Probing the role of microenvironment for microencapsulated Sacchromyces cerevisiae under osmotic stress

Cell encapsulation opens a new avenue to the oral delivery of genetically engineered microorganism for therapeutic purpose. Osmotic stress is one of the universal chemical stress factors in the application of microencapsulation technology. In order to understand the effect and mechanism of the encapsulated microenvironment on protecting cells from hyper-osmotic stress, yeast cells of Saccharomyces cerevisiae Y800 were encapsulated in calcium alginate micro-gel beads (MB), alginate-chitosan-alginate (ACA) solid core microcapsules (SCM), and ACA liquid core microcapsules (LCM), respectively. The stress-induced intracellular components and enzyme activity including trehalose, glycerol and super oxide dismutase (SOD) were measured. Free cell culture was used as control. The survival of encapsulated cells and the cells released from MB, SCM and LCM after osmotic shock induced by NaCl solution (1, 2 and 3M) was evaluated. An analysis method was established to probe the effect of encapsulated microenvironment on the cell tolerance to osmotic stress. The results showed that LCM gave rise to the highest level of intracellular trehalose and glycerol, and SOD activity, as well as the highest survival rate of encapsulated cells or cells released from microcapsule. It was demonstrated that LCM was able to induce the highest stress response and stress tolerance of cells, which was adapted during culture, while SCM failed. The theoretical analysis revealed that it was the liquid alginate matrix in microcapsule that played a central role in domesticating the cells to adapt to hyper-osmotic stress. This finding provides a very useful guideline to cell encapsulation.     

Effects of encapsulation of microorganisms on product formation during microbial fermentations

This paper reviews the latest developments in microbial products by encapsulated microorganisms in a liquid core surrounded by natural or synthetic membranes. Cells can be encapsulated in one or several steps using liquid droplet formation, pregel dissolving, coacervation, and interfacial polymerization. The use of encapsulated yeast and bacteria for fermentative production of ethanol, lactic acid, biogas, l-phenylacetylcarbinol, 1,3-propanediol, and riboflavin has been investigated. Encapsulated cells have furthermore been used for the biocatalytic conversion of chemicals. Fermentation, using encapsulated cells, offers various advantages compared to traditional cultivations, e.g., higher cell density, faster fermentation, improved tolerance of the cells to toxic media and high temperatures, and selective exclusion of toxic hydrophobic substances. However, mass transfer through the capsule membrane as well as the robustness of the capsules still challenge the utilization of encapsulated cells. The history and the current state of applying microbial encapsulation for production processes, along with the benefits and drawbacks concerning productivity and general physiology of the encapsulated cells, are discussed.     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.     

Transcriptomic responses of Synechocystis sp. PCC 6803 encapsulated in silica gel

Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors or from alkoxide precursors and incubated under constant light for 24?h prior to RNA extraction. Cultures suspended in liquid media were exposed to 500?mM salt stress and incubated under identical conditions for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change ≥1.5 and P value?相似文献   

Severe ethanol stress induces the preferential synthesis of mitochondrial disaggregase Hsp78 and formation of DUMPs in Saccharomyces cerevisiae

Severe ethanol stress (>9% v/v) induces pronounced translation repression in yeast cells. However, some proteins, which are exceptionally synthesized even under translation repression, play important roles in ethanol tolerance. These proteins are expected to provide important clues for elucidating the survival strategies of yeast cells under severe ethanol stress. In this study, we identified Hsp78 as a protein effectively synthesized under severe ethanol stress. As Hsp78 is involved in mitochondrial protein quality control, we investigated the effect of severe ethanol stress on mitochondrial proteins and found that Ilv2, Kgd1, and Aco1 aggregated with Hsp78 under severe ethanol stress, forming mitochondrial deposition sites for denatured proteins, called DUMPs (Deposits of Unfolded Mitochondrial Proteins). Aggregation of mitochondrial proteins and formation of DUMPs were accelerated in hsp78? cells compared with those in wild-type cells. During the recovery process after ethanol removal, aggregated Ilv2 and DUMP levels rapidly decreased in wild-type cells but were maintained for a long time (>180 min) in hsp78Δ cells. Furthermore, the frequency of respiration-deficient mutants caused by severe ethanol stress was higher in hsp78? cells than in wild-type cells. These results indicate that severe ethanol stress damaged mitochondrial proteins and that Hsp78 was preferentially synthesized to cope with the damage, thereby suppressing the rapid increase in aggregated protein levels under stress and achieving proper clearance of aggregated proteins during the recovery process. This study provides novel insights into the adverse effects of ethanol on mitochondria and yeast response to severe ethanol stress.     

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.     

Abiotic Stress Resistance,a Novel Moonlighting Function of Ribosomal Protein RPL44 in the Halophilic Fungus Aspergillus glaucus

Ribosomal proteins are highly conserved components of basal cellular organelles, primarily involved in the translation of mRNA leading to protein synthesis. However, certain ribosomal proteins moonlight in the development and differentiation of organisms. In this study, the ribosomal protein L44 (RPL44), associated with salt resistance, was screened from the halophilic fungus Aspergillus glaucus (AgRPL44), and its activity was investigated in Saccharomyces cerevisiae and Nicotiana tabacum. Sequence alignment revealed that AgRPL44 is one of the proteins of the large ribosomal subunit 60S. Expression of AgRPL44 was upregulated via treatment with salt, sorbitol, or heavy metals to demonstrate its response to osmotic stress. A homologous sequence from the model fungus Magnaporthe oryzae, MoRPL44, was cloned and compared with AgRPL44 in a yeast expression system. The results indicated that yeast cells with overexpressed AgRPL44 were more resistant to salt, drought, and heavy metals than were yeast cells expressing MoRPL44 at a similar level of stress. When AgRPL44 was introduced into M. oryzae, the transformants displayed obviously enhanced tolerance to salt and drought, indicating the potential value of AgRPL44 for genetic applications. To verify the value of its application in plants, tobacco was transformed with AgRPL44, and the results were similar. Taken together, we conclude that AgRPL44 supports abiotic stress resistance and may have value for genetic application.     

Production of heat shock protein is independent of cell cycle blockage in the yeast Saccharomyces cerevisiae.

In response to certain environmental stresses, cells display a response characterized by the production of heat shock proteins. In this study we showed that blockage of cells of the yeast Saccharomyces cerevisiae at specific points in the mitotic cell cycle was not in itself a stress that induced the production of heat shock proteins. Nevertheless, cell cycle blockage did not preclude a normal heat shock response in arrested cells subjected to elevated temperatures.     

Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

Identification of a drought-induced rice gene, OsSAP, that suppresses Bax-induced cell death in yeast

We identified rice genes that might be involved in drought stress tolerance by virtue of their anti-apoptotic activity. Potential anti-apoptosis related genes were identified by screening an Oryza sativa cDNA library derived from drought stressed tissues in a yeast functional assay. About 28 O. sativa cDNAs promoted yeast survival following engagement of Bax-induced apoptosis. An O. sativa cDNA encoding R12H780 was a highly conserved putative senescence-associated-protein (OsSAP). OsSAP was both highly and rapidly expressed in response to drought stress. Additionally, OsSAP was found to be localized to the mitochondria. Overall, OsSAP represents a new type of Bax suppressor related gene and endows multiple stress tolerance in yeast.     

A rapid flow cytometry assay for the relative quantification of protein encapsulation into bacterial microcompartments

Bacterial microcompartments (MCPs) are protein‐based organelles that have been suggested as scaffolds for creating in vivo nanobioreactors. One of the key steps towards engineering MCPs is to understand and maximize the encapsulation of enzymes into the lumen of the MCP. However, there are currently no high‐throughput methods for investigating protein encapsulation. Here, we describe the development of a rapid in vivo assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. In this assay, we fuse a degradation peptide to a MCP‐targeted fluorescence reporter and use flow cytometry to measure overall fluorescence from the encapsulated, protected reporter protein. Using this assay, we characterized various MCP‐targeting signal sequence mutants for their ability to encapsulate proteins and identified mutants that encapsulate a greater amount of protein than the wild type signal sequence. This assay is a powerful tool for reporting protein encapsulation and is an important step towards encapsulating metabolic enzymes into MCPs for synthetic biochemical pathways.     

Gene Expression Analysis of Cold and Freeze Stress in Baker's Yeast

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.     

Stress Tolerance Variations in Saccharomyces cerevisiae Strains from Diverse Ecological Sources and Geographical Locations

The budding yeast Saccharomyces cerevisiae is a platform organism for bioethanol production from various feedstocks and robust strains are desirable for efficient fermentation because yeast cells inevitably encounter stressors during the process. Recently, diverse S. cerevisiae lineages were identified, which provided novel resources for understanding stress tolerance variations and related shaping factors in the yeast. This study characterized the tolerance of diverse S. cerevisiae strains to the stressors of high ethanol concentrations, temperature shocks, and osmotic stress. The results showed that the isolates from human-associated environments overall presented a higher level of stress tolerance compared with those from forests spared anthropogenic influences. Statistical analyses indicated that the variations of stress tolerance were significantly correlated with both ecological sources and geographical locations of the strains. This study provides guidelines for selection of robust S. cerevisiae strains for bioethanol production from nature.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.