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eIF2α phosphorylation is constitutive in high-yielding Fps1 strains. Phosphorylation of eIF2α, as detected by immunoblot, is shown in amino-acid-starved and control cells: (+) represents cells cultured in growth medium supplemented with amino acids (control cells) whilst (−) represents cells cultured in growth medium lacking amino acids for 10 min (starved cells). yTHCBMS1 cultures were also supplemented with 0.5 μg/mL doxycycline. The upper panel (37 kDa) was detected with an anti-eIF2α antibody, whilst the lower panel (37 kDa) was detected with an anti-phospho-eIF2α antibody
Source publication
Background
We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5...
Citations
... It was possible to efficiently produce proteins as large as up to 1,500 residues in length [97]. Yeast can also be very easily genetically modified [11,94,98]. Thus, yeast protein expression has become a viable alternative to bacteria for large-scale eukaryotic protein production. ...
The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes.
... This agrees with the results above (Fig. 2) in which glycerol retention was higher in urea-SCJ while glycerol secretion was higher in DAP-SCJ, and suggests that Fps1, as the biggest contributor to glycerol retention [24], could also be regulated by the nitrogen source/NCR, in addition to the HOG and CWI pathways [45,47]. Nevertheless, while the expression of FPS1 is primarily considered as being constitutive [48], it can be regulated [24,47,49], namely in response to amino acids availability and osmotic stress. Whether DAP and/or urea can identically contribute to the regulation of FPS1/Fps1 is presently unknown. ...
Background
Applying very high gravity (VHG) fermentation conditions to the sugarcane juice (SCJ) bioethanol industry would improve its environmental and economic sustainability without the need for major infrastructure changes or investments. It could enable a decrease in the consumption of biological and natural resources (cane/land, water and energy) while maintaining acceptable production parameters. The present study attempts to demonstrate and characterise an effective industrially relevant SCJ-VHG fermentation process. ResultsAn industry-like SCJ-VHG bioethanol production process with 30 and 35 °Bx broth was employed to investigate the effects of both the yeast strain used and nitrogen source supplementation on process yield, process productivity, biomass viability, glycerol concentration and retention-associated gene expression. Process performance was shown to be variably affected by the different process conditions investigated. Highest process efficiency, with a 17% (w/v) ethanol yield and only 0.2% (w/v) sugar remaining unfermented, was observed with the Saccharomyces cerevisiae industrial strain CAT-1 in 30 °Bx broth with urea supplementation. In addition, efficient retention of glycerol by the yeast strain was identified as a requisite for better fermentation and was consistent with a higher expression of glycerol permease STL1 and channel FPS1. Urea was shown to promote the deregulation of STL1 expression, overcoming glucose repression. The consistency between Fps1-mediated ethanol secretion and ethanol in the extracellular media reinforces previous suggestions that ethanol might exit the cell through the Fps1 channel. Conclusions
This work brings solid evidence in favour of the utilisation of VHG conditions in SCJ fermentations, bringing it a step closer to industrial application. SCJ concentrated up to 30 °Bx maintains industrially relevant ethanol production yield and productivity, provided the broth is supplemented with a suitable nitrogen source and an appropriate industrial bioethanol-producing yeast strain is used. In addition, the work contributes to a better understanding of the VHG-SCJ process and the variable effects of process parameters on process efficiency and yeast strain response. Open image in new window
... Used as therapeutic proteins in medical field P. pastoris with strong and inducer promoter AOX1 Protein β-aminopeptidase BapA (Heyland et al., 2011) Used in protein hydrolysate industry. It helps in measuring the activity of the TCA cycle of yeast S. cerevisiae containing S and preS2-S genes under inducible condition Insulin protein Used for diabetic treatment in pharmaceutical industry S. cerevisiae strains with SIC Human hemoglobin (Liu et al., 2014) Used a s an oxygen carrier substitute in medical field S. cerevisiae Recombinant aquaporin Fps1 protein (FPS1), (Cartwright et al., 2017) Used glycerol uptake/efflux facilitator protein as drought and salinity response in Xenopus oocytes plant in agriculture. ...
Yeast is very well known eukaryotic organism for its remarkable biodiversity and extensive industrial applications. Saccharomyces cerevisiae is one of the most widely used microorganisms in biotechnology with successful applications in the biochemical production. Biological conversion with the focus on the different utilization of renewable feedstocks into fuels and chemicals has been intensively investigated due to increasing concerns on sustainability issues worldwide. Compared with its counterparts, Saccharomyces cerevisiae, the baker's yeast, is more industrially relevant due to known genetic and physiological background, the availability of a large collection of genetic tools, the compatibility of high-density and large-scale fermentation, and optimize the pathway for variety of products. Therefore, S. cerevisiae is one of the most popular cell factories and has been successfully used in the modern biotech industry to produce a wide variety of products such as ethanol, organic acids, amino acids, enzymes, and therapeutic proteins. This study explores how different sustainable solutions used to overcome various environmental effects on yeast. This work targets a broad matrix of current advances and future prospect in yeast biotechnology and discusses their application and potential in general.
In the face of flourishing industrialization and global trade, heavy metal and metalloid contamination of the environment is a growing concern throughout the world. The widespread presence of highly toxic compounds of arsenic, antimony, and cadmium in nature poses a particular threat to human health. Prolonged exposure to these toxins has been associated with severe human diseases, including cancer, diabetes, and neurodegenerative disorders. These toxins are known to induce analogous cellular stresses, such as DNA damage, disturbance of redox homeostasis, and proteotoxicity. To overcome these threats and improve or devise treatment methods, it is crucial to understand the mechanisms of cellular detoxification in metal and metalloid stress. Membrane proteins are key cellular components involved in the uptake, vacuolar/lysosomal sequestration, and efflux of these compounds; thus, deciphering the multilevel regulation of these proteins is of the utmost importance. In this review, we summarize data on the mechanisms of arsenic, antimony, and cadmium detoxification in the context of membrane proteome. We used yeast Saccharomyces cerevisiae as a eukaryotic model to elucidate the complex mechanisms of the production, regulation, and degradation of selected membrane transporters under metal(loid)-induced stress conditions. Additionally, we present data on orthologues membrane proteins involved in metal(loid)-associated diseases in humans.
Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.
