Yeast Cell Culture Research Articles

Biocalorimetry as a process analytical technology process analyser; robust in-line monitoring and control of aerobic fed-batch cultures of crabtree-negative yeast cells

Control of bioprocesses requires reliable and robust on- or in-line monitoring tools providing real-time information on process dynamics. Heat generation related to metabolic activity of living systems is currently gaining importance in bioprocess industry due to its non-invasive and essentially instantaneous characteristics. This study deals with monitoring and control of pure aerobic fed-batch cultures of three Crabtree-negative yeast strains, Kluyveromyces marxianus, Candida utilis and Pichia pastoris, based on in-line measured, metabolic heat flow signals. A high resolution biocalorimeter (BioRC1) was developed from a standard bench-scale heat flow calorimeter (RC1). The BioRC1 was equipped with in-line (dielectric spectroscopy, pH probe and dissolved oxygen probe) and at-line (exit gas analyser) sensors to characterise the growth behaviour of the yeast cells. Both metabolic heat flow and biomass profiles exhibited similar behaviour proving the significance of employing heat flow signal as a key-parameter for the system under investigation. A simple estimator for biomass concentration and specific growth rate was formulated based on heat flow values. In order to evaluate the potential of calorimetry as a reliable and powerful process monitoring tool, the robustness, reliability as well as the broad applicability of the developed estimators was assessed through comparison with off-line measurement techniques and showed promising results for general applicability with a wide range of bioprocesses.

Synchronization of Budding Yeast Cultures

AbstractSynchronized cell cultures that grow without limitations, e. g. by nutrients, are of interest in a broad range of applications. In research, synchronization would enable the correct identification of cell cycle dependent processes on the gene regulatory as well as on the metabolic level. The production and/or secretion rates of several heterologous proteins in Saccharomyces cerevisiae change during the cell cycle. In industrial applications, a continuous, synchronized culture would implicate the purification of fewer amounts of media. In this contribution a synchronization strategy for small cultures is developed based on a previously published cell culture model. The cells are synchronized by the manipulation of the levels of proteins that participate in the cell cycle, so that an additional purification step is not needed. The synchronization strategy is determined as the solution of an optimization problem. Due to the non-convexity of the problem a global search is performed by a memetic algorithm that consists of an evolution strategy and a local search by a combination of a continuous ant colony optimization and hill climbing. The operation strategy obtained by this in silico optimization can be seen as a first step towards the creation of fully synchronized budding yeast cell cultures.

Online impedance monitoring of yeast cell culture behaviors

Online impedance monitoring of cell suspensions can be performed with integrated microelectrode array in microfluidic devices. However, its accuracy is affected by the formation of cell aggregates on the surface of the electrode array. To make this technical more reliable, we deposited a thin film of agarose which is highly permeable to soluble ions on the electrodes. The growth behavior of yeast cells in YPD medium was studied for more than 24h and the recorded impedance spectra were used to determinate the cell metabolism induced changes of the medium resistance and the double layer capacitance as a function of time. Our results showed a much more important variation of the double layer capacitance, indicating the importance of online monitoring at low frequencies.

Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells ofMetschnikowia pulcherrima

The effect of freeze-drying using different lyoprotectants at different concentrations on the viability and biocontrol efficacy of Metschnikowia pulcherrima was evaluated. The effects of initial yeast cell concentration and culture age on viability were also considered. Yeast cells grown for 36 h were more resistant to freeze-drying than were 48 h cells. An initial concentration of 10⁸ cells·mL⁻¹ favoured the highest survival after freeze-drying. When maltose (25%, m/v) was used as protectant, a high cell viability was obtained (64.2%). Cells maintained a high viability after 6 months of storage at 4 °C. The biocontrol efficacy of freeze-dried cells was similar to the activity of fresh cells on 'Gala' apples and was slightly lower on 'Golden Delicious' apples. After optimizing freeze-drying conditions, the viability of M. pulcherrima cells was similar to that obtained in other studies. The results constitute a first step towards the commercial development of M. pulcherrima as a biocontrol agent.

Mitochondrial fission and fusion and their roles in the heart

Mitochondria are dynamic organelles that usually exist in extensive and interconnected networks that undergo constant remodeling through fission and fusion. These processes are governed by distinct sets of proteins whose mechanism and regulation we are only beginning to fully understand. Early studies on mitochondrial dynamics were performed in yeast and simple mammalian cell culture models that allowed easy visualization of these intricate networks. Equipped with this core understanding, the field is now expanding into more complex systems. Cardiac cells are a particularly interesting example because they have unique energetic and spatial demands that make the study of their mitochondria both challenging and potentially very fruitful. This review will provide an overview of mitochondrial fission and fusion as well as recent developments in the understanding of these processes in the heart.

MitoGenesisDB: an expression data mining tool to explore spatio-temporal dynamics of mitochondrial biogenesis

Mitochondria constitute complex and flexible cellular entities, which play crucial roles in normal and pathological cell conditions. The database MitoGenesisDB focuses on the dynamic of mitochondrial protein formation through global mRNA analyses. Three main parameters confer a global view of mitochondrial biogenesis: (i) time-course of mRNA production in highly synchronized yeast cell cultures, (ii) microarray analyses of mRNA localization that define translation sites and (iii) mRNA transcription rate and stability which characterize genes that are more dependent on post-transcriptional regulation processes. MitoGenesisDB integrates and establishes cross-comparisons between these data. Several model organisms can be analyzed via orthologous relationships between interspecies genes. More generally this database supports the ‘post-transcriptional operon’ model, which postulates that eukaryotes co-regulate related mRNAs based on their functional organization in ribonucleoprotein complexes. MitoGenesisDB allows identifying such groups of post-trancriptionally regulated genes and is thus a useful tool to analyze the complex relationships between transcriptional and post-transcriptional regulation processes. The case of respiratory chain assembly factors illustrates this point. The MitoGenesisDB interface is available at http://www.dsimb.inserm.fr/dsimb_tools/mitgene/.

Cdc42 and Vesicle Trafficking in Polarized Cells

Cdc42, a highly conserved small GTPase of the Rho family, acts as a molecular switch to modulate a wide range of signaling pathways. Vesicle trafficking and cell polarity are two processes Cdc42 is known to regulate. Although the trafficking and polarity machineries are each well understood, how they interact to cross-regulate each other in cell polarization is still a mystery. Cdc42 is an interesting candidate that may integrate these two networks within the cell. Here we review findings on the interplay between Cdc42 and trafficking in yeast, Caenorhabditis elegans, Drosophila and mammalian cell culture systems, and discuss recent advances in our understanding of the function of Cdc42 and two of its effectors, the WASp-Arp2/3 and Par complexes, in regulating polarized traffic. Work in yeast suggests that the polarized distribution of Cdc42, which acts here as a key polarity determinant, requires input from multiple processes including endocytosis and recycling. In metazoan cells, Cdc42 can regulate several steps in the biosynthetic as well as endocytotic and recycling pathways. The recent discovery that the Par polarity complex co-operates with Cdc42 in the regulation of endocytosis and recycling opens exciting possibilities for the integration of polarity protein function and endocytotic machinery.

The Comamonas testosteroni steroid biosensor system (COSS)—Reflection on other methods

Natural and synthetic steroid hormones are released uncontrolled into the environment and are considered as pollutants with regard to their endocrine activity and negative influence on all kind of organisms. Due to their widespread presence, endocrine activity even at low concentrations, and their potential adverse effects in both the environment and human health, there is an increasing need for the development of rapid, sensitive and quantitative techniques for measuring trace levels of these steroids. In addition to classical analytical methods like GC–MS, LC–MS and others, several techniques have been established that are based on human nuclear steroid receptors as reporter systems. However, many of these systems require human or yeast cell culture and are therefore time consuming and expensive, while others suffer from too low sensitivity or cover only one specific steroid compound. These are some of the main reasons that limit current techniques for environmental application. The remarkable ability of certain microorganisms to transform and degrade the steroid nucleus and to respond with the induced expression of steroid regulated genes lead us to explore, whether the steroid signalling machinery of Comamonas testosteroni could be used to construct a steroid sensoring system that is sensitive, rapid, easy to perform, and which could also be applied to detect environmental steroid mixtures at low concentrations. Both whole C. testosteroni mutant cells as well as the cytosol thereof were used as new and sensitive fluorescence based biosensor systems for the successful determination and quantification of a variety of different steroids. We could show that our COSS (Comamonas testosteroniSteroid Sensor) is able to detect testosterone, estradiol and cholesterol in concentrations of 29pg/mL, 0.027pg/mL, and 9.7pg/mL, respectively. The sensitivity of the COSS together with the fact that it is very fast, reproducible and can be used for high-throughput screening in a microplate format makes it suitable for the detection of single steroid hormones or steroid hormone mixtures in environmental samples at low costs. In summary, the COSS is able to detect steroid hormone effects at the molecular level through activation of bacterial steroid-sensing systems. In the future, it may be further developed as a useful tool for the integrative assessment of ecotoxicological potentials caused by hormonally active agents and endocrine-disrupting compounds.

Dispersion and Stability Optimization of TiO2 Nanoparticles in Cell Culture Media

Accurate evaluation of engineered nanomaterial toxicity requires not only comprehensive physical-chemical characterization of nanomaterials as produced, but also thorough understanding of nanomaterial properties and behavior under conditions similar to those used for in vitro and in vivo toxicity studies. In this investigation, TiO(2) nanoparticles were selected as a model nanoparticle and bovine serum albumin (BSA) was selected as a model protein for studying the effect of protein-nanoparticle interaction on TiO(2) nanoparticle dispersion in six different mammalian, bacteria, and yeast cell culture media. Great improvement in TiO(2) dispersion was observed upon the addition of BSA, even though the degree of dispersion varied from medium to medium and phosphate concentration in the cell culture media was one of the key factors governing nanoparticle dispersion. Fetal bovine serum (FBS) was an effective dispersing agent for TiO(2) nanoparticles in all six media due to synergistic effects of its multiple protein components, successfully reproduced using a simple "FBS mimic" protein cocktail containing similar concentrations of BSA, γ-globulin, and apo-transferrin.

A miniaturized continuous dielectrophoretic cell sorter and its applications

There is great interest in highly sensitive separation methods capable of quickly isolating a particular cell type within a single manipulation step prior to their analysis. We present a cell sorting device based on the opposition of dielectrophoretic forces that discriminates between cell types according to their dielectric properties, such as the membrane permittivity and the cytoplasm conductivity. The forces are generated by an array of electrodes located in both sidewalls of a main flow channel. Cells with different dielectric responses perceive different force magnitudes and are, therefore, continuously focused to different equilibrium positions in the flow channel, thus avoiding the need of a specific cell labeling as discriminating factor. We relate the cells' dielectric response to their output position in the downstream channel. Using this microfluidic platform that integrates a method of continuous-flow cell separation based on multiple frequency dielectrophoresis, we succeeded in sorting viable from nonviable yeast with nearly 100% purity. The method also allowed to increase the infection rate of a cell culture up to 50% of parasitemia percentage, which facilitates the study of the parasite cycle. Finally, we prove the versatility of our device by synchronizing a yeast cell culture at a particular phase of the cell cycle avoiding the use of metabolic agents interfering with the cells' physiology.

A Proteome-wide Analysis of Kinase-Substrate Network in the DNA Damage Response

The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.

Developmental and cellular functions of the ESCRT machinery in pluricellular organisms

ESCRTs (endosomal sorting complexes required for transport) were first discovered in yeast and are known to be required in the biogenesis of the MVB (multivesicular body). Most ESCRT research has been carried out in vitro using models such as yeast and mammalian cells in culture. The role of the ESCRTs genes in endosome maturation is conserved from yeast to mammals, but little is known about their function during development in multicellular organisms. Since ESCRTs play a leading role in regulating some cell signalling pathways by addressing receptors to the lysosome, it appears important to monitor ESCRT functions in multicellular models. The present review summarizes recent research on the developmental and cellular functions of the ESCRT in Caenorhabditis elegans, Drosophila melanogaster, Mus musculus or Arabidopsis thaliana.

Dual Fluorochrome Flow Cytometric Assessment of Yeast Viability

A novel staining protocol is reported for the assessment of viability in yeast, specifically the biocontrol yeast, Pichia anomala. Employing both the red fluorescent membrane potential sensitive oxonol stain DiBAC(4)(5) (Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol), a structural analog of the commonly used DiBAC(4)(3) (Bis-(1,3-dibutylbarbituric acid)trimethine oxonol), with one of the esterase dependent green fluorogenic probes such as CFDA-AM (5-Carboxyfluorescein diacetate, acetoxymethyl ester) or Calcein-AM (Calcein acetoxymethyl ester), a two-color flow cytometric method was developed, which yields rapid quantitative information on the vitality and vigor of yeast cell cultures. The method was validated by cell sorting and analysis of live, heat killed, and UV-treated yeast.

In Vivo Yeast Cell Morphogenesis Is Regulated by a p21-Activated Kinase in the Human Pathogen Penicillium marneffei

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37°C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37°C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37°C. Loss of pakB also leads to the inappropriate production of yeast cells at 25°C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.

So you Need a Protein - A Guide to the Production of Recombinant Proteins

The field of biotechnology owes a great deal to the ability to produce recombinant proteins, which can be made in far greater abundance than many native proteins, and are more easily quality controlled. There is a great need for individual proteins to be produced for research purposes. This review is aimed at researchers who are not experienced at protein expression, but find that they have a need to produce a recombinant protein. We detail the major expression systems that will be commonly used in the laboratory situation- bacterial, yeast and insect cell culture. The application of each, and the relative advantages/disadvantages are discussed.

High-throughput microfluidic system for monitoring diffusion-based monolayer yeast cell culture over long time periods

We present a simple and high-throughput microfluidic system for diffusion-based monolayer yeast cell culture monitoring. Yeast cells are patterned into the micro-cavity array with a suitable height (4 μm) that keeps the cells fixed in monolayer during the cell division. Different sizes of cavities and different repeating times of injection were tested in order to realize as many single-cell/cavity as possible. Single-cell/cavity has been achieved in about 40% of 100 parallel cavities. As a demonstration, we apply this technology to investigate budding yeast and fission yeast cultures and show that it permits single-cell resolution over many cellular generations. Our results show that the technique provides an easy way to study the phenotype of single yeast cell cycle or cell-cell communication in high-throughput microfluidic system.

Establishment of Human Lysozyme Mass Production System Using Insect Factory, Silkworm Larvae

To reduce the manufacturing cost of human lysozyme (HLY) and thus spread its use in medicine, we designed, synthesized, and expressed the HLY gene in silkworm larvae by using a baculovirus system. The production of HLY in silkworms was highly efficient. N-terminal sequencing and mass spectrometry showed that it was physicochemically identical to natural HLY. The lytic activity of recombinant HLY in the silkworm hemolymph was 115 and 154 times that in yeast and silkworm cell culture systems.

Semi-continuous ethanol production in bioreactor from whey with co-immobilized enzyme and yeast cells followed by pervaporative recovery of product – Kinetic model predictions considering glucose repression

Mathematical models for semi-continuous ethanolic fermentation in a whey medium employing co-immobilized Saccharomyces cerevisiae strain and β- d-galactosidase and for pervaporative product recovery from the resultant broth were developed. Kinetic parameters of biomass growth were estimated using the nonlinear least squares method on the basis of experimental data obtained from culture of suspended yeast cells grown in a mixture of glucose and galactose and during semi-continuous fermentation in a bioreactor in a whey medium with co-immobilized enzyme and yeast cells. Experimentally determined fluxes of ethanol and water from broth were applied for the estimation of kinetic constants of ethanol separation by pervaporation. The degree of sugar utilization and ethanol productivity in bioreactor as well as the time of ethanol separation needed to obtain the desired amount of product can be predicted using this model. The influence of selected operating parameters on the fermentation effectiveness was simulated based on the developed model.

Computational methods for yeast prion curing curves

If the chemical guanidine hydrochloride is added to a dividing culture of yeast cells in which some of the protein Sup35p is in its prion form, the proportion of cells that carry replicating units of the prion, termed propagons, decreases gradually over time. Stochastic models to describe this process of ‘curing’ have been developed in earlier work. The present paper investigates the use of numerical methods of Laplace transform inversion to calculate curing curves and contrasts this with an alternative, more direct, approach that involves numerical integration. Transform inversion is found to provide a much more efficient computational approach that allows different models to be investigated with minimal programming effort. The method is used to investigate the robustness of the curing curve to changes in the assumed distribution of cell generation times. Matlab code is available for carrying out the calculations.