The Presence of Actinidin (Cysteine Protease) and Recombinant Plasmids Carrying the Actinidin Gene Influence the Growth of Saccharomyces Cerevisiae

Actinidin is a protease found abundantly in the fruit of Actinidia chinensis or Kiwi fruit. The overproduction of this protein in microorganisms, especially using the yeast Saccharomyces cerevisiae, would be economically valuable as it would simplify the extraction and purification of the protein. It was observed, however, that the yeast growth rate was reduced by the presence of externally supplied actinidin in the growth medium. It was also observed that actinidin present in the yeast growth medium was partially degraded. Several actinidin-encoding gene variants have been cloned in a yeast expression and secretion vector. It was observed that different actinidin gene constructions influenced the growth rate of S. cerevisiae in complete media. Recombinant plasmids carrying only the mature actinidin-encoding DNA sequences reduced yeast transformability significantly and had the least stability. The results thus suggest that the presence of a recombinant plasmid carrying a gene of a potentially toxic protein may result in a deleterious effect on the host cell.     

Development of a yeast protein fragment complementation assay (PCA) system using dihydrofolate reductase (DHFR) with specific additives

A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein-protein interactions in vivo.     

The Role of Amino-Terminal and Carboxy-Terminal Extensions in the Processing and Translocation of a Plant Proteinase (Actinidin) Expressed in the Yeast Saccharomyces cerevisiae

Summary A plant proteinase gene naturally occuring in the Kiwi fruit plant (Actinidia chinensis) has been expressed in a yeast Saccharomyces cerevisiae. Different gene constructions consisting of different portions of the whole actinidin-encoding gene have been created and expressed using an expression-secretion yeast vector. It was observed that the amino- and carboxy-terminal extensions of the actinidin-encoding gene were required for the correct expression of the gene in yeast. A gene construction lacking both amino- and C-terminal extensions did not result in a detectable protein product. Similarly, a gene construction consisting of the amino-terminal extension plus mature actinidin-encoding DNA did not result in a detectable expression. However, intracellular expression was observed when a gene construction consisting of mature actinidin-encoding DNA plus C-terminal extension portion was employed. The expressed polypeptide was found however not to be correctly processed as it had a bigger size than the native actinidin. The correctly processed polypeptide was expressed intracellularly when the full-length actinidin cDNA was expressed in a vacuolar protease-proficient yeast strain. However, when a vacuolar protease-deficient yeast strain was employed, it was found that the precursor protein was not correctly processed, suggesting that the actinidin precursor had entered the vacuole and undergone proteolytic processing. The full-length actinidin cDNA consisted of the amino-terminal extension DNA, mature actinidin-encoding DNA, and C-terminal extension DNA. The results thus suggested that both amino- and C-terminal extensions were required for correct expression and processing of actinidin in yeast. The intracellular expression also suggested that the actinidin-encoding sequences contain intracellular targeting sequences which override the secretion signal included in the expression-secretion vector.     

Role of glutathione in detoxification of metal(loid)s by Saccharomyces cerevisiae

Cellular glutathione (GSH) was implicated in tolerance to potentially toxic metal(loid)s using two strains of Saccharomyces cerevisiae, a wild-type (sigma 1278b) and a GSH-deficient mutant strain (gshA-2). Both yeast strains exhibited no significant difference in tolerance to tellurite, zinc, cobalt, copper, manganese, nickel and chromate. There was no marked influence of glutathione on the accumulation of Te, Co, Cu, and Mn, although the absence of cellular glutathione significantly increased the cellular content of Zn and Ni, but greatly decreased Cr content without significant alteration of tolerance. These results indicated the independence of cellular glutathione activity from tolerance to Te, Zn, Co, Cu, Mn, Ni, and Cr. However, involvement of glutathione in Zn, Ni and Cr uptake is possible. The glutathione-deficient strain displayed a high sensitivity to selenite and cadmium in comparison to the wild-type strain of S. cerevisiae. The minimum inhibitory concentrations of Se and Cd for the glutathione-deficient strain were 980 +/- 13 and 32 +/- 4 microM, respectively, whereas the wild strain tolerated up to 4080 +/- 198 microM Se and 148 +/- 5 microM Cd. A relationship between tolerance and reduced cellular content of both Se and Cd was also shown: the mutant strain accumulated approximately three-fold more Se and two-fold more Cd than that accumulated by the wild-type strain. This suggests an influence of GSH on cellular uptake of Se and Cd, and also directly confirms the protective action of such a cellular thiol compound against Se and Cd toxicity.     

Removal of Cr(VI) from ground water by Saccharomyces cerevisiae

Chromium can be removed from ground water by the unicellular yeast, Saccharomyces cerevisiae. Local ground water maintains chromium as CrO₄ ^2- because of bicarbonate buffering and pH and E _h conditions (8.2 and +343 mV, respectively). In laboratory studies, we used commercially available, nonpathogenic S. cerevisiae to remove hexavalent chromium [Cr(VI)] from ground water. The influence of parameters such as temperature, pH, and glucose concentration on Cr(VI) removal by yeast were also examined. S. cerevisiae removed Cr(VI) under aerobic and anaerobic conditions, with a slightly greater rate occurring under anaerobic conditions. Our kinetic studies reveal a reaction rate (V_max) of 0.227 mg h^-1 (g dry wt biomass)^-1 and a Michaelis constant (Km) of 145 mg/l in natural ground water using mature S. cerevisiae cultures. We found a rapid (within 2 minutes) initial removal of Cr(VI) with freshly hydrated cells [55–67 mg h^-1 (g dry wt biomass)^-1] followed by a much slower uptake [0.6–1.1 mg h^-1 (g dry wt biomass)^-1] that diminished with time. A materials-balance for a batch reactor over 24 hours resulted in an overall shift in redox potential from +321 to +90 mV, an increase in the bicarbonate concentration (150–3400 mg/l) and a decrease in the Cr(VI) concentration in the effluent (1.9-0 mg/l).     

Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Summary Insertion of the transposable element Ty at the ADH4 locus results in increased levels of a new alcohol dehydrogenase (ADH) activity in Saccharomyces cerevisiae. The DNA sequence of this locus has been determined. It contains a long open reading frame which is not homologous to the other ADH isozymes that have been characterized in S. cerevisiae nor does it show obvious homology to Drosophila ADH. The hypothetical ADH does, however, show strong homology to the sequence of an iron-activated ADH from the bacterium Zymomonas mobilis. Thus ADH4 appears to encode an ADH structural gene which, along with the Zymomonas enzyme, may define a new family of alcohol dehydrogenases.Now The Plant Cell Research Institute, Inc., 6560 Trinity Court, Dublin, CA 94568, USA     

Bone morphogenetic protein (BMP)1-3 enhances bone repair

Small molecules that exhibit biological activity have contributed to the understanding of the molecular mechanisms of various biological phenomena. 5-Bromodeoxyuridine (BrdU) is a thymidine analogue that modulates various biological phenomena such as cellular differentiation and cellular senescence in cultured mammalian cells. Although BrdU is thought to function through changing chromatin structure and gene expression, its precise molecular mechanisms are not understood. To study the molecular mechanism for the action of BrdU, we have employed the yeast Saccharomycescerevisiae as a model system, and screened multi-copy suppressor genes that confer resistance to BrdU. Our genetic screen has revealed that expression of the N-terminal short fragment of TUP1, and also disruption of HDA1 or HOS1, histone deacetylases that interact with TUP1, conferred resistance to BrdU. These results suggest the implication of the chromatin proteins in the function of BrdU, and would provide novel clues to answer the old question of how BrdU modulates various biological phenomena.     

Use of a Novel Immobilization Yeast System for Winemaking

Penicillium was used to immobilize Saccharomyces cerevisiae, without using physico-chemical external supports, to form yeast biocapsules. The biocapsules, once the Penicillium was killed by the ethanol produced, were used in a grape must fermentation. Must fermentation was carried out for 160 h with the biocapsules and for 300 h with free yeast cells. Acetaldehyde (84 vs. 63 mg/l), isobutanol (217 vs. 194 mg/l), L-proline (7.7 vs. 6.5 mM) and aspartic acid (0.42 vs. 0 mM) in final wine were higher with the biocapsules than with free cells.     

Physiological aspects of growth and recombinant DNA stability inSaccharomyces cerevisiae

Despite the fact that plasmid stability in the yeastSaccharomyces cerevisiae is influenced by both genetical and physiological parameters most attention has been focussed on the former. Physiological factors affecting the stability of plasmids have been poorly characterized despite the need for such information in order to optimize the use ofS. cerevisiae as a host for recombinant protein production processes. The physiology of wild typeS. cerevisiae differs considerably when grown using different cultivation techniques. A limited amount of phenomenological data has been reported concerning plasmid instability effects under these different conditions and in this article these have been collected together with the intention of providing an overview to instability effects and to try and propose reasons as to how the physiological response to different growth conditions can be manifested as stability/instability effects.     

Genetic and cell biological aspects of the yeast vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase is a member of the third class of H⁺-pumping ATPase. A family of this type of H⁺-ATPase is now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. NineVMA genes that are indispensable for expression of the enzyme activity have been cloned and characterized in the yeastSaccharomyces cerevisiae. This review summarizes currently available information on theVMA genes and cell biological functions of theVMA gene products.     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

Expression and purification of ubiquitin-specific protease (UBP1) ofSaccharomyces cerevisiae in recombinantEscherichia coli

Truncated form of UBP1, an ubiquitin-specific protease ofSaccharomyces cerevisiae, was overexpressed inEscherichia coli. The hexahistidine residue (His₆) was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were 40°C and pH 8.0, respectively.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

TUF factor binds to the upstream region of the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae

Summary The upstream activation site of the pyruvate decarboxylase gene, PDC1, of Saccharomyces cerevisiae contains an RPG box, and mediates the increase in expression of a PDC1-lacZ fusion gene during growth on glucose. Oligonucleotide replacement experiments indicate that the RPG box functions as an absolute activator of expression, but other elements (possibly CTTCC repeats) are required for carbon source regulation, and maximal expression. Gel retardation and oligonucleotide competition experiments suggest that the DNA binding factor TUF interacts with the RPG box in the upstream region of PDC1. Binding of TUF factor is not carbon source dependent in in vitro experiments, and is probably not responsible for glucose induction of PDC1 expression.     

酒醅来源酵母菌合成异戊醇能力与途径解析

【背景】异戊醇是酵母菌在白酒发酵过程中通过氨基酸合成代谢途径和氨基酸分解代谢途径合成的主要高级醇,其含量影响白酒饮用的舒适度。【目的】分析和比较分离自浓香型白酒酒醅中的酵母菌合成异戊醇的能力,揭示酵母菌合成异戊醇的途径。【方法】从酒醅中分离具有异戊醇合成能力的酵母菌株,比较不同生长时期酵母菌合成异戊醇的能力,通过前体物代谢分析它们合成异戊醇的途径。【结果】分离自酒醅的5株酵母的异戊醇合成能力从强到弱依次为Naumovozyma castellii JP3-1、Saccharomyces cerevisiae JP3、Pichia fermentans JP22、Pichia kudriavzevii JP1和Naumovozyma dairenensis CBS421。这些酵母合成异戊醇的时期主要在对数生长期,N. castellii JP3-1、P. fermentans JP22和N. dairenensis CBS421在稳定生长期也合成异戊醇。S. cerevisiae JP3、N. castellii JP3-1和N. dairenensis CBS421在整个生长时期主要通过Harris途径合成异戊醇;P. kudriavzevii JP1在整个时期主要通过Ehrlich途径合成异戊醇;P. fermentans JP22在对数生长期通过Harris途径和Ehrlich途径合成异戊醇的能力接近,在稳定生长期主要通过Harris途径合成异戊醇。【结论】本研究揭示了酒醅来源5个属种酵母合成异戊醇的途径、能力与其生长时期的关系,研究结果可为解析浓香型白酒发酵过程异戊醇合成、积累机制及实施白酒发酵过程异戊醇合成的精准调控提供理论依据。     

Novel methods of genome shuffling in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome shuffling can improve complex phenotypes; however, there are several obstacles towards its broader applicability due to increased complexity of eukaryotic cells. Here, we describe novel, efficient and reliable methods for genome shuffling to increase ethanol production of Saccharomyces cerevisiae. Using yeast sexual and asexual reproduction by itself, mutant diploid cells were shuffled through highly efficient sporulation and adequate cross among the haploid cells, followed by selection on the special plates. The selected strain obtained after three round genome shuffling not only distinctly improved the resistance to ethanol, but also, increased ethanol yield by up to 13% compared with the control.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

Comparative Kinetic Effects of Mn (II), Mg (II) and the ATP/ADP Ratio on Phosphoenolpyruvate Carboxykinases from <Emphasis Type="Italic">Anaerobiospirillum succiniciproducens</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The kinetic affinity for CO₂ of phosphoenolpyruvate PEP⁵ carboxykinase from Anaerobiospirillum succiniciproducens, an obligate anaerobe which PEP carboxykinase catalyzes the carboxylation of PEP in one of the final steps of succinate production from glucose, is compared with that of the PEP carboxykinase from Saccharomyces cerevisiae, which catalyzes the decarboxylation of oxaloacetate in one of the first steps in the biosynthesis of glucose. For the A. succiniciproducens enzyme, at physiological concentrations of Mn²⁺ and Mg²⁺, the affinity for CO₂ increases as the ATP/ADP ratio is increased in the assay medium, while the opposite effect is seen for the S. cerevisiae enzyme. The results show that a high ATP/ADP ratio favors CO₂ fixation by the PEP carboxykinase from A. succiniciproducens but not for the S. cerevisiae enzyme. These findings are in agreement with the proposed physiological roles of S. cerevisiae and A. succiniciproducens PEP carboxykinases, and expand recent observations performed with the enzyme isolated from Panicum maximum (Chen et al. (2002) Plant Physiology 128: 160–164).     

Response of a parasitoid fly, Gymnosoma rotundatum (Linnaeus) (Diptera: Tachinidae) to the aggregation pheromone of Plautia stali Scott (Hemiptera: Pentatomidae) and its parasitism of hosts under field conditions

We studied the attraction of a tachinid fly, Gymnosoma rotundatum (Linnaeus) to the male-produced aggregation pheromone of the brown-winged green bug, Plautia stali Scott, its parasitism on the bug, and its seasonal occurrence in the field. The tachinid fly was continuously attracted to the aggregation pheromone from spring to autumn and utilized the bugs as hosts. Our field experiment to clarify the effect of the pheromone on parasitism demonstrated that parasitism occurred only in female bugs baited with synthetic aggregation pheromone and did not occur in females without the pheromone. The parasitoid flies therefore appeared to use the bug’s pheromone as a host-finding kairomone. The pheromone attracted not only female flies but also males. Male flies may increase their chance of encountering pheromone-attracted females by waiting near pheromone sources. The tachinid develops multiple generations in active hosts from spring to autumn and overwinters in dormant hosts. Thus, G. rotundatum seems to be highly adapted to using P. stali as its host, and it is a potentially important biological control agent of P. stali populations in the field.