High levels of inhibitors and organic acid. High ethanol concentration 7. Determination of Reactive Oxygen Species (ROS) and Detoxifying Enzymes: This trial was conducted in collaboration with Dr. Angela Beatriz Juárez in the Department of Biodiversity and Experimental Biology at University of Buenos Aires. For this, the Y8P parental strain and the Y8A adapted clone were used for the determination of EROs and detoxifying enzymes. For this purpose they were inoculated in 50 ml Erlenmeyer flasks with 10 ml of YPD medium (culture medium: Erlenmeyer volume 1: 5) and incubated at 28 ° C and 250 rpm for 24 h (pre-inoculum). Then, 2 ml of each pre-inoculum were taken and 250 ml Erlenmeyer was inoculated with 50 ml YPD medium and incubated again at 28 ° C and 250 rpm for 18 hours (inoculum). After such incubation, the number of cells per ml in Neubauer's chamber was determined. Subsequently, 5 ml aliquots of each strain were taken and placed into 15 ml Falcon tubes. The samples were centrifuged at 5000 rpm for 10 min, the supernatant was discarded and the cells resuspended in 5 ml of YP medium (1% yeast extract and 2% peptone) with H2O2 as a stress condition (control: no H2O2; Soft: 5 mM H2O2 and Strong Stress: 100 mM H2O2, all these conditions were maintained for 2 h at 28°C). All assays were performed in triplicate. Reactive oxygen species (ROS) were measured by fluorometry using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Zhang et al, 2003). Catalase activity (CAT) was determined by monitoring the degradation kinetics of H2O2 according to Aebi 1984. Glutathione-S-Transferase (GST) was evaluated by the detection of the reduced glutathione conjugation product (GSH) with the reagent 1-chloro-2, 4-dinitrobenzene (CDNB). This conjugation is catalyzed by GST, and the resulting conjugate (GS-DNB) was detected at 340 nm according to Habig WH et al, 1974. Finally, Superoxide dismutase (SOD) activity was determined according to Beauchamp and Fridovich, 1971. This method is based on the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT). Figure 4. Biochemical assays under low concentration of H2O2 5 mM and high concentration of H2O2 (100 mM). ROS (a), CAT (b), GST (c) and SOD (d). One-way ANOVA and Bonferroni multiple comparison post-test. *p <0.05, ** p <0.01. Resultados: Se observó que el nivel de EROs no cambió significativamente en el caso del clon adaptado Y8A bajo el tratamiento de H2O2 (Figura 4A). Por otra parte, se observó un incremento significativo en los niveles de EROs de estado de la cepa parental Y8P en ambas condiciones de estrés. Este resultado sugiere que la cepa parental Y8P fue más susceptibles al estrés oxidativo en tal condición de estrés en comparación con el clon adaptado Y8A que mantiene el nivel de equilibrio redox de la célula. Además, se observaron diferencias significativas en la actividad de la enzima CAT en la condición de control entre Y8P y Y8A (Figura 4A). Como resultado del aumento en los niveles de EROs, el clon adaptado elevó los niveles de actividad de la enzima CAT. Además, en el clon adaptado Y8A los niveles de actividad CAT se mantuvieron elevados cuando se compara con la cepa parental Y8P (Figura 4B). Probablemente, la actividad CAT en las condiciones de control y estrés fuerte podría conferir tolerancia a otras condiciones de estrés presentadas anteriormente en este trabajo (temperatura, etanol, osmótica, congelación, NaCl y ácido acético). De forma similar, la actividad de la enzima GST en el clon adaptado Y8A se mantuvo elevada tanto en el control como en la condición de baja concentración de H2O2 pero no en altas concentraciones Para la Y8P parental, los niveles de actividad GST no variaron a lo largo del experimento (Figura 4C). Los altos niveles en la actividad GST de la cepa adaptada también pueden estar involucrados en mantener los niveles de EROs (o bien los niveles más altos de actividad GST en la cepa adaptada también podrían explicar que los niveles de EROs se mantengan cercanos a los de la cepa en condiciones control). Finalmente, la actividad de la enzima SOD de la cepa parental y adaptada después de las condiciones de estrés no mostró diferencias significativas, por lo que esta enzima no estaría involucrada en la respuesta al estrés inducida por peróxido. Las enzimas SOD juegan un papel importante en la desintoxicación del anión superóxido generada en la cadena respiratoria mitocondrial (Herrero E. et al, 2008) pero la SOD no parece ser específica para la defensa contra H2O2 (Figura 4D). Las observaciones pueden sugerir que esta enzima no sería decisiva en esta tolerancia al estrés oxidativo. Estos resultados indicaron que el aumento de la tolerancia a las condiciones de estrés oxidativo observado en el clon adaptado Y8A, sería conferido por la alta actividad basal de la enzima desintoxicante GST y la enzima antioxidante CAT. 8. Profile of the bioethanol production in the presence of acetic acid: The alcoholic fermentations were carried out in 50 ml Erlenmeyers in YPD with different concentrations of acetic acid (0, 6 and 8 g/l). For the pre-inoculum, the cells were cultured ON in YPD and then inoculated into fresh new medium (inoculum). The initial cell concentration of the fermentation cultures was adjusted to a OD600nm of 0.5. Erlenmeyers were incubated at 28°C on an orbital shaker without shaking for 96 h. The concentration of bioethanol was determined enzymatically from supernatants of aliquots of the cultures. Using the kit provided by R-Biopharm (R-Biopharm AG, Darmstadt, Germany), based on the reaction of the enzyme alcohol dehydrogenase (ADH). Figure 5. Bioethanol production profile of Y8P and Y8A in different concentrations of acetic acid. Values are the average of three independent experiments. The bars represent the deviation of the mean. Results: Anaerobic fermentations were carried out at 28°C in YPD culture medium with different concentrations of acetic acid using the Y8A adapted clone and the Y8P parental strain. In the control condition, no significant differences were observed at the end of fermentation between Y8P and Y8A where similar amounts of ethanol were produced in all conditions (7.91 and 7.96 g/l1). In contrast, a significant increase in ethanol production was observed by the clone adapted from 53% and 25.5% in the conditions of 6 and 8 g/l after 48 hours after respectively. Conclusiones: It was demonstrated that the evolutionary engineering technique is a powerful tool to generate resistant clones to a wider range of stress conditions. Through the strategy of evolutionary adaptation in culture medium with acetic acid, a clone called Y8A was obtained with the capacity to tolerate and withstand a wider range of stress conditions (ethanol, temperature, NaCl, sorbitol, acetic acid, formic acid, gallic acid, hydrogen peroxide, frozen thawed) compared to the parent strain Y8P. The high basal activity of CAT and GST in the Y8A adapted clone could confer resistance to oxidative conditions. This increase in resistance of the Y8A adapted clone coincides with a significant increase in the production of bioethanol in the presence of acetic acid as a stress condition compared to the parent strain. “Involvement of CAT and GST enzyme in multiple stress tolerance of an evolutionary engineering S. cerivisiae strain” Gurdo, Nicolás1; Novelli, Guido2; Juárez, Ángela3;Ríos, María del Carmen3; Miguel Galvagno1,2. 1Instituto de Investigaciones Biotecnológicas “Dr. Rodolfo Ugalde” (IIB-UNSAM) – Universidad Nacional de San Martín (UNSAM) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, Buenos Aires, Argentina. 2Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Buenos Aires, Pabellón de Industrias, Ciudad Universitaria, CABA, Argentina. 3Departamento de Biodiversidad y Biología Experimental - IBBEA-CONICET y Departamento de Química Biológica Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales. Ciudad Universitaria, Buenos Aires, Argentina. INTRODUCCIÓN Introducción. Saccharomyces cerevisiae is the most used yeast for the production of second generation bioethanol (2GBE) from lignocellulosic residues, since its metabolism is widely known and also for its tolerance to multiple stress conditions during the fermentation stage. Throughout the bioethanol production process and especially in the alcoholic fermentation stage, the yeast cell undergoes different stress conditions such as elevated ethanol concentration, high temperatures, osmotic pressure and oxidative stress. In 2GBE production from lignocellulosic biomass, the following process is necessary: physicochemical pre-treatment of the biomass, enzymatic hydrolysis of the biomass, fermentation and eventual distillation to separate the ethanol. The objective of the physicochemical treatment is to release fermentable sugars able to be metabolized by the microorganism and exposing mainly the cellulose to the enzymatic hydrolysis. As a consequence of this process, toxic compounds (furfural, organic acids, hexose and pentose derivatives and also phenolic compounds) are generated in the culture medium, which have the capacity to inhibit cell growth and hence ethanol productivity. As a consequence of high cellular metabolism, reactive oxygen species (ROS) are generated, such as the radical superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (• OH), which generate an imbalance in the REDOX state of the cell. S. cerevisiae has a network of defense mechanisms that allow protection against oxidative stress. Primary defense includes enzymes that protect cells by removing ROS or sequestering metal ions, meanwhile, secondary defenses comprise enzymes that remove and repair damaged components. Primary defense mechanisms include antioxidant enzymes such as Catalase (CAT), Glutation S transferase (GST) and Superoxide dismutase (SOD). It is possible to increase the robustness of laboratory or industrial yeasts using mainly two approaches: a) genetic transformations (genetic or metabolic engineering) but generally industrial scale production values are not achieved, or b) predominantly phenotypic manipulations The evolutionary adaptations through evolutionary engineering technique. Evolutionary adaptation to the culture medium or inhibitor compound is a powerful strategy of subjecting strains to a constant and increasing selective pressure in order to increase resistance to different stress conditions. This work explores the bases of evolutionary adaptation and the physiological response of yeast Saccharomyces cerevisiae in a context of metabolic stress generated by the environmental conditions inherent in a second generation bioethanol production process. Adaptive Engineering – Methodology and Results Strains, culture media and reagents: The industrial osmotolerant strain Saccharomyces cerevisiae Y8 was used in evolutionary engineering experiments. The industrial strain and the clones obtained were maintained in YPD agar medium (yeast extract 10 g/l, peptone 20 g/l, glucose 20 g/l and agar 20 g/l). The liquid cultures were made in YPD medium. All pre-cultures were incubated 14 hours in YPD medium at 28°C and 250 rpm on a rotary shaker in Erlenmeyer flasks with a 5: 1 ratio (flask volume: medium volume). The experimental cultures were inoculated in the same medium and under the same conditions of temperature and agitation as the pre-cultures, during the times corresponding to each test. 2. Evolutionary adaptation in acetic acid: From the osmotolerant strain of Saccharomyces cerevisiae Y8, batch cultures repeated were performed in YPD medium with increasing concentrations of acetic acid at 28 ° C and 250 rpm. When the batch culture reached a cell concentration ≈ 10 DO600nm, which corresponds to the intermediate exponential stage, passages of 100 μl were made to the successive batch, increasing the concentration of acetic acid from 3 g/l until no growth was observed, according to methodology described by Wallace-Salinas (Wallace-Salinas et al., 2013). In the last batch culture, the resistant population was stored in glycerol 20% v/v at -80°C: 3. Selection of a clone resistant to acetic acid: After adaptation in medium with acetic acid, the stocks were inoculated in the presence of acetic acid using plates of YPD with 5 g/l of acetic acid. The first colony/clone to appear was selected for subsequent trials. The selected clone was named Y8A as the parent strain Y8P. 4. Evaluation of innate tolerance to acetic acid: Y8P parental strain and Y8A clone were evaluated in different concentrations of acetic acid (6, 8 and 10 g/l) at different times (2, 4 and 6 hours) . CFU / ml were quantified in the assays. The experiments were performed at 28 ° C. Results: The Y8P parental strain and the Y8A adapted clone were evaluated in the presence of increasing concentrations of acetic acid (6, 8 and 10 g/l) at different times (2, 4 and 6 h). A rapid decrease in CFU/ml was observed at concentrations of 8 and 10 g/l acetic acid for the parent strain. In contrast, the Y8A-adapted clone resisted conditions with higher acid concentration and longer exposure time (Figure 1). In all conditions tested, the survival rate was higher in the Y8A-adapted clone than in the Y8P parental strain. This result indicated that acetic acid tolerance of Y8A (defined as percent survival) improved during the process of adaptive evolution in acetic acid. 5. Cell growth assesment in medium with acetic acid: Y8P strain and Y8A clone were cultured in triplicate in 100 ml Erlenmeyer flasks with 20 ml of YPD culture medium at 28°C and 250 rpm with an initial inoculum of DO600nm/ml = 1. Acetic acid was added to each culture at concentrations of 0, 6, 8 and 10 g/l and cell growth was evaluated by biomass measurement (DO600nm). Results: Batch cultures in presence of different concentrations of acetic acid (0, 6, 8 and 10 g/l) analyzed the behavior over 96 hours of the Y8P strain and the Y8A adapted clone. In Figure 2, control conditions (without acetic acid) and 10 g/l acetic acid did not show significant differences between both clones analyzed. However, the adapted Y8A clone reached higher biomass levels compared to the parental clone after 48 hours in the YPD medium containing 8 g/l acetic acid and at 72 hours in the medium with 6 g/l acetic acid. 6. Assays under different stress conditions: Y8P strain and Y8A clone were cultured for 14 hours in 5 ml of YPD medium. 500 μl of each culture was inoculated into 100 ml Erlenmeyer flasks containing 20 ml of YPD and incubated for 4 h at 28°C and 250 rpm. Cell concentration (DO600nm) was monitored and the corresponding volume was taken to obtain a final concentration of cells of DO600nm=1. Cells were washed with 0.1M potassium phosphate buffer (pH=6) and the following treatments were performed: Ethanol (10% v/v for 1 h), acetic acid (5 g/l for 2 h), temperature (42°C for 2 h), sorbitol (3 M for 3 h), frozen-thawed (2 cycles of 24 h), formic acid (20 mM for 30 min), NaCl (1.5 M for 4 h), gallic acid (15 mM for 4 h) and H2O2 (5 mM - 1 h). The stress source was removed by washing the culture with 0.1M potassium phosphate buffer (pH=6) and serial dilutions were made to the tenth (100-10-4). 100 μl of the appropriate dilution was incoulated in YPD agar and the plates were incubated at 28°C for 48 h. In addition, 10 μl of YPD agar in a drop form (spot assay) was plated from each of the dilutions. Results: In order to carry out these experiments, a set of stress was chosen to imitate different damages that can be suffered by the yeast cell. The first set of stress studied were high temperature, ethanol, hydrogen peroxide, sorbitol and frozen-thawed. The second set of stress studied corresponds to inhibitors that may be present in the lignocellulosic hydrolyzate used for fermentation. The inhibitors studied were: acetic acid, formic acid and gallic acid and as another parameter of toxicity the NaCl. The studies were carried out with cells in exponential phase and the percentage of survival after the treatments in the different conditions of stress was compared. The Y8A clone obtained by evolutionary engineering showed a greater tolerance against all stress conditions tested except for formic acid. It should be noted that these results coincide with the principle of co-tolerance to different types of stress found in S. cerevisiae (Lewis et al., 1997). 1 2 3 4 5 6 Osmotic Pressure Oxidative Stress High temperature High levels of inhibitors and organic acid. High ethanol concentration Figure 1. Growth of yeast cells under different concentrations of acetic acid (0, 6, 8 and 10 g/l) and exposure time (2, 4 and 6 h). After the acetic acid treatments, the Y8P parent strain and the Y8A adapted clone were serially diluted, and 10 μl of each 100-10-4 dilution (left to right) were placed in YPD plates at 28°C for 48 hours. Figure 2. Cell growth of Y8P and Y8A in YPD medium with increasing concentrations of acetic acid (6, 8 and 10 g./l): Control. 6 g/l Acetic acid. 8 g/l Acetic acid. 10 g/l Acetic acid. The results represent the mean values ± SD of three independent experiments. Figure 3. Survival percentages of the Y8P and Y8A after being subjected to different stress conditions. Heat (42 ° C for 2 h), ethanol (10% v. v-1 for 1 h), osmotic (3M for 3 h), freeze-thaw (two cycle of 24 h), oxidative (5 mM-1 h). Acetic acid (5 g. l-1 for 6 h), formic acid (20 mM for 30 min), NaCl (1.5 M for 4 h) and gallic acid (15 mM for 4 h). 8 7 Y8P Y8A