Yeast Saccharomyces Kluyveri Research Articles

A New Expression Vector for Production of Enzymes in the Yeast Saccharomyces (Lachancea) Kluyveri

We overexpressed and purified enzymes involved in the pyrimidine catabolic pathway in the yeast Saccharomyces (Lachancea) kluyveri. A new vector was therefore designed, providing the first specific expression system in Saccharomyces kluyveri. The URC1 gene was overexpressed and a soluble protein obtained and successfully purified using the C-terminally added His-tag. Our system will be used for further studies of the structure and function of the enzymes belonging to the URC pyrimidine degradation pathway.

Saccharomyces kluyverias a model organism to study pyrimidine degradation

The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.

A Recruited Protease is Involved in Catabolism of Pyrimidines

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-β-alanine to β-alanine, is catalyzed by two β-alanine synthase (βASase, EC 3.5.1.6) subfamilies. We show that the “prototype” eukaryote βASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-βA compared with a representative of fungal βASases, the yeast Saccharomyces kluyveri βASase, which has a high K m value (71 mM). S. kluyveri βASase is specifically inhibited by dipeptides and tripeptides, and the apparent K i value of glycyl-glycine is in the same range as the substrate K m. We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal βASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.

Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

Clinical, biochemical and genetic findings in two siblings with a dihydropyrimidinase deficiency

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and it catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. To date, only nine individuals have been reported suffering from a complete DHP deficiency. We report two siblings presenting with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the siblings had a severe delay in speech development and white matter abnormalities, whereas the other one was free of symptoms. Analysis of the DHP gene ( DPYS) showed that both patients were compound heterozygous for the missense mutation 1078T > C (W360R) in exon 6 and a novel missense mutation 1235G > T (R412M) in exon 7. Heterologous expression of the mutant enzymes in Escherichia coli showed that both missense mutations resulted in a mutant DHP enzyme without residual activity. Analysis of the crystal structure of eukaryotic DHP from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum suggests that the W360R and R412M mutations lead to structural instability of the enzyme which could potentially impair the assembly of the tetramer.

The Crystal Structures of Dihydropyrimidinases Reaffirm the Close Relationship between Cyclic Amidohydrolases and Explain Their Substrate Specificity

In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 angstroms, respectively. Both enzymes have a (beta/alpha)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller beta-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the beta-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-beta-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.

Cloning and functional characterization of a fatty acid synthase component FAS2 gene from Saccharomyces kluyveri

A gene coding the alpha subunit of fatty acid synthase (FAS2) was isolated from the budding yeast Saccharomyces kluyveri. Nucleotide sequence analysis indicated that this gene, termed Sk-FAS2, coded a protein having an amino acid sequence 83% identical to the FAS2 protein of S. cerevisiae (Sc-FAS2). The Sk-FAS2 gene was able to functionally complement an S. cerevisiae fas2 disruptant. This Sk-FAS2-expressing strain was found to produce larger amounts of C18 than C16, in contrast to the Sc-FAS2-expressing fas2 strain. In addition, fusion genes of Sk-FAS2 and Sc-FAS2 were transformed into a fas2-disrupted strain of S. cerevisiae, and fatty acid analysis of these transformants suggested that the region containing the acyl carrier protein and beta-ketoacyl reductase domains of yeast FAS2 protein play an important role in determining carbon chain length of fatty acids.

Production of Cerebroside from Beet Molasses by the Yeast Saccharomyces kluyveri

Twenty-two strains classified as Saccharomyces kluyveri were grown on beet molasses as the principal carbon source to select a strain for the production of a glycosphingolipid cerebroside. Six strains accumulated relatively high amounts of cerebroside (> 0.40 mg [g dry weight]−1) and grew well in an Erlenmeyer flask. Thirty strains were isolated from single spores of strain CBS 4800 after sporulation and further tested for cerebroside contents. Strain SP-25 accumulated greater amounts than the parental strain (0.59 mg [g dry weight]−1) and was finally selected and cultured in a 5 L jar fermentor. In a comparison with a conventional baking strain of S. cerevisiae, the selected strain SP-25 propagated more rapidly in conjunction with depressed synthesis of ethanol. The amount of cerebroside produced from 1 kg of sugar in molasses was estimated to be 0.15 g. The constituents of cerebroside accumulated in the yeast cells were principally 2-hydroxystearic acid as a fatty acid, 9-methyl-trans-4, trans-8-sphingadienine as a sphingoid base, and glucose as a hexose.

Crystallization and X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri.

Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 A at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2(1) (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 A, beta = 91.4 degrees), with one homotetramer per asymmetric unit.

Production of fungal α-amylase by Saccharomyces kluyveri in glucose-limited cultivations

Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. α-Amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 μ plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active α-amylase in both cases. The S. cerevisiae 2 μ plasmid was found to be stable in S. kluyveri as evaluated by a constant α-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted α-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h −1 (4.8–5.7 mg (g dry weight) −1). At a dilution rate of 0.3 h −1 the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l −1 and the α-amylase concentration reached 320 mg l −1. Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.

Two different dihydroorotate dehydrogenases from yeast Saccharomyces kluyveri

Genes for two structurally and functionally different dihydroorotate dehydrogenases (DHODHs, EC 1.3.99.11), catalyzing the fourth step of pyrimidine biosynthesis, have been previously found in yeast Saccharomyces kluyveri. One is closely related to the Schizosaccharomyces pombe mitochondrial family 2 enzymes, which use quinones as direct and oxygen as the final electron acceptor. The other one resembles the Saccharomyces cerevisiae cytosolic family 1A fumarate-utilizing DHODH. The DHODHs from S. kluyveri, Sch. pombe and S. cerevisiae, were expressed in Escherichia coli and compared for their biochemical properties and interaction with inhibitors. Benzoates as pyrimidine ring analogs were shown to be selective inhibitors of cytosolic DHODs. This unique property of Saccharomyces DHODHs could appoint DHODH as a species-specific target for novel anti-fungal therapeutics.

Two different dihydroorotate dehydrogenases from yeast Saccharomyces kluyveri

Two different dihydroorotate dehydrogenases from yeast Saccharomyces kluyveri

Crystallization and preliminary X-ray analysis of β-alanine synthase from the yeastSaccharomyces kluyveri

In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.

Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties.

Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-beta-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes. Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap. Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones.

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri.

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant.

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.

Intraspecific Divergence of Saccharomyces kluyveri as Revealed by the Nucleotide Sequences of 18S-28S rRNA Spacer Regions and α-Galactosidase MEL Genes

In the five strains classified as the yeast Saccharomyces kluyveri, several substitutions were observed in the two internal transcribed spacer regions between 18S and 28S rRNA. A PCR reaction with primers targeted to the MEL1 gene of Saccharomyces cerevisiae amplified fragments of the expected size, and those sequences showed significant divergence in the strains of S. kluyveri.

The yeast Saccharomyces kluyveri as a recipient eukaryote in transkingdom conjugation: behavior of transmitted plasmids in transconjugants

The prokaryote Escherichia coli successfully conjugated with the eukaryote Saccharomyces kluyveri, which is relatively distant from the species S. cerevisiae. To achieve this transkingdom conjugation, we constructed three types of conjugative plasmids, namely integrative, replicative, and centromere vectors, for S. cerevisiae. By transfer of any of the three plasmids from E. coli, an S. kluyveri Ura- mutant was converted to the Ura+ phenotype. This phenotype was easily lost under nonselective conditions. Southern analysis of the transconjugants clearly indicated the presence of the plasmids in many different structures and sizes.

Molecular monitoring of the transcriptional activation of the yeast Saccharomyces kluyveri mating pheromone signal transduction by using FUS1-lacZ fusion gene

To analyse the molecular mechanism by which a mating pheromone signal is transmitted in Saccharomyces kluyveri, the S. cerevisiae FUS1-lacZ fusion gene was introduced into S. kluyveri cells and its transcriptional activation was investigated. The expression of FUS1-lacZ fusion product was cell-type-specifically induced by pheromone treatment in S. kluyveri. The result suggests that S. kluyveri pheromone signal may be transduced by a mechanism similar to that in S. cerevisiae. This system may provide a model for analysing the molecular mechanism of the mating reaction in Saccharomyces kluyveri.

Transformation of the yeast Saccharomyces kluyveri by Saccharomyces cerevisiae-based plasmids

For the transformation of the yeast Saccharomyces kluyveri, ura3 mutants were obtained by 5-fluoro-orotic acid selection. By utilizing the method based on treatment of intact cells with alkali cations, the ura3 strains of S. kluyveri were transformed by Saccharomyces cerevisiae-based plasmids. In the transformed cells, a S. cerevisiae centromere-based plasmid was stably replicated autonomously. Thus, this system will permit the study of gene expression and its regulation in S. kluyveri in relationship to that in S. cerevisiae.