”

Section: Introductionmentioning

confidence: 99%

An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis

Schaffrath

Meacock

2001

Yeast

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The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses. Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB. As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro. Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity. ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication. Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme. Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis.

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Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

Cited by 144 publications

References 180 publications

Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

Induction of the gal Pathway and Cellulase Genes Involves No Transcriptional Inducer Function of the Galactokinase in Hypocrea jecorina

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis

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