Engenharia genômica de linhagem industrial de Saccharomyces cerevisiae visando melhorar a tolerância ao etanol

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2014.Durante a produção industrial de álcool combustível, um dos fatores limitantes no processo é o estresse promovido às células de levedura pelas altas concentrações de etanol presentes no meio. Este trabalho teve por objetivo incrementar a tolerância ao etanol de uma linhagem industrial de Saccharomyces cerevisiae através da modificação da região promotora de dois genes: o gene TRP1 envolvido na síntese de triptofano, ou o gene MSN2 que codifica para um fator de transcrição que regula a resposta ao estresse geral em S. cerevisiae. As linhagens recombinantes foram desenvolvidas usando técnicas de engenharia genômica baseadas em PCR, para inserir o promotor forte e constitutivo PADH1 na região promotora dos genes alvo. Embora a linhagem industrial utilizada (CAT-1) seja diplóide, apenas um dos alelos do gene alvo foi modificado. Após confirmação por PCR das modificações realizadas no genoma das linhagens recombinantes, a sobre-expressão dos genes foi quantificada por qRT-PCR. Os resultados mostraram que a linhagem de levedura industrial é mais tolerante ao etanol (podendo crescer em até 14% do álcool), quando comparado com uma linhagem de laboratório (nessa última 10% de etanol inibe totalmente o crescimento). Em ambas a linhagens a sobre-expressão do gene TRP1 claramente incrementou a tolerância ao etanol. As linhagens industriais sobre-expressando o gene MSN2, ou uma versão truncada do gene (MSN2-T, sem os primeiros 48 aminoácidos), mostraram-se igualmente mais tolerantes ao etanol. A linhagem que sobre-expressa o gene MSN2 mostrou-se também mais resistente ao estresse salino, mas mais sensível ao estresse oxidativo, enquanto que a linhagem que sobre-expressa o gene MSN2-T foi mais resistente a esse último estresse. A análise dos níveis intracelulares de espécies reativas de oxigênio (ROS) revelou que o estresse provocado pelo etanol aumenta significativamente os níveis de ROS nas células, sendo que ocorreu uma redução na quantidade desses compostos nas linhagens modificadas no gene MSN2. Em seguida, foi investigado se essa melhora no crescimento celular na presença de altas concentrações de etanol leva a produtividades mais elevadas de etanol durante a fermentação de altas concentrações (200 g/L) de sacarose, inclusive na presença de diferentes concentrações estressantes de etanol no início das fermentações. Os resultados revelaram que não ocorreram diferenças significativas entre a linhagem industrial CAT-1 e as linhagens recombinantes nos diferentes parâmetros fermentativos analisados, tanto em processos de bateladaXsimples quanto na batelada alimentada, com reciclo celular. O estresse alcoólico afetou principalmente o consumo dos monossacarídeos (glicose e frutose) produzidos na hidrólise da sacarose. Em conclusão, os resultados sugerem que uma maior tolerância ao etanol (através das modificações genômicas realizadas neste trabalho), não significa necessariamente uma maior produção de etanol pela linhagem industrial de S. cerevisiae.Abstract : During fuel alcohol industrial production, one of the limiting factors in the process is the stress promoted to yeast cells by high ethanol concentrations in the medium. This study aimed to improve the ethanol tolerance of an industrial Saccharomyces cerevisiae strain by modifying the promoter region of two genes: the TRP1 gene involved in the synthesis of tryptophan, or the MSN2 gene encoding for a transcription factor that regulates the general stress response in S. cerevisiae. The recombinant yeast strains were developed using PCR-based genomic engineering techniques, to insert the strong and constitutive PADH1 promoter in the promoter region of the target genes. Although the industrial strain used (CAT-1) is diploid, only one allele of the target gene was modified. After confirmation by PCR of the changes made in the genome of the recombinant strains, gene overexpression was quantified by qRT-PCR. Our results show that the industrial yeast strain is more tolerant to ethanol (being able to grow in up to 14% alcohol), when compared to a laboratory strain (10% ethanol completely inhibited growth of these cells). In both types of strain overexpressing the TRP1 gene clearly improved ethanol tolerance. The industrial strains overexpressing the MSN2 gene, or a truncated version of the gene (MSN2-T, without the first 48 amino acids), were also more ethanol tolerant. The strain that overexpresses the MSN2 gene was also more resistant to a salinity stress, but more sensitive to an oxidative stress, while the strain that overexpresses MSN2-T was more resistant to this last stress. The analysis of the intracellular levels of reactive oxygen species (ROS) revealed that stress promoted by ethanol significantly increases the levels of ROS in cells, and there was a reduction in the amount of these compounds in the strains modified in the MSN2 gene. Afterwards, it was investigated whether this improvement in the cellular growth under high ethanol concentrations leads to higher ethanol productivity during fermentation of high (200 g/L) sucrose concentrations, even in the presence of different stressful ethanol concentrations at the beginning of fermentation. The results revealed that there were no significant differences between the industrial strain CAT-1 and the recombinant strains in the different fermentation parameters analyzed, both in simple batch and fed batch processes with cell recycle. The alcoholic stress affected mainly the consumption of monosaccharides (glucose and fructose) produced by the hydrolysis of sucrose. In conclusion, the results suggest that increased tolerance toXIIethanol (through genomic modifications carried out in this work), does not necessarily mean a higher ethanol production by an industrial S. cerevisiae strain

Larvicidal effects of endophytic and basidiomycete fungus extracts on Aedes and Anopheles larvae (Diptera, Culicidae)

Introduction: In vitro bioassays were performed to access the larvicidal activity of crude extracts from the endophytic fungus Pestalotiopsis virgulata (Melanconiales, Amphisphaeriaceae) and the saprophytic fungus Pycnoporus sanguineus (Basidiomycetes, Polyporaceae) against the mosquitoes Aedes aegypti and Anopheles nuneztovari. Methods: The extracts were tested at concentrations of 100, 200, 300, 400 and 500ppm. Ethyl acetate mycelia (EAM) extracts and liquid culture media (LCM) from Pe. virgulata and Py. sanguineus were tested against third instar larvae of Ae. aegypti and An. nuneztovari. Results: The larvicidal activity of the EAM extracts from Pe. virgulata against Ae. aegypti had an LC50=101.8ppm, and the extract from the basidiomycete fungus Py. sanguineus had an LC50=156.8ppm against the Ae. aegypti larvae. The Pe. virgulata extract had an LC50=16.3ppm against the An. nuneztovari larvae, and the Py. sanguineus extract had an LC50=87.2ppm against these larvae. Conclusions: These results highlight the larvicidal effect of EAM extracts from the endophyte Pe. virgulata against the two larval mosquitoes tested. Thus, Pe. virgulata and Py. sanguineus have the potential for the production of bioactive substances against larvae of these two tropical disease vectors, with An. nuneztovari being more susceptible to these extracts

Avaliação da atividade enzimática de fungos isolados do Bioma Amazônico / Evaluation of the enzymatic activity of fungi isolated from the Amazonian Biome

A geração de energia renovável é uma das maneiras de solucionar o acúmulo deresíduos agroindustriais. A estratégia de sucesso para produzir enzimas celulolíticas inclui seleção de microrganismos e melhorias nas condições do processo fermentativo. Este trabalho descreve aanálise das atividades enzimáticas de celulases usando diferentes fontes de substratos a partir de fungos degradantes de biomassa da floresta Amazônica. As fermentações foram realizadas em agitadores a 150 rpm, 30 °C durante 240 h. Foram determinadas três atividades enzimáticas: CMCase, FPase e ?-glicosidase. O substrato comercial AVICEL® apresentou as maiores atividades enzimáticas, principalmente após 96 horas de fermentação, tanto para a cepa 511 quanto 519

Process Mining for Six Sigma

Process mining offers a set of techniques for gaining data-based insights into business processes from event logs. The literature acknowledges the potential benefits of using process mining techniques in Six Sigma-based process improvement initiatives. However, a guideline that is explicitly dedicated on how process mining can be systematically used in Six Sigma initiatives is lacking. To address this gap, the Process Mining for Six Sigma (PMSS) guideline has been developed to support organizations in systematically using process mining techniques aligned with the DMAIC (Define-Measure-Analyze-Improve-Control) model of Six Sigma. Following a design science research methodology, PMSS and its tool support have been developed iteratively in close collaboration with experts in Six Sigma and process mining, and evaluated by means of focus groups, demonstrations and interviews with industry experts. The results of the evaluations indicate that PMSS is useful as a guideline to support Six Sigma-based process improvement activities. It offers a structured guideline for practitioners by extending the DMAIC-based standard operating procedure. PMSS can help increasing the efficiency and effectiveness of Six Sigma-based process improving efforts. This work extends the body of knowledge in the fields of process mining and Six Sigma, and helps closing the gap between them. Hence, it contributes to the broad field of quality management

Interactions between plant hormones and heavy metals responses

Heavy metals are natural non-biodegradable constituents of the Earth’s crust that accumulate and persist indefinitely in the ecosystem as a result of human activities. Since the industrial revolution, the concentration of cadmium, arsenic, lead, mercury and zinc, amongst others, have increasingly contaminated soil and water resources, leading to significant yield losses in plants. These issues have become an important concern of scientific interest. Understanding the molecular and physiological responses of plants to heavy metal stress is critical in order to maximize their productivity. Recent research has extended our view of how plant hormones can regulate and integrate growth responses to various environmental cues in order to sustain life. In the present review we discuss current knowledge about the role of the plant growth hormones abscisic acid, auxin, brassinosteroid and ethylene in signaling pathways, defense mechanisms and alleviation of heavy metal toxicity

Interactions between plant hormones and heavy metals responses

Heavy metals are natural non-biodegradable constituents of the Earth’s crust that accumulate and persist indefinitely in the ecosystem as a result of human activities. Since the industrial revolution, the concentration of cadmium, arsenic, lead, mercury and zinc, amongst others, have increasingly contaminated soil and water resources, leading to significant yield losses in plants. These issues have become an important concern of scientific interest. Understanding the molecular and physiological responses of plants to heavy metal stress is critical in order to maximize their productivity. Recent research has extended our view of how plant hormones can regulate and integrate growth responses to various environmental cues in order to sustain life. In the present review we discuss current knowledge about the role of the plant growth hormones abscisic acid, auxin, brassinosteroid and ethylene in signaling pathways, defense mechanisms and alleviation of heavy metal toxicity

Increasing Ethanol Tolerance and Ethanol Production in an Industrial Fuel Ethanol <i>Saccharomyces cerevisiae</i> Strain

The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well-characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work, we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel–ethanol producing strain: the overexpression of the TRP1 or MSN2 genes, or the overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae