Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

Background: Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. Results: OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform-near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). Conclusions: OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level. © 2013 Chandel et al; licensee BioMed Central Ltd.

Produção de etanol a 40-42ºC em uma co-cultura de Saccharomyces cerevisiae e Issatchenkia orientalis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Imobilização de invertase comercial e de Saccharomyces cerevisiae em sabugo de milho e bagaço de cana-de-açúcar

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise de interação funcional de elF5A com a tradução, repressão da tradução e degradação de mRNA em Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise de propriedades topológicas das redes biológicas integradas da Escherichia coli e da Saccharomyces cerevisiae

Biological processes are complex and possess emergent properties that can not be explained or predict by reductionism methods. To overcome the limitations of reductionism, researchers have been used a group of methods known as systems biology, a new interdisciplinary eld of study aiming to understand the non-linear interactions among components embedded in biological processes. These interactions can be represented by a mathematical object called graph or network, where the elements are represented by nodes and the interactions by edges that link pair of nodes. The networks can be classi- ed according to their topologies: if node degrees follow a Poisson distribution in a given network, i.e. most nodes have approximately the same number of links, this is a random network; if node degrees follow a power-law distribution in a given network, i.e. small number of high-degree nodes and high number of low-degree nodes, this is a scale-free network. Moreover, networks can be classi ed as hierarchical or non-hierarchical. In this study, we analised Escherichia coli and Saccharomyces cerevisiae integrated molecular networks, which have protein-protein interaction, metabolic and transcriptional regulation interactions. By using computational methods, such as MathematicaR , and data collected from public databases, we calculated four topological parameters: the degree distribution P(k), the clustering coe cient C(k), the closeness centrality CC(k) and the betweenness centrality CB(k). P(k) is a function that calculates the total number of nodes with k degree connection and is used to classify the network as random or scale-free. C(k) shows if a network is hierarchical, i.e. if the clusterization coe cient depends on node degree. CC(k) is an indicator of how much a node it is in the lesse way among others some nodes of the network and the CB(k) is a pointer of how a particular node is among several ...(Complete abstract click electronic access below)

Avaliação fenotípica e genotípica de segregantes de uma linhagem industrial de Saccharomyces cerevisiae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cwc24p Is a General Saccharomyces cerevisiae Splicing Factor Required for the Stable U2 snRNP Binding to Primary Transcripts

Splicing of primary transcripts is an essential process for the control of gene expression. Specific conserved sequences in premature transcripts are important to recruit the spliceosome machinery. The Saccharomyces cerevisiae catalytic spliceosome is composed of about 60 proteins and 5 snRNAs (U1, U2, U4/U6 and U5). Among these proteins, there are core components and regulatory factors, which might stabilize or facilitate splicing of specific substrates. Assembly of a catalytic complex depends on the dynamics of interactions between these proteins and RNAs. Cwc24p is an essential S. cerevisiae protein, originally identified as a component of the NTC complex, and later shown to affect splicing in vivo. In this work, we show that Cwc24p also affects splicing in vitro. We show that Cwc24p is important for the U2 snRNP binding to primary transcripts, co-migrates with spliceosomes, and that it interacts with Brr2p. Additionally, we show that Cwc24p is important for the stable binding of Prp19p to the spliceosome. We propose a model in which Cwc24p is required for stabilizing the U2 association with primary transcripts, and therefore, especially important for splicing of RNAs containing non- consensus branchpoint sequences.

In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism

Abstract Background The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p. Results In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain. Conclusions These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

Background Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. Results OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform–near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). Conclusions OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases’ ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level.

Studies on the {\it in vivo} function of a phosphatidylinositol transfer protein from {\it Saccharomyces cerevisiae}

Phosphatidylinositol transfer proteins (PI-TP's) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However the in vivo function of these proteins is unknown. In this thesis we have used a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino terminal sequence of the PI-TP from Saccharomyces cerevisiae, was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Yeast strains transformed with the positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5$\sp\prime$ terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Non-viable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid borne copy of the wild type gene. Sequencing of the entire PIT1 gene has revealed that the PIT1 gene is identical to the SEC14 gene. The sec14 ts mutant which exhibits conditional defects at the Golgi stage of protein secretion, is also temperature sensitive for PI-TP activity in vitro. These findings represent the first instance in which a physiological function has been assigned to any phospholipid transfer protein. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

Funding: Wellcome Trust, 070247/Z/03/A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Efeito do substrato e das condições de tratamento do fermento sobre a fermentação etanólica contaminada com leveduras Saccharomyces cerevisiae selvagens e Lactobacillus fermentum

Pouco se sabe sobre o efeito do substrato e a interação entre as leveduras selvagens e bactérias do gênero Lactobacillus na fermentação alcoólica, pois os estudos tem se concentrado na avaliação dos efeitos da contaminação por um ou outro contaminante separadamente. Diante disso, este trabalho teve como objetivos estudar o efeito do substrato e das condições de tratamento do fermento sobre as fermentações contaminadas com ambos os micro-organismos, leveduras S. cerevisiae selvagens (três linhagens apresentando colônias rugosas e células dispostas em pseudohifas) e Lactobacillus fermentum, tendo a linhagem industrial de S. cerevisiae PE-2 como levedura do processo. Foram realizadas fermentações em batelada em mosto de caldo e de melaço, sem reciclo e com reciclo celular, utilizando tanto a cultura pura da linhagem PE-2 quanto as culturas mistas com as linhagens rugosas e ou L. fermentum. Foram avaliadas modificações no tratamento ácido do fermento, visando o controle do crescimento dos contaminantes sem afetar a levedura do processo. Em seguida, foram conduzidas fermentações contaminadas e não contaminadas submetidas ao tratamento ácido combinado com adição de etanol, tanto em caldo quanto em melaço, utilizando-se PE-2, uma das linhagens rugosas e L. fermentum. A atividade da invertase extracelular foi também avaliada em ambos os substratos para os micro-organismos estudados, em condições de crescimento. Concluiu-se que o tipo de substrato de fermentação, caldo de cana ou melaço, influenciou o desempenho da linhagem industrial PE-2 assim como afetou o desenvolvimento das contaminações com as leveduras rugosas S. cerevisiae na presença ou ausência da bactéria L. fermentum, em fermentações sem reciclo celular. O efeito da contaminação foi mais evidente quando se utilizou caldo de cana do que melaço como substrato, no caso da contaminação com leveduras rugosas, e o inverso no caso da contaminação com L. fermentum. O efeito da contaminação sobre a eficiência fermentativa foi maior na presença da levedura rugosa do que com a bactéria, e a contaminação dupla (tanto com a levedura rugosa quanto com a bactéria) não teve efeito maior sobre a eficiência fermentativa do que a contaminação simples, por um ou por outro micro-organismo isoladamente, especialmente na fermentação em batelada com reciclo celular, independentemente do substrato. Nas fermentações com reciclo de células, o efeito do substrato foi menos evidente. O controle do crescimento das linhagens rugosas pode ser realizado modificando o tratamento ácido normalmente realizado na indústria, seja pela adição de etanol à solução ácida ou pelo abaixamento do pH, dependendo da linhagem rugosa. O tratamento combinado baixo pH (2,0) + 13% etanol afetou a fisiologia da linhagem industrial, trazendo prejuízos à fermentação com reciclo celular, com pequeno controle sobre o crescimento da levedura rugosa e causando morte celular à L. fermentum. A diferença na atividade invertásica entre as linhagens rugosas e industrial de S. cerevisiae pode ser a responsável pela fermentação lenta apresentada pelas linhagens rugosas quando presentes na fermentação, sendo não significativa a influência do substrato sobre a atividade dessa enzima.

Serum and salivary IgA antibody responses to Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans in orofacial granulomatosis and Crohn's disease

Orofacial granulomatosis (OFG) is a condition of unknown aetiology with histological and, in some cases, clinical association with Crohn's disease (CD). However, the exact relationship between OFG and CD remains uncertain. The aim of this study was to determine whether OFG could be distinguished immunologically from CD by comparing non-specific and specific aspects of humoral immunity in serum, whole saliva and parotid saliva in three groups of patients: (a) OFG only (n = 14), (b) those with both oral and gut CD (OFG + CD) (n = 12) and (c) CD without oral involvement (n = 22) and in healthy controls (n = 29). Non-specific immunoglobulin (IgA, SigA, IgA subclasses and IgG) levels and antibodies to whole cells of Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans were assayed by enzyme-linked immunosorbent assay (ELISA) in serum, whole saliva and parotid saliva. Serum IgA and IgA1 and IgA2 subclasses were raised in all patient groups (P < 0.01). Salivary IgA (and IgG) levels were raised in OFG and OFG + CD (P < 0.01) but not in the CD group. Parotid IgA was also raised in OFG and OFG + CD but not in CD. The findings suggest that serum IgA changes reflect mucosal inflammation anywhere in the GI tract but that salivary IgA changes reflect involvement of the oral cavity. Furthermore, the elevated levels of IgA in parotid saliva suggest involvement of the salivary glands in OFG. Serum IgA antibodies to S. cerevisiae were raised markedly in the two groups with gut disease while serum IgA (or IgG) antibodies to C. albicans were elevated significantly in all three patient groups (P < 0.02). No differences were found with antibodies to S. mutans. Whole saliva IgA antibodies to S. cerevisiae (and C. albicans) were raised in the groups with oral involvement. These findings suggest that raised serum IgA antibodies to S. cerevisiae may reflect gut inflammation while raised SIgA antibodies to S. cerevisiae or raised IgA or IgA2 levels in saliva reflect oral but not gut disease. Analysis of salivary IgA and IgA antibodies to S. cerevisiae as well as serum antibodies in patients presenting with OFG may allow prediction of gut involvement.