Browsing by Author "Casella, Sergio"

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    Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production
    (BioMed Central, 2013-11) Favaro, Lorenzo; Basaglia, Marina; Trento, Alberto; Van Rensburg, Eugene; Garcia-Aparicio, Maria; Van Zyl, Willem H.; Casella, Sergio
    Background Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance. Results Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse. Conclusions This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.
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    Metabolomic alterations do not induce metabolic burden in the industrial yeast M2n[pBKD2-Pccbgl1]-C1 engineered by multiple δ-integration of a fungal β-glucosidase gene
    (Frontiers Media, 2019-11-28) Favaro, Lorenzo; Cagnin, Lorenzo; Corte, Laura; Roscini, Luca; De Pascale, Fabio; Treu, Laura; Campanaro, Stefano; Basaglia, Marina; Van Zyl, Willem H.; Casella, Sergio; Cardinali, Gianluigi
    In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple d-integration of the b-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the d-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple d-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.