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Effects of S-adenosyl-l -methionine on membrane components and ethanol tolerance in Saccharomyces cerevisiae
New Biotechnology · Volume 25S · September 2009
concomitantly when otsBA genes were expressed and thereby degradation of trehalose. It was found that trehalase activity in the cells was the highest under the high osmotic pressure. However, the activity of expressed trehalase was strongly inhibited by the validamycin A and thus addition of trehalase inhibitor, validamycin A into medium significantly enhanced the production of trehalose under the osmotic stress. In M9 medium supplemented with glycerol, the amount of trehalose accumulated in the cells or excreted into the medium was examined in medium with or without validamycin A under normal or high osmotic condition. The effect of gene dosage was also investigated. doi:10.1016/j.nbt.2009.06.790
ABSTRACTS
shunt and lactate dehydrogenase were mainly active in the arcA strain; showing a 5- and 12.7-fold increase when compared to the wild-type strain, respectively. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in mutant strains. Taken together, the results show that there is a shared control on glucose catabolism under restricted oxygen supply by the Arc and Cre two-component systems of E. coli. Manipulating metabolic fluxes through deletion of global regulators involved in carbon catabolism and redox control could be an interesting tool to modulate central metabolism for biotechnological purposes. doi:10.1016/j.nbt.2009.06.791
4.1.09 4.1.10 Metabolic flux analysis of Escherichia coli ArcA and CreB mutants reveals shared control of carbon catabolism under microaerobic conditions of growth P.I. Nikel 1,2,3,∗ , J. Zhu 4 , K.Y. San 4 , M.A. Galvagno 1,5 , B.S. Méndez 2 , G.N. Bennett 1
Effects of S-adenosyl-L-methionine on membrane components and ethanol tolerance in Saccharomyces cerevisiae S.W. Lee ∗ , E.S. Choi, M.K. Oh Korea University, Seoul, Republic of Korea
1
Instituto de Investigaciones Biotecnológicas, UNSAM-CONICET, San Martín, Argentina 2 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina 3 Department of Biochemistry and Cell Biology, Rice University, Houston, TX, United States 4 Department of Bioengineering, Rice University, Houston, TX, United States 5 Departamento de Ingeniería Química, Facultad de Ingeniería, UBA, Buenos Aires, Argentina
Escherichia coli has several elaborate sensing mechanisms for response to oxygen and carbon source availability in the environment. The CreBC (carbon source responsive) and ArcAB (aerobic respiration control) two-component signal transduction systems are responsible for regulation of carbon source utilization and intracellular redox control, respectively. However, very little is known about how both systems concomitantly modulate central metabolic fluxes. In this work, the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions of growth was assessed by means of 13 C-labeling experiments in chemostat cultures of a wild-type strain, creB and arcA single mutants, and a creB arcA double mutant. Continuous cultures were conducted at D = 0.10 h−1 under carbon-limited conditions using glucose as the carbon source, and microaerobic conditions were attained by sparging the bioreactor with a mixture of 2.5% O2 in N2 . It was found that all experimental strains metabolized glucose mainly through the Embden—Meyerhoff—Parnas pathway and mutant strains had significantly lower fluxes in both the oxidative and non-oxidative pentose phosphate pathways (ca. 75% reduction). Significant differences were also found at the pyruvate branching point. Both pyruvate—formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis, and their activity seemed to be dependent on both ArcAB and CreBC. Strains carrying the creB deletion showed a ca. 2-fold higher biomass yield on glucose when compared to the wild-type strain and its arcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate
Ethanol tolerance is an essential factor of metabolic engineering for bioethanol production because ethanol inhibits the growth, fermentation and viability of cell. However, the research to improve ethanol tolerance of bioethanol producing microorganism has not been effectively proceeded because the cellular toxicity of ethanol and its mechanism has been barely known. We postulated that ethanol effect on membrane fluidity is major factor of cellular toxicity by ethanol. S-Adenosyl-L-methionine (SAM), as a universal methyl donor, is consumed in biosynthetic pathway of ergosterol and phosphatidylcholine which are key components in membrane. We discovered SAM accumulating strain of Saccharomyces cerevisiae showed improved ethanol tolerance and its composition of ergosterol and phosphatidylcholine decreased. We concluded that SAM effects on membrane can improve the ethanol tolerance of cell by modification of membrane fluidity. doi:10.1016/j.nbt.2009.06.792
4.1.11 Effects of aminooxyacetate on alanine and aspartate aminotransferase activity in Sparus aurata J.D. González ∗ , I. Metón, M.C. Salgado, A. Caballero, F. Fernández, I.V. Baanante Universitat de Barcelona, Barcelona, Spain
To study the metabolic effects caused by inhibition of alanine aminotransferase (ALT), a key enzyme involved in protein metabolism, we analysed the action of aminooxyacetate (AOA) on the carnivorous fish gilthead sea bream (Sparus aurata). The in vitro effect of AOA on ALT and aspartate aminotransferase (AST) enzyme activities was studied in liver crude extracts from Sparus aurata. The effect of AOA in vivo was analysed in fish after either intraperitoneal injection with different amounts of AOA or feeding www.elsevier.com/locate/nbt S327
concomitantly when otsBA genes were expressed and thereby degradation of trehalose. It was found that trehalase activity in the cells was the highest under the high osmotic pressure. However, the activity of expressed trehalase was strongly inhibited by the validamycin A and thus addition of trehalase inhibitor, validamycin A into medium significantly enhanced the production of trehalose under the osmotic stress. In M9 medium supplemented with glycerol, the amount of trehalose accumulated in the cells or excreted into the medium was examined in medium with or without validamycin A under normal or high osmotic condition. The effect of gene dosage was also investigated. doi:10.1016/j.nbt.2009.06.790
ABSTRACTS
shunt and lactate dehydrogenase were mainly active in the arcA strain; showing a 5- and 12.7-fold increase when compared to the wild-type strain, respectively. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in mutant strains. Taken together, the results show that there is a shared control on glucose catabolism under restricted oxygen supply by the Arc and Cre two-component systems of E. coli. Manipulating metabolic fluxes through deletion of global regulators involved in carbon catabolism and redox control could be an interesting tool to modulate central metabolism for biotechnological purposes. doi:10.1016/j.nbt.2009.06.791
4.1.09 4.1.10 Metabolic flux analysis of Escherichia coli ArcA and CreB mutants reveals shared control of carbon catabolism under microaerobic conditions of growth P.I. Nikel 1,2,3,∗ , J. Zhu 4 , K.Y. San 4 , M.A. Galvagno 1,5 , B.S. Méndez 2 , G.N. Bennett 1
Effects of S-adenosyl-L-methionine on membrane components and ethanol tolerance in Saccharomyces cerevisiae S.W. Lee ∗ , E.S. Choi, M.K. Oh Korea University, Seoul, Republic of Korea
1
Instituto de Investigaciones Biotecnológicas, UNSAM-CONICET, San Martín, Argentina 2 Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina 3 Department of Biochemistry and Cell Biology, Rice University, Houston, TX, United States 4 Department of Bioengineering, Rice University, Houston, TX, United States 5 Departamento de Ingeniería Química, Facultad de Ingeniería, UBA, Buenos Aires, Argentina
Escherichia coli has several elaborate sensing mechanisms for response to oxygen and carbon source availability in the environment. The CreBC (carbon source responsive) and ArcAB (aerobic respiration control) two-component signal transduction systems are responsible for regulation of carbon source utilization and intracellular redox control, respectively. However, very little is known about how both systems concomitantly modulate central metabolic fluxes. In this work, the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions of growth was assessed by means of 13 C-labeling experiments in chemostat cultures of a wild-type strain, creB and arcA single mutants, and a creB arcA double mutant. Continuous cultures were conducted at D = 0.10 h−1 under carbon-limited conditions using glucose as the carbon source, and microaerobic conditions were attained by sparging the bioreactor with a mixture of 2.5% O2 in N2 . It was found that all experimental strains metabolized glucose mainly through the Embden—Meyerhoff—Parnas pathway and mutant strains had significantly lower fluxes in both the oxidative and non-oxidative pentose phosphate pathways (ca. 75% reduction). Significant differences were also found at the pyruvate branching point. Both pyruvate—formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis, and their activity seemed to be dependent on both ArcAB and CreBC. Strains carrying the creB deletion showed a ca. 2-fold higher biomass yield on glucose when compared to the wild-type strain and its arcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate
Ethanol tolerance is an essential factor of metabolic engineering for bioethanol production because ethanol inhibits the growth, fermentation and viability of cell. However, the research to improve ethanol tolerance of bioethanol producing microorganism has not been effectively proceeded because the cellular toxicity of ethanol and its mechanism has been barely known. We postulated that ethanol effect on membrane fluidity is major factor of cellular toxicity by ethanol. S-Adenosyl-L-methionine (SAM), as a universal methyl donor, is consumed in biosynthetic pathway of ergosterol and phosphatidylcholine which are key components in membrane. We discovered SAM accumulating strain of Saccharomyces cerevisiae showed improved ethanol tolerance and its composition of ergosterol and phosphatidylcholine decreased. We concluded that SAM effects on membrane can improve the ethanol tolerance of cell by modification of membrane fluidity. doi:10.1016/j.nbt.2009.06.792
4.1.11 Effects of aminooxyacetate on alanine and aspartate aminotransferase activity in Sparus aurata J.D. González ∗ , I. Metón, M.C. Salgado, A. Caballero, F. Fernández, I.V. Baanante Universitat de Barcelona, Barcelona, Spain
To study the metabolic effects caused by inhibition of alanine aminotransferase (ALT), a key enzyme involved in protein metabolism, we analysed the action of aminooxyacetate (AOA) on the carnivorous fish gilthead sea bream (Sparus aurata). The in vitro effect of AOA on ALT and aspartate aminotransferase (AST) enzyme activities was studied in liver crude extracts from Sparus aurata. The effect of AOA in vivo was analysed in fish after either intraperitoneal injection with different amounts of AOA or feeding www.elsevier.com/locate/nbt S327