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Exploring the new threonine aldolases with broad donor specificity
New Biotechnology · Volume 31S · July 2014
PA-26 Microorganisms respond in different ways to oscillations in large-scale bioreactors: Conclusions from scale-down approaches Peter Neubauer 1,∗ , Anja Lemoine 2 , Sergej Trippel 2 , Eva Brand 2 , Robert Spann 2 , Dennis Runge 2 , Ping Lu 2 , Basant El Kady 2 , Christian Reitz 2 , Stefan Junne 2 1
2
TU Berlin, Chair of Bioprocess Engineering TU Berlin
Gradients of various growth related parameters are observed in large scale bioreactors by the limited mass transfer and are enhanced by the application of the substrate limited fed-batch technology. Consequently, cells are exposed to oscillations, which cause a specific physiological adaptation. Our studies with different scale-down approaches indicate that microorganisms adopt with different strategies to oscillations, which is closely connected to their response to oxygen limitation. While Escherichia coli, similar to Saccharomyces cerevisiae reacts with increased glycolytic fluxes, Bacillus subtilisdecreases the maximum glucose uptake capacity. In contrast, Corynebacterium glutamicum shows a high robustness to oscillations. In all cases, the oscillations influenced the pools of various amino acids, which are closely connected to the central carbon metabolism. When we connected the two-compartment scaledown bioreactor with the rapid sampling unit Bioscope to use 13 C-labelling for a study of the metabolic fluxes in E. coli, we observed that the preferred route towards the synthesis of branchchain amino acids after a pulse depends on the history of growth conditions. The presented methodology, which also includes other novel analytical instruments, provides a bridge between systems biology for the investigation of the cellular regulatory networks under industrially relevant conditions. http://dx.doi.org/10.1016/j.nbt.2014.05.1812
PA-27 Exploring the new threonine aldolases with broad donor specificity Kateryna Lypetska (Fesko) ∗ , Gernot Strohmeier, Rolf Breinbauer Graz University of Technology
Threonine aldolases have great biotechnological potential, as they catalyze the formation of unnatural amino acids with high enantioselectivity. The enzymes have been efficiently applied for the aldol condensation of an aldehyde and glycine to produce Land D--hydroxy-␣-amino acids.[1] Aldolases are tolerant towards acceptor aldehyde, but they are quite rigid for the amino acid donor. In our previous work we have isolated two natural threonine aldolases, which were able to accept alanine and serine as donor.[2] Here we present the identification and characterization of a range of L- and D-threonine aldolases with broad donor specificity. The kinetic properties and the substrate specificity of new
BIOCATALYSIS
enzymes were investigated and the biocatalytic method for the stereoselective synthesis of a-quaternary a-amino acids was developed. Acknowledgements: The research leading to these results has received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n◦ 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. References [1].(a) Steinreiber J, Fesko K, Reisinger C, Schurmann M, van Assema F, Wolberg M, Mink D, Griengl H. Tetrahedron 2007;63:918–26; (b) Steinreiber J, Fesko K, Mayer C, Reisinger C, Schürmann M, Griengl H. Tetrahedron 2007;63:8088–93. [2].Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H. Angew Chem 2010;122:125–8. Angew. Chem. Int. Ed. 2010, 49, 121–124.
http://dx.doi.org/10.1016/j.nbt.2014.05.1813
PA-28 The effect of salt-preconditioning Torulaspora delbrueckii cells on fermentation performance Stilianos Logothetis 1,∗ , Fotini Drosou 1 , Arhodoula Hatzilazarou 1 , Panagiotis Tataridis 1 , Anastasios Kannelis 2 , Elias Nerantzis 1 , Graeme Walker 2 1
2
TEI of Athens Department of Enology and Spirit Technology Abertay University of Dundee
Abstract This paper concerns research into the influence of salt on physiology of the yeast, Torulaspora delbrouekii. Specifically, the work focused on how NaCl affected the growth, viability and fermentation performance of this yeast in laboratory-scale experiments. One of the main findings of the research presented involved the influence of salt “preconditioning” of yeasts which represents a method of pre-culturing cells in the presence of salt in an attempt to improve subsequent fermentation performance. Such an approach resulted in preconditioned T. delbruekii yeasts having an improved capability to ferment high-sugar containing media (up to 30% w/v of glucose) with increased cell viability and with elevated levels of produced ethanol. Salt-preconditioning most likely influenced the stress-tolerance of yeasts by inducing the synthesis of key metabolites such as trehalose and glycerol which act to improve cells’ ability to withstand osmostress and ethanol toxicity. Overall, this research has demonstrated that a relatively simple method designed to physiologically adapt yeast cells–by salt-preconditioning–can have distinct advantages for alcohol fermentation processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1814
www.elsevier.com/locate/nbt S87
PA-26 Microorganisms respond in different ways to oscillations in large-scale bioreactors: Conclusions from scale-down approaches Peter Neubauer 1,∗ , Anja Lemoine 2 , Sergej Trippel 2 , Eva Brand 2 , Robert Spann 2 , Dennis Runge 2 , Ping Lu 2 , Basant El Kady 2 , Christian Reitz 2 , Stefan Junne 2 1
2
TU Berlin, Chair of Bioprocess Engineering TU Berlin
Gradients of various growth related parameters are observed in large scale bioreactors by the limited mass transfer and are enhanced by the application of the substrate limited fed-batch technology. Consequently, cells are exposed to oscillations, which cause a specific physiological adaptation. Our studies with different scale-down approaches indicate that microorganisms adopt with different strategies to oscillations, which is closely connected to their response to oxygen limitation. While Escherichia coli, similar to Saccharomyces cerevisiae reacts with increased glycolytic fluxes, Bacillus subtilisdecreases the maximum glucose uptake capacity. In contrast, Corynebacterium glutamicum shows a high robustness to oscillations. In all cases, the oscillations influenced the pools of various amino acids, which are closely connected to the central carbon metabolism. When we connected the two-compartment scaledown bioreactor with the rapid sampling unit Bioscope to use 13 C-labelling for a study of the metabolic fluxes in E. coli, we observed that the preferred route towards the synthesis of branchchain amino acids after a pulse depends on the history of growth conditions. The presented methodology, which also includes other novel analytical instruments, provides a bridge between systems biology for the investigation of the cellular regulatory networks under industrially relevant conditions. http://dx.doi.org/10.1016/j.nbt.2014.05.1812
PA-27 Exploring the new threonine aldolases with broad donor specificity Kateryna Lypetska (Fesko) ∗ , Gernot Strohmeier, Rolf Breinbauer Graz University of Technology
Threonine aldolases have great biotechnological potential, as they catalyze the formation of unnatural amino acids with high enantioselectivity. The enzymes have been efficiently applied for the aldol condensation of an aldehyde and glycine to produce Land D--hydroxy-␣-amino acids.[1] Aldolases are tolerant towards acceptor aldehyde, but they are quite rigid for the amino acid donor. In our previous work we have isolated two natural threonine aldolases, which were able to accept alanine and serine as donor.[2] Here we present the identification and characterization of a range of L- and D-threonine aldolases with broad donor specificity. The kinetic properties and the substrate specificity of new
BIOCATALYSIS
enzymes were investigated and the biocatalytic method for the stereoselective synthesis of a-quaternary a-amino acids was developed. Acknowledgements: The research leading to these results has received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n◦ 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. References [1].(a) Steinreiber J, Fesko K, Reisinger C, Schurmann M, van Assema F, Wolberg M, Mink D, Griengl H. Tetrahedron 2007;63:918–26; (b) Steinreiber J, Fesko K, Mayer C, Reisinger C, Schürmann M, Griengl H. Tetrahedron 2007;63:8088–93. [2].Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H. Angew Chem 2010;122:125–8. Angew. Chem. Int. Ed. 2010, 49, 121–124.
http://dx.doi.org/10.1016/j.nbt.2014.05.1813
PA-28 The effect of salt-preconditioning Torulaspora delbrueckii cells on fermentation performance Stilianos Logothetis 1,∗ , Fotini Drosou 1 , Arhodoula Hatzilazarou 1 , Panagiotis Tataridis 1 , Anastasios Kannelis 2 , Elias Nerantzis 1 , Graeme Walker 2 1
2
TEI of Athens Department of Enology and Spirit Technology Abertay University of Dundee
Abstract This paper concerns research into the influence of salt on physiology of the yeast, Torulaspora delbrouekii. Specifically, the work focused on how NaCl affected the growth, viability and fermentation performance of this yeast in laboratory-scale experiments. One of the main findings of the research presented involved the influence of salt “preconditioning” of yeasts which represents a method of pre-culturing cells in the presence of salt in an attempt to improve subsequent fermentation performance. Such an approach resulted in preconditioned T. delbruekii yeasts having an improved capability to ferment high-sugar containing media (up to 30% w/v of glucose) with increased cell viability and with elevated levels of produced ethanol. Salt-preconditioning most likely influenced the stress-tolerance of yeasts by inducing the synthesis of key metabolites such as trehalose and glycerol which act to improve cells’ ability to withstand osmostress and ethanol toxicity. Overall, this research has demonstrated that a relatively simple method designed to physiologically adapt yeast cells–by salt-preconditioning–can have distinct advantages for alcohol fermentation processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1814
www.elsevier.com/locate/nbt S87