A potential histone-chaperone activity for the MIER1 histone deacetylase complex.

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here, we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and could potentially deposit histone octamer onto nucleosome-depleted regions of DNA.

Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases.

Oxidative stress appears to be a key feature of many neurodegenerative diseases either as a cause or consequence of disease. A range of molecules are subject to oxidation, but in particular, proteins are an important target and measure of oxidative stress. Proteins are subject to a range of oxidative modifications at reactive cysteine residues, and depending on the level of oxidative stress, these modifications may be reversible or irreversible. A range of experimental approaches has been developed to characterize cysteine oxidation of proteins. In particular, mass spectrometry-based proteomic methods have emerged as a powerful means to identify and quantify cysteine oxidation sites on a proteome scale; however, their application to study neurodegenerative diseases is limited to date. Here we provide a guide to these approaches and highlight the under-exploited utility of these methods to measure oxidative stress in neurodegenerative diseases for biomarker discovery, target engagement and to understand disease mechanisms.

Exposure to 1-Butanol Exemplifies the Response of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius to Solvent Stress.

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80°C and pH 2 to 3. Due to its unique physiological properties, allowing life at environmental extremes, and the recent availability of genetic tools, this extremophile has received increasing interest for biotechnological applications. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius toward the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred at up to 1.5% (vol/vol) 1-butanol, while planktonic growth was observed at up to 1% (vol/vol) 1-butanol. Confocal laser-scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy, three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in the absence and presence of 1-butanol. In response to 1% (vol/vol) 1-butanol, transcript levels of genes encoding motility and cell envelope structures, as well as membrane proteins, were reduced. Cell division and/or vesicle formation were upregulated. Furthermore, changes in immune and defense systems, as well as metabolism and general stress responses, were observed. Our findings show that the extreme lifestyle of S.acidocaldarius coincided with a high tolerance to organic solvents. This study provides what may be the first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldariusIMPORTANCEArchaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius, which is adapted to the two extremes of low pH and high temperature, to study its tolerance and robustness as well as its global cellular response toward organic solvents, as exemplified by 1-butanol. We were able to identify biofilm formation as a primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes, such as cell division and cellular defense systems, thus suggesting a global response for the protection at the population level.

Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials.

Significant technical advancements in phosphopeptide enrichment have enabled the identification of thousands of p-peptides (mono and multiply phosphorylated) in a single experiment. However, it is still not possible to enrich all p-peptide species in a single step. A range of new techniques and materials has been developed, with the potential to provide a step-change in phosphopeptide enrichment. The first half of this review contains a tutorial for new potential phosphoproteomic researchers; discussing the key steps of a typical phosphoproteomic experiment used to investigate canonical phosphorylation sites (serine, threonine and tyrosine). The latter half then show-cases the latest developments in p-peptide enrichment including: i) Strategies to mitigate non-specific binding in immobilized metal ion affinity chromatography and metal oxide affinity chromatography protocols; ii) Techniques to separate multiply phosphorylated peptides from monophosphorylated peptides (including canonical from non-canonical phosphorylated peptides), or to simultaneously co-enrich other post-translational modifications; iii) New hybrid materials and methods directed towards enhanced selectivity and efficiency of metal-based enrichment; iv) Novel materials that hold promise for enhanced phosphotyrosine enrichment. A combination of well-understood techniques and materials is much more effective than any technique in isolation; but the field of phosphoproteomics currently requires benchmarking of novel materials against current methodologies to fully evaluate their utility in peptide based proteoform analysis.

Early Response of Sulfolobus acidocaldarius to Nutrient Limitation.

In natural environments microorganisms encounter extreme changes in temperature, pH, osmolarities and nutrient availability. The stress response of many bacterial species has been described in detail, however, knowledge in Archaea is limited. Here, we describe the cellular response triggered by nutrient limitation in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. We measured changes in gene transcription and protein abundance upon nutrient depletion up to 4 h after initiation of nutrient depletion. Transcript levels of 1118 of 2223 protein coding genes and abundance of approximately 500 proteins with functions in almost all cellular processes were affected by nutrient depletion. Our study reveals a significant rerouting of the metabolism with respect to degradation of internal as well as extracellular-bound organic carbon and degradation of proteins. Moreover, changes in membrane lipid composition were observed in order to access alternative sources of energy and to maintain pH homeostasis. At transcript level, the cellular response to nutrient depletion in S. acidocaldarius seems to be controlled by the general transcription factors TFB2 and TFEß. In addition, ribosome biogenesis is reduced, while an increased protein degradation is accompanied with a loss of protein quality control. This study provides first insights into the early cellular response of Sulfolobus to organic carbon and organic nitrogen depletion.

Natural Mutagenesis-Enabled Global Proteomic Study of Metabolic and Carbon Source Implications in Mutant Thermoacidophillic Archaeon Sulfolobus solfataricus PBL2025.

The thermoacidophilic crenarchaeon Sulfolobus solfataricus has been widely used as a model organism for archaeal systems biology research. Investigation using its spontaneous mutant PBL2025 provides an effective metabolic baseline to study subsequent mutagenesis-induced functional process shifts as well as changes in feedback inhibitions. Here, an untargeted metabolic investigation using quantitative proteomics and metabolomics was performed to correlate changes in S. solfataricus strains P2 against PBL2025 and under both glucose and tryptone. The study is combined with pathway enrichment analysis to identify prominent proteins with differential stoichiometry. Proteome level quantification reveals that over 20% of the observed overlapping proteome is differentially expressed under these conditions. Metabolic-induced differential expressions are observed along the central carbon metabolism, along with 12 other significantly regulated pathways. Current findings suggest that PBL2025 is able to compensate through the induction of carbon metabolism, as well as other anabolic pathways such as Val, Leu and iso-Leu biosynthesis. Studying protein abundance changes after changes in carbon sources also reveals distinct differences in metabolic strategies employed by both strains, whereby a clear down-regulation of carbohydrate and nucleotide metabolism is observed for P2, while a mixed response through down-regulation of energy formation and up-regulation of glycolysis is observed for PBL2025. This study contributes, to date, the most comprehensive network of changes in carbohydrate and amino acid pathways using the complementary systems biology observations at the protein and metabolite levels. Current findings provide a unique insight into molecular processing changes through natural (spontaneous) metabolic rewiring, as well as a systems biology understanding of the metabolic elasticity of thermoacidophiles to environmental carbon source change, potentially guiding more efficient directed mutagenesis in archaea.

An iTRAQ characterisation of the role of TolC during electron transfer from Shewanella oneidensis MR-1.

Anodophilic bacteria have the ability to generate electricity in microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigated the anode-specific responses of Shewanella oneidensis MR-1, an exoelectroactive Gammaproteobacterium, using for the first time iTRAQ and 2D-LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture. The regulated dataset produced was enriched in membrane proteins. Proteins shown to be more abundant in anaerobic electroactive anodic cells included efflux pump TolC and an uncharacterised tetratricopeptide repeat (TPR) protein, whilst the TonB2 system and associated uncharacterised proteins such as TtpC2 and DUF3450 were more abundant in microaerobic planktonic cells. In order to validate the iTRAQ data, the functional role for TolC was examined using a δTolC knockout mutant of S. oneidensis. Possible roles for the uncharacterised proteins were identified using comparative bioinformatics. We demonstrate that employing an insoluble extracellular electron acceptor requires multiple proteins involved in cell surface properties. All MS and processed data are available via ProteomeXchange with identifier PXD004090.

Protein phosphorylation and its role in archaeal signal transduction.

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies.

Quantitative analysis of UV-A shock and short term stress using iTRAQ, pseudo selective reaction monitoring (pSRM) and GC-MS based metabolite analysis of the cyanobacterium Nostoc punctiforme ATCC 29133.

A quantitative proteomics and metabolomics analysis was performed using iTRAQ, HPLC and GC-MS in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 to understand the effect of short and long term UV-A exposure. Changes in the proteome were measured for short-term stress (4-24h) using iTRAQ. Changes in the photosynthetic pigments and intracellular metabolites were observed at exposures of up to 7days (pigments) and up to 11days (intracellular metabolites). To assess iTRAQ measurement quality, pseudo selected reaction monitoring (pSRM) was used, with this confirming underestimation of protein abundance levels by iTRAQ. Our results suggest that short term UV-A radiation lowers the abundance of PS-I and PS-II proteins. We also observed an increase in abundance of intracellular redox homeostasis proteins and plastocyanin. Additionally, we observed statistically significant changes in scytonemin, Chlorophyll A, astaxanthin, zeaxanthin, and ß-carotene. Assessment of intracellular metabolites showed significant changes in several, suggesting their potential role in the Nostoc's stress mitigation strategy. Cyanobacteria under UV-A radiation have reduced growth due to intensive damage to essential functions, but the organism shows a defense response by remodeling bioenergetics pathway, induction of the UV protection compound scytonemin and increased levels of proline and tyrosine as a mitigation response. BIOLOGICAL SIGNIFICANCE: The effect of UV-A radiation on the proteome and intracellular metabolites of N. punctiforme ATCC 29133 including photosynthetic pigments has been described. We also verify the expression of 13 iTRAQ quantified protein using LC-pSRM. Overall we observed that UV-A radiation has a drastic effect on the photosynthetic machinery, photosynthetic pigments and intracellular amino acids. As a mitigation strategy against UV-A radiation, proline, glycine, and tyrosine were accumulated.

Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.

A cool tool for hot and sour Archaea: proteomics of Sulfolobus solfataricus.

In recent years, much progress has been made in proteomic studies to unravel metabolic pathways and basic cellular processes. This is especially interesting for members of the Archaea, the third domain of life. Archaea exhibit extraordinary features and many of their cultivable representatives are adaptable to extreme environments. Archaea harbor many unique traits besides bacterial attributes, such as size, shape, and DNA structure and eukaryal characteristics like information processing. Sulfolobus solfataricus P2, a thermoacidophilic archaeal representative, is a well-established model organism adapted to low-pH environments (pH 2-3) and high temperatures (80°C). The genome has a size of 3 Mbp and its sequence has been deciphered. Approximately 3033 predicted open reading frames have been identified and the genome is characterized by a great number of diverse insertion sequence elements. In unraveling the organisms' metabolism and lifestyle, proteomic analyses have played a major role. Much effort has been directed at this organism and is reviewed here. With the help of proteomics, unique metabolic pathways were resolved in S. solfataricus, targets for regulatory protein phosphorylation identified, and cellular responses upon virus infection as well as oxidative stress analyzed.

Quantitative proteomic analysis of the exoelectrogenic bacterium Arcobacter butzleri ED-1 reveals increased abundance of a flagellin protein under anaerobic growth on an insoluble electrode.

Exoelectrogens have the ability to generate electricity in mediator-less microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigate the anode-specific responses of Arcobacter butzleri ED-1, the first identified exoelectrogenic Epsilonproteobacterium. iTRAQ and 2D-LC MS/MS driven proteomics were used to compare protein abundances in A. butzleri ED-1 when generating an electronegative potential (-225 mV) in an anaerobic half-cell - either growing as an electrogenic biofilm or suspended in the liquid medium - versus a microaerobic culture. This is the first quantitative proteomic study concentrating on growth of an exoelectrogen during current generation. From 720 proteins identified and quantified (soluble and insoluble sub-proteomes), statistical analysis reveals 75 differentially-expressed proteins. This dataset was enriched in proteins regulating energy and intermediary metabolism, electron and protein transport. Flagellin up-regulation was concomitant with electron transport in the anodic cells, while decreased abundance of a methyl-accepting chemotaxis protein suggested that flagella were involved in communication with the anode surface and electrogenesis, rather than motility. Two novel cytochromes potentially related to electron transport were up-regulated in anaerobic cultures. We demonstrate that employing an insoluble extracellular electron acceptor for anaerobic growth regulates multiple proteins involved in cell surface properties, electron transport and the methylcitrate cycle.

Bioinformatic study of the relationship between protein regulation and sequence properties.

Although protein expression and regulation have been intensively studied, a complete picture of its mechanisms is still to be drawn. Analysis of high-throughput quantitative proteomics data provides a way to better understand protein regulation. Here, we introduce a bioinformatic analysis method to correlate protein regulation with individual amino acid patterns. We compare the amino acid composition between groups of regulated and unregulated proteins and investigate the correlation between codon usage patterns and protein regulation levels in two Sulfolobus species in "biofilm vs planktonic" experiments. The identified amino acids can then be associated with the regulation of specific gene functions. Strikingly, our analysis shows that functional categories of regulated proteins with similar composition and codon usage pattern of specific amino acids behave similarly. This finding can contribute to a better understanding of protein and gene expression regulation and could find applications in gene optimisation.

Protein profiling of the secretome of FcεRI activated RBL-2H3.1 cells.

BACKGROUND: Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. OBJECTIVE: The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. METHODS: A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identification. RESULTS: Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified ~53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines (e.g. MCP-2, PF-4, CSF-1 and TGF-ß1) are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. CONCLUSION: In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes.

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

Macromolecular fingerprinting of sulfolobus species in biofilm: a transcriptomic and proteomic approach combined with spectroscopic analysis.

Microorganisms in nature often live in surface-associated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ~3.4 and ~1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development.

A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia.

Tannerella forsythia is a Gram-negative anaerobe that is one of the most prominent inhabitants of the sub-gingival plaque biofilm, which is crucial for causing periodontitis. We have used iTRAQ proteomics to identify and quantify alterations in global protein expression of T. forsythia during growth in a biofilm. This is the first proteomic study concentrating on biofilm growth in this key periodontal pathogen, and this study has identified several changes in protein expression. Moreover, we introduce a rigorous statistical method utilising peptide-level intensities of iTRAQ reporters to determine which proteins are significantly regulated. In total, 348 proteins were identified and quantified with the expression of 44 proteins being significantly altered between biofilm and planktonic cells. We identified proteins from all cell compartments, and highlighted a marked upregulation in the relative abundances of predicted outer membrane proteins in biofilm cells. These included putative transport systems and the T. forsythia S-layer proteins. These data and our finding that the butyrate production pathway is markedly downregulated in biofilms indicate possible alterations in host interaction capability. We also identified upregulation of putative oxidative stress response proteins, and showed that biofilm cells are 10 to 20 fold more resistant to oxidative stress. This may represent an important adaptation of this organism to prolonged persistence and immune evasion in the oral cavity.

Quantitative proteomic analysis of Sulfolobus solfataricus membrane proteins.

A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30% of the total number of proteins. Using a modified iTRAQ protocol for membrane protein analysis, of the 284 proteins detected, 246 proteins were identified as membrane proteins, while using an original iTRAQ protocol, 147 proteins were detected with only 133 proteins being identified as membrane proteins. Furthermore, 97.2% of proteins identified in the modified protocol contained more than 2 distinct peptides compared to the original workflow. The successful application of this modified protocol offers a potential technique for quantitatively analyzing membrane-associated proteomes of organisms in the archaeal kingdom. The combination of 3 different iTRAQ experiments resulted in the detection of 395 proteins (>or=2 distinct peptides) of which 373 had predicted membrane properties. Approximately 20% of the quantified proteins were observed to exhibit >or=1.5-fold differential expression at temperatures well below the optimum for growth.

The proteomic response of Saccharomyces cerevisiae in very high glucose conditions with amino acid supplementation.

Ethanol yield by Saccharomyces cerevisiae in very high glucose (VHG) media with an amino acid supplement was investigated. Amino acid supplementation led to positive cell responses, including reduced lag time and increased cell viability in VHG media. A quantitative shotgun proteomic analysis was used to understand how amino acid supplemented S. cerevisiae responds to high osmotic conditions. iTRAQ data revealed that most proteins involved in glycolysis and pentose phosphate pathways were up-regulated under high glucose shock. Reactivation of amino acid metabolism was also observed at the end of the lag phase. The relative abundance of most identified proteins, including aminoacyl-tRNA biosynthesis proteins, and heat-shock proteins, remained unchanged in the hours immediately following application of glucose shock. However, the expression of these proteins increased significantly at the end of the lag phase. Furthermore, the up-regulation of trehalose and glycogen biosynthesis proteins, first maintaining then latterly increasing glycolysis pathway activity was also observed. This was verified by enhanced ethanol yields at 10 and 12 h (0.43 and 0.45 g ethanol/g glucose) compared to 2 h (0.32 g ethanol/g glucose). These data combined with relevant metabolite measurements demonstrates that enhanced ethanol fermentation under VHG conditions can be achieved with the aid of amino acid supplementation.

Proteomic analysis of calcium alginate-immobilized Saccharomyces cerevisiae under high-gravity fermentation conditions.

Saccharomyces cerevisiae KAY446 cells immobilized in calcium alginate gel, and supplemented with additional amino acids, were successfully used in enhancing ethanol production. This combination succeeded in improving the ethanol yield and reducing the fermentation time. The ethanol yield under these conditions was 0.40 g of ethanol/g of glucose, with a final ethanol concentration of 118 g/L after 72 h. This is compared to yields with immobilized cells alone of 0.35 g of ethanol/g of glucose and freely suspended cells with no amino acid supplementation of 0.30 g of ethanol/g of glucose, under the same VHG conditions. The maximum specific ethanol production rates were 0.98, 0.73, and 0.61 g (g dry weight) (-1) h (-1) for immobilized cells under VHG conditions with and without amino acid supplementation and free cells, respectively. A proteomic analysis showed significant stimulation of many pathways during fermentation under these conditions, including the Ras/cAMP, glycolysis, starch, and sucrose pathways, amino acids biosynthesis, and aminoacyl-tRNA synthetases. The upregulation of ribosomal, heat-shock proteins and proteins involved in cell viability confirmed that protein biosynthesis was accelerated and revealed likely mechanisms for improving cellular viability.