Difference between the cell wall roughnesses of mothers and daughters of Saccharomyces cerevisiae subjected to high pressure stress.

High hydrostatic pressure (HHP) stress generates cellular responses similar to those to other stresses that yeasts endure in fermentation tanks. Structural and spatial compaction of molecules, as well as weakening and stretching of plasma membranes and cell walls, are often observed and have a significant influence on the fermentative process. Atomic force microscopy (AFM) yields accurate data on the morphological characteristics of yeast cell walls, providing important insights for the development of more productive yeast strains. Saccharomyces cerevisiae cell wall assessment using AFM in the intermittent contact reading mode using a silicon cantilever, before and after application of a pressure of 100 MPa for 30 min, demonstrated that mother and daughter cells have different responses. Daughter cells were more sensitive to the effects of HHP, presenting lower average Ra (arithmetic roughness), Rz (ten-point average roughness), and Rq (root-mean-square roughness) after exposure to high pressure. Better adaptation to stress in mother cells leads to higher cell wall resistance and, therefore, to better protection.

Complete genome sequence and analysis of a Saccharomyces cerevisiae strain used for sugarcane spirit production.

Distillation of fermented sugarcane juice produces both rum and cachaça, significant sources of revenue in Brazil and elsewhere. In this study, we provide a genomic analysis of a Saccharomyces cerevisiae strain isolated from a cachaça distillery in Brazil. We determined the complete genome sequence of a strain with high flocculation capacity, high tolerance to ethanol, osmotic and heat shock stress and high fermentation rates and compared the sequence with that of the reference S288c genome as well as those of two other cachaça strains. Single-nucleotide polymorphism analysis identified alterations in genes involved in nitrogen and organic compound metabolism, integrity of organelles and ion homeostasis. The strain exhibited fragmentation of several flocculation genes relative to the reference genome, as well as loss of a stop codon in the FLO8 gene, which encodes a transcription factor required for FLO gene expression. The strain contained no genes not present in the reference genome strain but did lack several genes, including asparaginase genes, maltose utilization loci, and several genes from the tandem array of the DUP240 family. The three cachaça strains lacked different sets of genes, but the asparaginase genes and several of the DUP240 genes were common deficiencies. This study provides new insights regarding the selective pressure of sugarcane fermentation on the genome of yeast strains and offers additional genetic resources for modern synthetic biology and genome editing tools.

Battle of Three: The Curious Case of Papaya Sticky Disease.

Among the most serious problems in papaya production are the viruses associated with papaya ringspot and papaya sticky disease (PSD). PSD concerns producers worldwide because its symptoms are extremely aggressive and appear only after flowering. As no resistant cultivar is available, several disease management strategies have been used in affected countries, such as the use of healthy seeds, exclusion of the pathogen, and roguing. In the 1990s, a dsRNA virus, papaya meleira virus (PMeV), was identified in Brazil as the causal agent of PSD. However, in 2016 a second virus, papaya meleira virus 2 (PMeV2), with an ssRNA genome, was also identified in PSD plants. Only PMeV is detected in asymptomatic plants, whereas all symptomatic plants contain both viral RNAs separately packaged in particles formed by the PMeV capsid protein. PSD also affects papaya plants in Mexico, Ecuador, and Australia. PMeV2-like viruses have been identified in the affected plants, but the partner virus(es) in these countries are still unknown. In Brazil, PMeV and PMeV2 reside in laticifers that promote spontaneous latex exudation, resulting in the affected papaya fruit's sticky appearance. Genes modulated in plants affected by PSD include those involved in reactive oxygen species and salicylic acid signaling, proteasomal degradation, and photosynthesis, which are key plant defenses against PMeV complex infection. However, the complete activation of the defense response is impaired by the expression of negative effectors modulated by the virus. This review presents a summary of the current knowledge of the Carica papaya-PMeV complex interaction and management strategies.

Differences in gene modulation in Saccharomyces cerevisiae indicate that maturity plays an important role in the high hydrostatic pressure stress response and resistance.

There is a strong relationship between the regulatory pathways to oxidative stress, longevity, and aging. High hydrostatic pressure (HHP) induces oxidative stress and activates cellular defense mechanisms. The understanding of these mechanisms is a strategy to delay damage associated with aging. Addressing resistance to stress and aging in Saccharomyces cerevisiae is a well-accepted approach since pathways involved in energy balance, damage accumulation and stress response are preserved among eukaryotes. The purpose of this study was to correlate the environmental stress response to cell maturity. HHP stress response on S. cerevisiae mother and daughter cells was evaluated through survival, reactive oxygen species (ROS) accumulation and gene expression. Mature cells were yeasts that had budded and originated at least one descendant, and young cells were the ones that did not form a bud. Mature cells were more resistant to HHP, although they showed a decrease in expression of antioxidants enzymes genes, and a higher intracellular levels of ROS. Young cells had less resistance to HHP despite a tendency of positively regulating these same antioxidant encoders. The TOR1 gene, related to aging and apoptosis, was unchanged in mother cells and showed a tendency toward increased expression in daughter cells submitted to HHP. The gene modulation differences of the mother and daughter cells indicates that maturity plays an important role in the HHP stress response and resistance. Thus, even accumulating high levels of ROS, mature cells were more tolerant to HHP stress and survived better, despite aging.

Second-Generation Bioethanol from Coconut Husk.

Coconut palm (Cocos nucifera) is an important commercial crop in many tropical countries, but its industry generates large amounts of residue. One way to address this problem is to use this residue, coconut husk, to produce second-generation (2G) ethanol. The aim of this review is to describe the methods that have been used to produce bioethanol from coconut husk and to suggest ways to improve different steps of the process. The analysis performed in this review determined that alkaline pretreatment is the best choice for its delignification potential. It was also observed that although most reported studies use enzymes to perform hydrolysis, acid hydrolysis is a good alternative. Finally, ethanol production using different microorganisms and fermentation strategies is discussed and the possibility of obtaining other added-value products from coconut husk components by using a biorefinery scheme is addressed.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

KEY MESSAGE: Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.

High hydrostatic pressure leads to free radicals accumulation in yeast cells triggering oxidative stress.

Saccharomyces cerevisiae is a unicellular organism that during the fermentative process is exposed to a variable environment; hence, resistance to multiple stress conditions is a desirable trait. The stress caused by high hydrostatic pressure (HHP) in S. cerevisiae resembles the injuries generated by other industrial stresses. In this study, it was confirmed that gene expression pattern in response to HHP displays an oxidative stress response profile which is expanded upon hydrostatic pressure release. Actually, reactive oxygen species (ROS) concentration level increased in yeast cells exposed to HHP treatment and an incubation period at room pressure led to a decrease in intracellular ROS concentration. On the other hand, ethylic, thermic and osmotic stresses did not result in any ROS accumulation in yeast cells. Microarray analysis revealed an upregulation of genes related to methionine metabolism, appearing to be a specific cellular response to HHP, and not related to other stresses, such as heat and osmotic stresses. Next, we investigated whether enhanced oxidative stress tolerance leads to enhanced tolerance to HHP stress. Overexpression of STF2 is known to enhance tolerance to oxidative stress and we show that it also leads to enhanced tolerance to HHP stress.

High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain.

High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries.

High hydrostatic pressure activates transcription factors involved in Saccharomyces cerevisiae stress tolerance.

A number of transcriptional control elements are activated when Saccharomyces cerevisiae cells are submitted to various stress conditions, including high hydrostatic pressure (HHP). Exposure of Saccharomyces cerevisiae cells to HHP results in global transcriptional reprogramming, similar to that observed under other industrial stresses, such as temperature, ethanol and oxidative stresses. Moreover, treatment with a mild hydrostatic pressure renders yeast cells multistress tolerant. In order to identify transcriptional factors involved in coordinating response to high hydrostatic pressure, we performed a time series microarray expression analysis on a wild S. cerevisiae strain exposed to 50 MPa for 30 min followed by recovery at atmospheric pressure (0.1 MPa) for 5, 10 and 15 min. We identified transcription factors and corresponding DNA and RNA motifs targeted in response to hydrostatic pressure. Moreover, we observed that different motif elements are present in the promoters of induced or repressed genes during HHP treatment. Overall, as we have already published, mild HHP treatment to wild yeast cells provides multiple protection mechanisms, and this study suggests that the TFs and motifs identified as responding to HHP may be informative for a wide range of other biotechnological and industrial applications, such as fermentation, that may utilize HHP treatment.

Influence of cellular fatty acid composition on the response of Saccharomyces cerevisiae to hydrostatic pressure stress.

High hydrostatic pressure (HHP) interferes with cellular membrane structure. The orientation of lipid molecules is changed, especially in the vicinity of proteins, leading to decreased membrane fluidity. Adaptation to HHP requires increased membrane fluidity, often achieved through a rise in the proportion of unsaturated fatty acids. In this work, a desaturase-deficient Saccharomyces cerevisiae mutant strain (OLE1 gene deletion) was grown in media supplemented with fatty acids differing in size and number of unsaturations and submitted to pressure up to 200 MPa for 30 min. Desaturase-deficient yeast supplemented with palmitoleic acid demonstrated increased sensitivity to pressure compared to cells supplemented with oleic acid or a proportionate mixture of both acids. In contrast, yeast cells grown with linoleic and linolenic acids were more piezoresistant than cells treated with oleic acid. Furthermore, growth with palmitoleic acid led to higher levels of lipid peroxidation. Intracellular trehalose during HHP treatment increased cell tolerance to pressure. However, when trehalose remained extracellular cells were sensitised to pressure. Therefore, fatty acid composition and trehalose content might play a role in the protection of the cell membrane from oxidative damage produced by HHP, confirming that alteration in cell membrane fluidity is correlated with pressure resistance in yeast.

Biotechnological properties of distillery and laboratory yeasts in response to industrial stresses.

The stress sensitivity of different wild-type strains was evaluated, as well as the response of cells arrested at different cell cycle positions to high hydrostatic pressure (HPP). HHP was chosen both for its importance in food decontamination and assessment of its suitability as a model for stress in general and understanding the yeast stress response. Studies were conducted with four industrial strains and four laboratory wild-type yeast strains (two haploid and two diploid) that differed in their backgrounds. Fundamental differences were found between the laboratory and industrial populations. Industrial strains were clearly more sensitive to hydrostatic pressure and ethanol stresses than the laboratory strains. However, ethanol production was higher in industrial strains than laboratory strains. Furthermore, no correlation was observed between ploidy and stress resistance. Yeast cells arrested in the G1 phase led to an enhancement in pressure tolerance compared to unarrested, G2 arrested, and S arrested cells. Moreover, cells arrested in the S phase were more sensitive to hydrostatic pressure than cells arrested in the G2 phase. Again, industrial strains were more sensitive than laboratory strains. HHP responses of industrial yeasts correlated well with both ethanol concentration and temperature stress, which suggests that it would be a useful model stress.

High hydrostatic pressure and the cell membrane: stress response of Saccharomyces cerevisiae.

The brewing and baking yeast Saccharomyces cerevisiae is a useful eukaryotic model of stress response systems whose study could lead to the understanding of stress response mechanisms in other organisms. High hydrostatic pressure (HHP) exerts broad effects upon yeast cells, interfering with cell membranes, cellular architecture, and the processes of polymerization and denaturation of proteins. In this review, we focus on the effect of HHP on the S. cerevisiae cell membrane and describe the main signaling pathways involved in the pressure response.

Antimicrobial activity and potential use of monoterpenes as tropical fruits preservatives

Banana, papaya and pineapple are the most consumed tropical fruits in the world, being Brazil one of the main producers. Fungi Colletotrichum musae, Colletotrichum gloeosporioides and Fusarium subglutinans f.sp. ananas cause severe post harvest diseases and losses in fruits quality. The aim of this work was to evaluate the effectiveness of five monoterpenes to inhibit the mycelial growth and conidia germination of these three phytopathogens. The monoterpenes citral, citronellal, L-carvone, isopullegol and a-pinene were diluted in ethanol to final concentrations from 0.2 to 1 percent. All monoterpenes were found to inhibit the growth of the three studies fungi in a dose-dependent manner. Citral was the most effective of the oils tested and showed potent fungicidal activity at concentrations above 0.5 percent. Also, in vivo evaluation with these tropical fruits demonstrated the efficiency of citral to inhibit fungal growth. These results indicate the potential use of citral as a natural pesticide control of post-harvest fruit diseases.

Banana, mamão e abacaxi são as frutas tropicais mais consumidas no mundo, sendo o Brasil um dos principais produtores. Os fungos Colletotrichum musae, Colletotrichum gloeosporioides e Fusarium subglutinans f.sp ananas são os principais causadores de doenças e perdas em pós-colheita de frutas. A proposta deste estudo foi avaliar a eficácia de cinco monoterpenos em inibir o crescimento micelial e a germinação dos conídios destes três fitopatógenos. Os monoterpenos citral, citronelal, L-carvona, isopulegol e a-pineno foram diluídos em etanol à concentração final de 0,2 a 1 por cento. Todos os monoterpenos testados inibiram os três fungos estudados de maneira dose-dependente. Citral foi o mais efetivo dos óleos testados e apresentou uma potente atividade fungicida em concentrações acima de 0,5 por cento. Mais ainda, avaliação in vivo com estas frutas tropicais demonstrou a eficácia de citral como inibidor do crescimento fúngico. Estes resultados indicam o uso em potencial de citral como um pesticida natural no controle das doenças em pós-colheita de frutas tropicais.

Antimicrobial activity and potential use of monoterpenes as tropical fruits preservatives.

Banana, papaya and pineapple are the most consumed tropical fruits in the world, being Brazil one of the main producers. Fungi Colletotrichum musae, Colletotrichum gloeosporioides and Fusarium subglutinans f.sp. ananas cause severe post harvest diseases and losses in fruits quality. The aim of this work was to evaluate the effectiveness of five monoterpenes to inhibit the mycelial growth and conidia germination of these three phytopathogens. The monoterpenes citral, citronellal, L-carvone, isopullegol and α-pinene were diluted in ethanol to final concentrations from 0.2 to 1%. All monoterpenes were found to inhibit the growth of the three studies fungi in a dose-dependent manner. Citral was the most effective of the oils tested and showed potent fungicidal activity at concentrations above 0.5%. Also, in vivo evaluation with these tropical fruits demonstrated the efficiency of citral to inhibit fungal growth. These results indicate the potential use of citral as a natural pesticide control of post-harvest fruit diseases.