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Pathway thermodynamics: Metabolomics integrated pathway analysis
S186
Abstracts / New Biotechnology 33S (2016) S1–S213
are able to bind proteins as co-expression leads to an increase in signal intensity compared to protein expression without scaffold. http://dx.doi.org/10.1016/j.nbt.2016.06.1363
P29-10 Quick and stable generation of multi-enzyme pathway in yeasts Martina Geier 1,∗ , Pia Fauland 1 , Thomas Vogl 2 , Anton Glieder 2 1 2
ACIB GmbH, Austria TU Graz, Austria
With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement synthetic or natural pathways into microorganisms. For this purpose it is not sufficient to only functionally produce the single pathway components, but to achieve a balanced expression throughout the pathway to avoid bottlenecks and to obtain high titres of the desired product. In addition, the resulting strains need to be genetically stable for their employment in industrial processes. We have evaluated different strategies to implement a multienzyme pathway into the methylotrophic yeast Pichia pastoris: The repeated use of the same regulatory elements for pathway expression – as it is commonly used up to now – resulted in genetically instable strains. However, employing a diverse set of promoters and terminators did not only circumvent this problem, but also allowed to individually control the expression of single pathway proteins. Another approach was to exploit self-processing 2A sequences for polycistronic pathway expression. This strategy features many advantages such as compact pathway design, quick assembly and the generation of stable strains. In addition, we showed that these 2A sequences can be employed for the coordinate expression of at least nine genes and offer possibilities for pathway engineering and balancing. Acknowledgements: The research leading to these results has received funding from the Innovative Medicines Initiative Joint Undertaking project CHEM21 under grant agreement n◦ 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and EFPIA companies’ in kind contribution. http://dx.doi.org/10.1016/j.nbt.2016.06.1364
P29-11 Potential applications of ␥-glutamyl transferases in synthetic biology Tilmann Kuenzl ∗ , Sven Panke ETH Zurich, Switzerland ␥-Glutamyltransferases (GGTs) are evolutionary conserved enzymes that are found in all organisms ranging from bacteria to mammals. Besides their natural role in glutathione metabolism GGTs gained attention as clinical markers as well as enzymes in biotechnological processes. One of the most interesting features of GGTs is their relaxed substrate specificity and their ability to hydrolyze ␥-glutamyl compounds with bulky substituents which makes them an interesting candidate for applications in a synthetic or xenobiology context. However, all bacterial GGTs described so far are extracellular enzymes and therefore not well suited for intracellular applications.
In this work we expressed GGT variants from different host organisms in Escherichia coli and tested them regarding their activity, substrate range and cellular localization. Through genetic modifications we were able to change the cellular localization from the periplasmic space to the cytoplasm while retaining high catalytic activity. Having a GGT variant that is active exclusively in the cytoplasm combined with the promiscuity of this enzyme family opens up interesting possibilities to apply these enzymes in synthetic biology as well as in xenobiology. We could demonstrate that it is possible to import different compounds into the cytoplasm of E. coli as part of a ␥-glutamyl compound and subsequently unload them with the help of our modified GGT variant. These results offer a promising solution for many synthetic biology applications that are so far hampered by insufficient uptake of potentially interesting compounds. http://dx.doi.org/10.1016/j.nbt.2016.06.1365
Systems biology P30-1 Pathway thermodynamics: Metabolomics integrated pathway analysis ˜ Navarro ∗ , Matthias Gerstl, Christian David Alejandro Pena Jungreuthmayer, Jürgen Zanghellini University of Natural Resources and Life Sciences, Vienna, Austria The laws of thermodynamics represent fundamental constraints for organisms. To derive these constraints the cellular metabolome is particularly useful as it determines the Gibbs energy surface, which allows us to draw conclusions on the directionality and feasibility of reactions and whole pathways. Thus we developed thermodynamic elementary flux mode analysis (tEFMA): a method that integrates the cellular metabolome into a metabolic pathway analysis and allows us to uniquely identify all thermodynamically feasible pathways. However, not all of these pathways can be combined into thermodynamically feasible flux distributions. In fact, we show that only a few out of all feasible pathways are biologically relevant and can be combined into thermodynamically feasible flux distributions. These pathways can be grouped into different sets of pathways, called largest, thermodynamically consistent (LTC) sets. We identify all of these LTC sets in E. coli and show that only a single LTC set is biologically relevant and is able to describe commonly available phenotypic data. This set is characterized by its ability to maximize biomass and ATP production, consistent with evolutionary interpretations of cell behavior. Moreover this LTC set unambiguously explains the experimentally observed behavior of glutamate dehydrogenase. In conclusion, we find that an LTC set fully determines all thermodynamically feasible capabilities of an organism and allows for a computationally efficient, unbiased, systems-level analysis of metabolism that delivers significant biological insight. http://dx.doi.org/10.1016/j.nbt.2016.06.1366
Abstracts / New Biotechnology 33S (2016) S1–S213
are able to bind proteins as co-expression leads to an increase in signal intensity compared to protein expression without scaffold. http://dx.doi.org/10.1016/j.nbt.2016.06.1363
P29-10 Quick and stable generation of multi-enzyme pathway in yeasts Martina Geier 1,∗ , Pia Fauland 1 , Thomas Vogl 2 , Anton Glieder 2 1 2
ACIB GmbH, Austria TU Graz, Austria
With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement synthetic or natural pathways into microorganisms. For this purpose it is not sufficient to only functionally produce the single pathway components, but to achieve a balanced expression throughout the pathway to avoid bottlenecks and to obtain high titres of the desired product. In addition, the resulting strains need to be genetically stable for their employment in industrial processes. We have evaluated different strategies to implement a multienzyme pathway into the methylotrophic yeast Pichia pastoris: The repeated use of the same regulatory elements for pathway expression – as it is commonly used up to now – resulted in genetically instable strains. However, employing a diverse set of promoters and terminators did not only circumvent this problem, but also allowed to individually control the expression of single pathway proteins. Another approach was to exploit self-processing 2A sequences for polycistronic pathway expression. This strategy features many advantages such as compact pathway design, quick assembly and the generation of stable strains. In addition, we showed that these 2A sequences can be employed for the coordinate expression of at least nine genes and offer possibilities for pathway engineering and balancing. Acknowledgements: The research leading to these results has received funding from the Innovative Medicines Initiative Joint Undertaking project CHEM21 under grant agreement n◦ 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and EFPIA companies’ in kind contribution. http://dx.doi.org/10.1016/j.nbt.2016.06.1364
P29-11 Potential applications of ␥-glutamyl transferases in synthetic biology Tilmann Kuenzl ∗ , Sven Panke ETH Zurich, Switzerland ␥-Glutamyltransferases (GGTs) are evolutionary conserved enzymes that are found in all organisms ranging from bacteria to mammals. Besides their natural role in glutathione metabolism GGTs gained attention as clinical markers as well as enzymes in biotechnological processes. One of the most interesting features of GGTs is their relaxed substrate specificity and their ability to hydrolyze ␥-glutamyl compounds with bulky substituents which makes them an interesting candidate for applications in a synthetic or xenobiology context. However, all bacterial GGTs described so far are extracellular enzymes and therefore not well suited for intracellular applications.
In this work we expressed GGT variants from different host organisms in Escherichia coli and tested them regarding their activity, substrate range and cellular localization. Through genetic modifications we were able to change the cellular localization from the periplasmic space to the cytoplasm while retaining high catalytic activity. Having a GGT variant that is active exclusively in the cytoplasm combined with the promiscuity of this enzyme family opens up interesting possibilities to apply these enzymes in synthetic biology as well as in xenobiology. We could demonstrate that it is possible to import different compounds into the cytoplasm of E. coli as part of a ␥-glutamyl compound and subsequently unload them with the help of our modified GGT variant. These results offer a promising solution for many synthetic biology applications that are so far hampered by insufficient uptake of potentially interesting compounds. http://dx.doi.org/10.1016/j.nbt.2016.06.1365
Systems biology P30-1 Pathway thermodynamics: Metabolomics integrated pathway analysis ˜ Navarro ∗ , Matthias Gerstl, Christian David Alejandro Pena Jungreuthmayer, Jürgen Zanghellini University of Natural Resources and Life Sciences, Vienna, Austria The laws of thermodynamics represent fundamental constraints for organisms. To derive these constraints the cellular metabolome is particularly useful as it determines the Gibbs energy surface, which allows us to draw conclusions on the directionality and feasibility of reactions and whole pathways. Thus we developed thermodynamic elementary flux mode analysis (tEFMA): a method that integrates the cellular metabolome into a metabolic pathway analysis and allows us to uniquely identify all thermodynamically feasible pathways. However, not all of these pathways can be combined into thermodynamically feasible flux distributions. In fact, we show that only a few out of all feasible pathways are biologically relevant and can be combined into thermodynamically feasible flux distributions. These pathways can be grouped into different sets of pathways, called largest, thermodynamically consistent (LTC) sets. We identify all of these LTC sets in E. coli and show that only a single LTC set is biologically relevant and is able to describe commonly available phenotypic data. This set is characterized by its ability to maximize biomass and ATP production, consistent with evolutionary interpretations of cell behavior. Moreover this LTC set unambiguously explains the experimentally observed behavior of glutamate dehydrogenase. In conclusion, we find that an LTC set fully determines all thermodynamically feasible capabilities of an organism and allows for a computationally efficient, unbiased, systems-level analysis of metabolism that delivers significant biological insight. http://dx.doi.org/10.1016/j.nbt.2016.06.1366