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Production of natural and semisynthetic cardenolides – A synthetic biology approach
S184
Abstracts / New Biotechnology 33S (2016) S1–S213
P29-2 Establishment of synthetic microcompartments in Corynebacterium glutamicum Isabel Huber ∗ , Meike Baumgart, Julia Frunzke Forschungszentrum Jülich, Germany The integration of synthetic heterologous pathways into chassis organisms is often associated with the intracellular accumulation of the final product or the appearance of toxic intermediates. Eukaryotic cells have evolved a wide range of different organelles to encapsulate specific metabolic pathways within the cell to avoid interference with other cytoplasmic processes. Whereas most bacteria lack compartmentalization, some species use protein-coated microcompartments (BMCs) as distinct reaction chambers. The objective of the work is the establishment of synthetic BMCs in Corynebacterium glutamicum which allow the encapsulation of heterologous pathways within this important industrial platform organism. The correct self-assembly of the shell proteins (PduABJKNUT) from Citrobacter freundii in C. glutamicum was investigated by the heterologous expression of combinations of several or all shell proteins under consideration of different protein stoichiometries. The most promising variant exhibited distinct fluorescent foci in the presence of BMC shell proteins and eYFP equipped with an Nterminal BMC targeting peptide. These findings demonstrated the successful heterologous production of BMC shells and the delivery of heterologously produced proteins into the compartment lumen. A SsrA degradation tag fusion to the C-terminus of eYFP confirmed the protection of BMC encapsulated eYFP from cytosolic proteases. Altogether, these data provide a promising starting point for an application of BMCs as synthetic nano-bioreactors in C. glutamicum. http://dx.doi.org/10.1016/j.nbt.2016.06.1357
P29-3 Production of natural and semisynthetic cardenolides – A synthetic biology approach Jennifer Munkert 1,∗ , Daniel Geiger 1 , Nadine Meitinger 2 , Christoph Rieck 1 , Jan Petersen 2 , Wolfgang Kreis 1 1 2
Pharmaceutical Biology, Germany FAU Erlangen, Germany
Cardenolides are drugs used to treat congestive heart failure. More recently their antiproliferative action was brought into focus (Nolte et al., 2015). They are still extracted from plants grown in the field, as their chemical structure impedes chemical synthesis, with Digitalis lanata (Dl) being the most important source. Though attempts have been made in the past to produce cardenolides by plant cell tissue culture, farming of foxglove still remains the sole source of cardenolides. We are about to engineer yeast and E. coli for producing different cardenolides. We combine enzyme discovery, enzyme engineering, as well as pathway optimization to realize this project. Cardenolide agylcone formation from a sterol precursor requires the following steps: (1) sterol side chain cleavage, (2) pregnenolone 3-O-dehydrogenation, (3) isoprogesterone 3,4 isomerisation, (4) progesterone 5-reduction, (5) pregnane3,20-dione 3-keto reduction, (6) pregnane 21-hydroxylation, (7) pregnane 14-hydroxylation, (8) malonyl 21-O-hemiester formation and 9 butenolide ring formation (Clemente et al., 2011). Basically, we intend to follow and adapt the strategy reported for hydrocortisone biosynthesis in yeast (Szczebara et al., 2003),
however, using plant genes in several places instead of where mammalian genes have been used. The proof of principle has been demonstrated in modules converting pregnenolone to 21hydroxyprogesterone in yeast and pregnenolone to 5-pregnanes in E. coli. These modules are developed further by generating a vector construct containing a steroid-21-hydroxylase from bovine, 3-hydroxysteroid dehydrogenase and a shaped progesterone 5reductase from plant. In vivo studies using the different modules as well as studies to shape step (4) for improved co-substrate usage are in progress. http://dx.doi.org/10.1016/j.nbt.2016.06.1358
P29-4 Spanning the boundaries: Expanding the cross-bacterial toolbox David Bauwens Ghent University, Belgium Advances in the field of synthetic biology and metabolic engineering greatly rely on the development of tools to alter, incorporate or delete genetic information. Despite tremendous progress in recent years; developing extensive genetic parts, tools and libraries, to reroute the flow of carbon towards a product of interest, that progress has been developed mostly for well-studied textbook organisms like Escherichia coli. Because of the paucity of a genetic toolbox for majority of the bacterial kingdom, metabolic engineers generally restrict themselves to well-known workhorses, when selecting the preferred production host. Whereas, the choice of production host should be based on features like growth-requirements and precursor pool sizes, and not on the practicability of tools or part libraries. Thereto, in order to disclose the bacterial kingdom for biotechnological applications, the field lacks a device to engineer a variety of microbial production hosts using a generic toolbox with standardized parts and tools. In this perspective, an outset was established for such a generic toolbox by creating a cross-bacterial expression tool. This tool consists of an expression vector that is maintainable in various bacterial systems, and a promoter library, enabling tunable expression in diverse prokaryotes. Since the level of expression for every promoter in the promoter library coincides across bacterial species, the work-flow grants the ability to optimize the expression for a gene or even a complete pathway in one organism, and transfer it to another organism, allowing a comparison between production, and circumventing the need for tedious host-by-host expression optimization. http://dx.doi.org/10.1016/j.nbt.2016.06.1359
P29-5 Signal integration and decision making in Escherichia coli: A biological implementation of negative feedback control systems Fabio Annunziata ∗ , Gianfranco Fiore, Antoni Matyjaszkiewicz, Claire Grierson, Lucia Marucci, Mario di Bernardo, Nigel J. Savery University of Bristol, United Kingdom The ability to sense, integrate and react to different signals is of vital importance for living organisms. At the cellular level signal integration is achieved via molecular interactions, intertwined in feedback loops. In this way cells sense and compare extracellular environmental cues with intracellular conditions, in order to
Abstracts / New Biotechnology 33S (2016) S1–S213
P29-2 Establishment of synthetic microcompartments in Corynebacterium glutamicum Isabel Huber ∗ , Meike Baumgart, Julia Frunzke Forschungszentrum Jülich, Germany The integration of synthetic heterologous pathways into chassis organisms is often associated with the intracellular accumulation of the final product or the appearance of toxic intermediates. Eukaryotic cells have evolved a wide range of different organelles to encapsulate specific metabolic pathways within the cell to avoid interference with other cytoplasmic processes. Whereas most bacteria lack compartmentalization, some species use protein-coated microcompartments (BMCs) as distinct reaction chambers. The objective of the work is the establishment of synthetic BMCs in Corynebacterium glutamicum which allow the encapsulation of heterologous pathways within this important industrial platform organism. The correct self-assembly of the shell proteins (PduABJKNUT) from Citrobacter freundii in C. glutamicum was investigated by the heterologous expression of combinations of several or all shell proteins under consideration of different protein stoichiometries. The most promising variant exhibited distinct fluorescent foci in the presence of BMC shell proteins and eYFP equipped with an Nterminal BMC targeting peptide. These findings demonstrated the successful heterologous production of BMC shells and the delivery of heterologously produced proteins into the compartment lumen. A SsrA degradation tag fusion to the C-terminus of eYFP confirmed the protection of BMC encapsulated eYFP from cytosolic proteases. Altogether, these data provide a promising starting point for an application of BMCs as synthetic nano-bioreactors in C. glutamicum. http://dx.doi.org/10.1016/j.nbt.2016.06.1357
P29-3 Production of natural and semisynthetic cardenolides – A synthetic biology approach Jennifer Munkert 1,∗ , Daniel Geiger 1 , Nadine Meitinger 2 , Christoph Rieck 1 , Jan Petersen 2 , Wolfgang Kreis 1 1 2
Pharmaceutical Biology, Germany FAU Erlangen, Germany
Cardenolides are drugs used to treat congestive heart failure. More recently their antiproliferative action was brought into focus (Nolte et al., 2015). They are still extracted from plants grown in the field, as their chemical structure impedes chemical synthesis, with Digitalis lanata (Dl) being the most important source. Though attempts have been made in the past to produce cardenolides by plant cell tissue culture, farming of foxglove still remains the sole source of cardenolides. We are about to engineer yeast and E. coli for producing different cardenolides. We combine enzyme discovery, enzyme engineering, as well as pathway optimization to realize this project. Cardenolide agylcone formation from a sterol precursor requires the following steps: (1) sterol side chain cleavage, (2) pregnenolone 3-O-dehydrogenation, (3) isoprogesterone 3,4 isomerisation, (4) progesterone 5-reduction, (5) pregnane3,20-dione 3-keto reduction, (6) pregnane 21-hydroxylation, (7) pregnane 14-hydroxylation, (8) malonyl 21-O-hemiester formation and 9 butenolide ring formation (Clemente et al., 2011). Basically, we intend to follow and adapt the strategy reported for hydrocortisone biosynthesis in yeast (Szczebara et al., 2003),
however, using plant genes in several places instead of where mammalian genes have been used. The proof of principle has been demonstrated in modules converting pregnenolone to 21hydroxyprogesterone in yeast and pregnenolone to 5-pregnanes in E. coli. These modules are developed further by generating a vector construct containing a steroid-21-hydroxylase from bovine, 3-hydroxysteroid dehydrogenase and a shaped progesterone 5reductase from plant. In vivo studies using the different modules as well as studies to shape step (4) for improved co-substrate usage are in progress. http://dx.doi.org/10.1016/j.nbt.2016.06.1358
P29-4 Spanning the boundaries: Expanding the cross-bacterial toolbox David Bauwens Ghent University, Belgium Advances in the field of synthetic biology and metabolic engineering greatly rely on the development of tools to alter, incorporate or delete genetic information. Despite tremendous progress in recent years; developing extensive genetic parts, tools and libraries, to reroute the flow of carbon towards a product of interest, that progress has been developed mostly for well-studied textbook organisms like Escherichia coli. Because of the paucity of a genetic toolbox for majority of the bacterial kingdom, metabolic engineers generally restrict themselves to well-known workhorses, when selecting the preferred production host. Whereas, the choice of production host should be based on features like growth-requirements and precursor pool sizes, and not on the practicability of tools or part libraries. Thereto, in order to disclose the bacterial kingdom for biotechnological applications, the field lacks a device to engineer a variety of microbial production hosts using a generic toolbox with standardized parts and tools. In this perspective, an outset was established for such a generic toolbox by creating a cross-bacterial expression tool. This tool consists of an expression vector that is maintainable in various bacterial systems, and a promoter library, enabling tunable expression in diverse prokaryotes. Since the level of expression for every promoter in the promoter library coincides across bacterial species, the work-flow grants the ability to optimize the expression for a gene or even a complete pathway in one organism, and transfer it to another organism, allowing a comparison between production, and circumventing the need for tedious host-by-host expression optimization. http://dx.doi.org/10.1016/j.nbt.2016.06.1359
P29-5 Signal integration and decision making in Escherichia coli: A biological implementation of negative feedback control systems Fabio Annunziata ∗ , Gianfranco Fiore, Antoni Matyjaszkiewicz, Claire Grierson, Lucia Marucci, Mario di Bernardo, Nigel J. Savery University of Bristol, United Kingdom The ability to sense, integrate and react to different signals is of vital importance for living organisms. At the cellular level signal integration is achieved via molecular interactions, intertwined in feedback loops. In this way cells sense and compare extracellular environmental cues with intracellular conditions, in order to