Screening and identification of cellulase producing yeast-like microorganisms from Brazilian biomes

The main goals of the present study included the screening and identification of cellulase producing wild yeasts, isolated from samples collected from different Brazilian biomes. They were selected according to their capabilities of degrading carboxymethyl cellulose (CMC) and micro-crystalline cellulose (SERVACEL®), as single carbon sources in solid medium. After the step of solid medium selection, yeast cells were grown in liquid medium containing cellulose (SERVACEL®); in shake flasks at temperature of 30°C and 150 rpm agitation for 288 h. Three specific activities were evaluated: endoglucanase (CMCase), total activity (filter paper activity), and cellobiase. From a total of 390 strains of wild yeasts previously isolated, 16 strains performed cellulose hydrolysis, verified by the colorless halo in the solid medium. Among these 16 strains, 5 stood out as presenting higher levels of enzyme activity. The following step, screening in liquid medium, indicated only one strain as a potential producer of cellulases, named as AAJ6, for which the highest hydrolytic activity on carboxymethyl cellulose (0.33 U/ml) and filter paper (0.039 U/ml) was recorded. Afterwards, this wild yeast strain (AAJ6) was molecularly identified by sequencing the ITS1-5.8S-ITS2 and D1/D2 domains of the subunit (26 S) ribosomal DNA. Sequencing resulted in the identification of this strain as yeast-like fungus Acremonium strictum.Keywords: Acremonium strictum, screening, identification, yeast-like, cellulase

Perspective: Indigenous sugarcane yeast strains as ideal biological platforms for the delivery of next generation biorefining technologies

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)In the industrial process used to convert sugarcane juice to bioethanol, the yeast cells responsible for the fermentation are often exposed to severe biotic and abiotic stresses. These include the constant entry of microbial contaminants into the system, prolonged cell recycling, ethanol toxicity, and osmotic, oxidative, and temperature stresses. In the past decade, several indigenous yeast strains have been isolated in Brazil that combine excellent adaptation to this hostile environment and high performance in bioethanol fermentation. Recent genetic studies have demonstrated that these strains have heterogeneous genome architectures, and established a strong link between this genomic complexity and their adaptation to the industrial environment. We propose a role for these highly adapted organisms as biological platforms for the delivery of a wide range of future biorefining technologies. By adopting genetic manipulation strategies that do not interfere with their genomic complexity, it should be possible create new yeast strains that are much more likely to succeed in large scale industrial applications.11213348689Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)ETH BioenergiaConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [PITE3 - 07/58336-3

Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-speciric antioxidant), was investigated in the oxidative stress response of Saccharomyces ceravisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H2O2 in Cells, which could be related to their elevated sensitivity to oxidative stress. In. fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H2O2. In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.509343043

Caste-specific gene expression in the stingless bee Melipona quadrifasciata - Are there common patterns in highly eusocial bees?

Caste polyphenism is a multifaceted phenomenon, most evident in the marked differences in reproductive capacity and longevity between queens and workers. The mechanisms underlying caste differentiation and division of labor are mainly addressed in the honey bee, and recently have been studied at the molecular level. Yet, generalizations drawn from studies on this model organism require validation by comparative studies. We choose Melipona quadrifasciata, a sister-group species to honey bees, to investigate differences in gene expression between newly emerged adult queens and workers. RNA extracts were subjected to a differential display protocol (DDRT-PCR). The putative differentially expressed genes, for which annotation was available, were validated by RT-PCR and hybridization. Differential expression was observed for myosin, projectin, kettin, cytochrome P450, Rab11 and Sas10. Except for kettin, all of these were overexpressed in the worker caste. Projectin and kettin could play roles in caste-specific flight muscle organization. The putative Rab11 and Sas10 homolog genes could be involved in fertility-related cell signaling and in longevity-related gene silencing, respectively. Cytochrome P450 overexpression in Melipona workers corroborates similar findings in the honey bee, thus indicating a common function in the social insect caste syndrome.51435235

Yeast oxidative stress response - Influences of cytosolic thioredoxin peroxidase I and of the mitochondrial functional state

We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability. First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic thioredoxin peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death. This cTPxI-dependent protection could be related to its dual functions, as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2. We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells to improve their antioxidant defense. On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2 treatment and reduced the H2O2-mediated activation of some genes otherwise observed, impairing cell defense and viability. The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder. Yap1 cellular distribution was not affected by cTpxI deficiency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms of Yap1 activation/deactivation. Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dysfunction due to its functional versatility as an antioxidant, chaperone and modulator of gene expression.273480581

Regulation of mitochondrial thioredoxin peroxidase I expression by two different pathways: One dependent on cAMP and the other on heme

Mitochondrial isoform of thioredoxin peroxidase (mTPx I) is an antioxidant protein recently described in Saccharomyces cerevisiae. Here we characterized pathways that lead to mTPx I induction in two situations: growth in media containing low glucose concentrations and treatment with peroxides. The induction of mTPx I by growth on low glucose concentrations was dependent on cAMP and on the transcription factors Msn2p/Msn4p as demonstrated by northern blot experiments using yeast strains with deletion of MSN2 and MSN4 genes and also using a strain permeable to cAMP. mTPx I expression was also induced by peroxides in a time- and dose-dependent manner and varied with the carbon source present in the media. Deletion of HAP1 or inhibition of heme synthesis abolished induction of mTPx I by H2O2 on cells which were grown in media containing glucose, indicating that Hap1p is involved in the regulation of this process. mTPx I was induced by H2O2 on glycerol/ethanol-containing media, but we could not associate any transcription factor with this phenomenon. Finally, mTPx I also induced by t-butyl hydroperoxide in a Hap1p-independent manner. In conclusion, mTPx I expression is under a complex regulatory network, which involves, at least, two signaling pathways: one sensing the carbon source (which is signalized by cAMP) and the other sensing the intracellular redox state (which is signalized by heme). (C) 2002 Elsevier Science Inc.32327828

Raman spectroscopy and chemometrics for on-line control of glucose fermentation by Saccharomyces cerevisiae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)This work presents the use of Raman spectroscopy and chemometrics for on-line control of the fermentation process of glucose by Saccharomyces cerevisiae. In a first approach, an on-line determination of glucose, ethanol, glycerol, and cells was accomplished using multivariate calibration based on partial least squares (PLS). The PLS models presented values of root mean square error of prediction (RMSEP) of 0.53, 0.25, and 0.02% for glucose, ethanol and glycerol, respectively, and RMSEP of 1.02 g L-1 for cells. In a second approach, multivariate control charts based on multiway principal component analysis (MPCA) were developed for detection of fermentation fault-batch. Two multivariate control charts were developed, based on the squared prediction error (Q) and Hotelling's T2. The use of the Q control chart in on-line monitoring was efficient for detection of the faults caused by temperature, type of substrate and contamination, but the T2 control chart was not able to monitor these faults. On-line monitoring by Raman spectroscopy in conjunction with chemometric procedures allows control of the fermentative process with advantages in relation to reference methods, which require pretreatment, manipulation of samples and are time consuming. Also, the use of multivariate control charts made possible the detection of faults in a simple way, based only on the spectra of the system. (C) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 201228615981604Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Transposable elements in Coffea (Gentianales : Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK beta-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted.279438540