Teses e Dissertações
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/1
Teses e dissertações defendidas no contexto dos programas de pós graduação da Instituição.
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Item Assessment of metabolic regulation and stress response in oleaginous yeasts by systems biology approaches(Universidade Federal de Viçosa, 2024-01-30) Almeida, Eduardo Luís Menezes de; Silveira, Wendel Batista da; http://lattes.cnpq.br/3157480507007265The current environmental crises promoted by the extensive use of oil have highlighted the necessity of alternative and sustainable production chains. Lipids and fatty acids from oleaginous yeasts produced from lignocellulosic hydrolysates are promising sources for oleochemicals. Hence, selecting and developing robust yeast strains that can grow and produce lipids in lignocellulosic hydrolysates is pivotal to broaden their applicability and viability. Lipomyces starkeyi is an oleaginous yeast capable of growing and producing lipids using a diverse range of carbon sources. Its growth and lipid production have been demonstrated in lignocellulosic biomasses. Papiliotrema laurentii can also assimilate sugars derived from agricultural wastes, such as glucose and xylose from lignocellulosic biomasses, and convert them into high lipid amounts. However, wild P. laurentii strains are highly sensitive to acetic acid, one of the primary inhibitors in lignocellulosic hydrolysates. The understanding of multifactorial stress responses and yeast physiology can be facilitated by the application of holistic approaches, including biological networks and high-throughput data. Therefore, we hypothesized that the use of systems biology approaches, including genome-scale metabolic models (GEMs) and transcriptomics, as well as their integration, can support us to understand the metabolism and stress responses of oleaginous yeasts and identify suitable targets for metabolic engineering. Herein, we propose the first GEM of L. starkeyi, lista-GEM. We reconstructed lista-GEM using two high-quality oleaginous yeast models as templates and curated it to reflect the metabolism of L. starkeyi. The simulated phenotypes and predicted flux distributions were in good accordance with experimental data. Then, we predicted targets to improve lipid production in glucose, xylose, and glycerol. Enzymes related to lipid synthesis in the endoplasmic reticulum, such as stearoyl-CoA desaturase, fatty-acyl-CoA synthase, diacylglycerol acyltransferase, and glycerol-3-phosphate acyltransferase, were the main targets to improve lipid production. Glycolytic genes were also predicted as targets for overexpression. Pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase, adenylate kinase, inorganic diphosphatase, and triose-phosphate isomerase were predicted only when glycerol was the carbon source. Hence, lista-GEM provides multiple metabolic engineeringtargets to improve lipid production by L. starkeyi using carbon sources from agricultural and industrial wastes. Furthermore, we combined transcriptome and genome-scale metabolic modeling to deepen our understanding regarding the targets of acetic acid stress, as well as the adaptive responses in P. laurentii. Acetic acid stress promoted global expression changes and most repressed genes were related to transcriptional and translational processes. Under stress, the sensitive strain induced DNA mismatch repair mechanisms and meiosis, while the tolerant strain negatively regulated autophagy and the cell cycle. The tolerant strain induced processes responsible for increasing the intracellular pH (e.g., arginase, ornithine metabolism, urea cycle), detoxification of toxic compounds (e.g., glutathione metabolism), and proton efflux. The tolerant strain also presented a remarkable NAD(P)H pool in the metabolic modeling analysis, which might support the reducing power required by tolerance mechanisms. Otherwise, the sensitive strain induced genes related to cell wall biogenesis and cobalamin synthesis. Overall, the genes and pathways described herein as tolerant-related can be useful in future metabolic engineering strategies to improve the tolerance of P. laurentii to weak acids, boosting its application in lignocellulosic-based biorefineries. Keywords: Metabolic modeling; Transcriptomics; Non-Saccharomyces.Item New Papiliotrema laurentii UFV-1 strains with improved acetic acid tolerance selected by adaptive laboratory evolution(Universidade Federal de Viçosa, 2021-03-17) Almeida, Eduardo Luís Menezes de; Silveira, Wendel Batista da; http://lattes.cnpq.br/3157480507007265Depletion of fossil fuels and increase in greenhouse gas emissions have boosted the development of new technologies for biodiesel production. Oil extracted from soybeans is the major source for Brazilian biodiesel production (69.8%); nevertheless, its utilization as feedstock requires arable land, water, and nutrients that could be utilized for food crops and conversion to native vegetation. These drawbacks can be circumvented by using yeast oil for biodiesel production. The oleaginous yeast Papiliotrema laurentii can accumulate a high amount of lipids and metabolize lignocellulose-derived sugars. Due to the recalcitrant nature of lignocellulosic biomasses, a pretreatment step is required. Nevertheless, acid pretreatment, the most used in lignocellulosic biomasses, leads to the formation of toxic compounds that can inhibit yeast growth. Among them, acetic acid is the most abundant, and in its undissociated form diffuses through the cell membrane and dissociates in the cytosol, disrupting cell homeostasis. To circumvent the inhibitor effect, detoxification processes are applied to remove or reduce their concentrations. However, the detoxification strategies applied are usually insufficient to reduce the acetic acid concentration. For this, oleaginous yeasts capable of tolerating acetic acid are of interest. Recently, our research team isolated and characterized a P. laurentii able to achieve the highest lipid contents from xylose as the sole carbon source. Nevertheless, we observed in this work that its growth is severely impaired by acetic acid (1.0 g/L). Therefore, we applied Adaptive Laboratory Evolution (ALE) to select strains of P. laurentii UFV-1 tolerant to acetic acid. We selected and characterized three Acetic acid Tolerant Strains (ATS). All strains evolved displayed the tolerance phenotype (able to grow in the presence of 1.5 g/L of acetic acid) after 398 generations being exposed to increasing concentrations of acetic acid (0.7, 0.9, and 1.5 g/L). However, different phenotypes emerged alongside. Although the acetic acid tolerance presented by ATS II was, along with ATS I, the highest observed in this work, it displayed trade-offs in the absence of the acid. as its lipid productivity, biomass and specific growth rate decreased. ATS I and III showed physiological parameters similar to the parental strain (lipid and biomass production, and sugar uptake) in stress absence. However, the ATS III, in contrast to ATS I, did not display the oleaginousviii phenotype (<20% g lipids/ g DW) when challenged with 1.75 g/L of acetic acid. Therefore, ATS I was the most promising strain, showing tolerance to acetic acid and oleaginous phenotype in all conditions evaluated. Keywords: Yeast. Oleaginous. Inhibitors. Lignocellulosic biomass.