Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Degradation of DNA during the autolysis of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The autolysis of yeast cells has practical implications in the production of fermented foods and beverages and flavourants for food processing. Protein and RNA degradation during yeast autolysis are well described but the fate of DNA is unclear. Yeast cells (Saccharomyces cerevisiae) were autolysed by incubating suspensions at 30–60°C (pH 7.0), and at pH 4.0–7.0 (40°C) for 10–14 days. Up to 55% of total DNA was degraded, with consequent leakage into the extracellular environment of mainly 3′- and 5′-deoxyribonucleotides, and lesser amounts of polynucleotides. The rate and extent of DNA degradation, composition of the DNA degradation products and DNase activity were affected by temperature and pH. The highest amount of DNA degradation occurred at 40°C and pH 7.0, where the highest DNase activity was recorded. DNase activity was lowest at 60°C and pH 4.0, where the proportion of polynucleotides in the degradation products was higher. Electronic Publication     

Construction of a novel kind of expression plasmid by homologous recombination in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R-IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.     

Evaluation of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> as a source of yeast autolysates

Cells of Kluyveromyces marxianus FII 510700 and Saccharomyces cerevisiae CBS 1907 were autolysed in phosphate buffer, pH 4.5, for a maximum of 10 days to compare chemical changes that occur in the carbohydrate, protein, amino acid and nucleic acid content. Approximately 2.2–3% carbohydrate, 9.5–12% protein, 0.6–1.0% DNA and 6–7% RNA were recovered in the autolysates. The main amino acids were β-alanine, phenylalanine, cysteine, methionine, glutamic acid and isoleucine. No significant differences in the yeast autolysates of K. marxianus and S. cerevisiae were observed. Consequently, K. marxianus produced from lactose-based media has potential as a source of yeast autolysates used in the food industry. Electronic Publication     

Synchronization of cell cycle of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by using a cell chip platform

Cell synchrony is a critical requirement for the study of eukaryotic cells. Although several chemical and genetic methods of cell cycle synchronization are currently available, they have certain limitations, such as unnecessary perturbations to cells. We developed a novel cell cycle synchronization method that is based on a cell chip platform. The budding yeast, Saccharomyces cerevisiae, is a simple but useful model system to study cell biology and shares many similar features with higher eukaryotic cells. Single yeast cells were individually captured in the wells of a specially designed cell chip platform. When released from the cell chip, the yeast cells were synchronized, with all cells in the G1 phase. This method is non-invasive and causes minimal chemical and biological damage to cells. The capture and release of cells using cells chips with microwells of specific dimensions allows for the isolation of cells of a particular size and shape; this enables the isolation of cells of a given phase, because the size and shape of yeast cells vary with the phase of the cell cycle. To test the viability of synchronized cells, the yeast cells captured in the cell chip platform were assessed for response to mating pheromone (α-factor). The synchronized cells isolated using the cell chip were capable of mediating the mating signaling response and exhibited a dynamic and robust response behavior. By changing the dimensions of the well of the cell chip, cells of other cell cycle phases can also be isolated.     

Domain engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg ^–1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.     

Nutrient-specific effects in the coordination of cell growth with cell division in continuous cultures of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cell cycle progression of Saccharomyces cerevisiae cells was monitored in continuous cultures limited for glucose or nitrogen. The G1 cell cycle phase, before initiation of DNA replication, did not exclusively expand when growth rate decreased. Especially during nitrogen limitation, non-G1 phases expanded almost as much as G1. In addition, cell size remained constant as a function of growth rate. These results contrast with current views that growth requirements are met before initiation of DNA replication, and suggest that distinct nutrient limitations differentially impinge on cell cycle progression.     

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.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

Methods designed for the identification and characterization of<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> chromatin assembly mutants in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model systemSaccharomyces cerevisiae. Modifications to anin vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s):RSP5, and a subunit of the Anaphase Promoting Complex (APC),APC5. Additional modifications are described that allow for a rapid analysis and anin vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that thein vitro andin vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks. Published: July 3, 2003     

A database of microsatellite genotypes for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A system for genotyping Saccharomyces cerevisiae is described based on a multiplex of ten microsatellite loci and the MAT locus. A database of genotypes has been developed for 246 yeast strains, including a large set of commercial wine yeasts, as well as 35 sequenced natural isolates currently being sequenced. The latter allow us, for the first time, to make direct comparisons of the relationship between DNA sequence data and microsatellite-based genotypes. The genotyping system provides a rapid and valuable system for strain identification as well as studying population genetics of S. cerevisiae.     

Ultrastructure of yeast cell <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> after amiodarone treatment

The ultrastrcutre of Saccharomyces cerevisiae cells (wild-type and ysp2 mutant cells) was studied after amiodarone treatment. Amiodarone is used as a pharmaceutical substance for treating a number of diseases; however, it is known that amiodarone causes structural and functional disturbances in patient tissues. Here, the peculiarities of the amiodarone effect are studied in Saccharomyces cerevisiae yeast, in which amiodarone has been shown to cause apoptosis. Electron-microscopic study of yeast cells after amiodarone treatment reveals a significant increase in the number of lipid particles, which can lead to the formation of a structural complex by interacting with cell membranous organelles. Amiodarone causes the appearance of small and slightly swollen mitochondria. Chromatin displacement to the periphery of the nucleus, nuclear sectioning, and nuclear envelope disturbances are observed in the cells under these conditions. The detected changes int eh ultrastructure of the cell in Saccharomyces cerevisiae are considered to be a specific response to phospholipidosis and apoptosis caused by amiodarone.     

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.     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Gene <Emphasis Type="Italic">RAD31</Emphasis> is identical to gene <Emphasis Type="Italic">MEC1</Emphasis> of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The earlier identified gene RAD31 was mapped on the right arm of chromosome II in the region of gene MEC1 localization. Epistatic analysis demonstrated that the rad31 mutation is an allele of the MEC1 gene, which allows further designation of the rad31 mutation as mec1-212. Mutation mec1-212, similar to deletion alleles of this gene, causes sensitivity to hydroxyurea, disturbs the check-point function, and suppresses UV-induced mutagenesis. However, this mutation significantly increases the frequency of spontaneous canavanine-resistance mutations induced by disturbances in correcting errors of DNA replication and repair, which distinguishes it from all identified alleles of gene MEC1.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Genome-Wide Screening of Aluminum Tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome-wide screening has identified 37 Al-tolerance genes in Saccharomyces cerevisiae. These genes can be roughly categorised into three groups on the basis of function, i.e., genes related to vesicle transport processes, signal transduction pathways, and protein mannosylation. The largest group is composed of genes related to vesicle transport processes; severe Al sensitivity was found in yeast strains lacking these genes. The retrograde transport of endosome-derived vesicles back to the Golgi apparatus is an important factor in determining the Al tolerance of the vesicle transport system. The PKC1-MAPK cascade signalling pathway is important in the Al tolerance of signal transduction. The lack of the gene implicated in this process leads to weakened cell wall architecture, rendering the yeast Al-sensitive. Alternatively, Al might attack the cell wall and/or plasma membrane, and, as signalling is prevented in cells devoid of the genes related to signalling processes, the cells may be unable to alleviate the damage. The genes for protein mannosylation are also associated with Al tolerance, demonstrating the importance of cell wall architecture. These genes are involved in cell integrity processes. An erratum to this article is available at .