Signal integration and decision making in Escherichia coli: A biological implementation of negative feedback control systems

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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 5␤reductase 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

react and maintain cellular homeostasis, in a process called negative feedback control. In this kind of control system the molecular interactions and feedback loops are grouped in different modules, similar to an engineering feedback control system, able to act synergistically in order to achieve signal sensing (akin to sensing modules), signal integration (akin to comparator modules), decision making (akin to controller modules) and adaptation or reaction to the original signal (akin to actuation modules). The ability to design and implement synthetic gene networks for negative feedback control can have a huge impact in all applications where a cellular process needs to be regulated and controlled in relation to external conditions (e.g. bacterial fermentation, gene expression regulation, cell differentiation). Here we propose an autonomous synthetic controller, completely embedded in Escherichia coli cells, able to adapt intracellular gene expression levels to different cues. The biological controller is composed of four modules (resembling the negative feedback system), developed using a combination of rational biological design and mathematical simulations, and validated in batch cultures and microfluidic experiments. We predict that the system will assure high specificity and precision in signal integration, modularity and robustness, constituting a powerful tool to elicit and control cellular behaviors. http://dx.doi.org/10.1016/j.nbt.2016.06.1360

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P29-7 Preparation of chondroitin derivatives for the recognition of neurotrophic factor Agatha Bastida ∗ , Raúl Benito-Arenas, Ester Martinez, Eduardo Garcia-Junceda, Julia Revuelta, Alfonso Fernandez-Mayoralas CSIC, Spain An approximation for the repair of spinal cord injury (SCI) is the generation of new neural cells from precursors that produce neurites to establish connections with the already existing cells. In recent years it has been described that analogs of chondroitin sulfate (CS) are capable of promoting the growth of neurites in culture. It is known that the sulfation pattern of glycosaminoglycans, encodes the information required to regulate neurite growth. Sulfatases catalyze hydrolysis of sulfate groups from a broad range of substrate molecules including small organic compounds to large macromolecules such as glycosaminpglycans (GAGs). Therefore, we had over-expressed two glycosaminoglycan sulfatases from Bacteroides thetaiotaomicron to selectively remove sulfate groups from the analogues of Chondroitin sulfate derivatives. So, we have described the synthesis of chondroitin (C-0S), persufate chondroitin (C-PS), trisulfate chondroitin (C-TS), disulfate chondroitin (C-DS) and monosulfate chondroitin (C-MS) derivatives. Acknowledgements: We thank the Spanish Ministerio de Ciencia e Innovación (MAT2015-65184-C2-2-R, and CTQ201345538-P). http://dx.doi.org/10.1016/j.nbt.2016.06.1362

Biosynthetic approach to combine early steps of cardenolide biosynthesis in Saccharomyces cerevisiae Daniel Geiger ∗ , Jan Petersen, Nadine Metinger, Jennifer Munkert, Christoph Rieck, Wolfgang Kreis FAU Erlangen-Nuernberg, Germany Synthetic biology provides a promising access to cardenolides by using Saccharomyces cerevisiae as microbial production host for their synthesis starting from a simple sugar source. To realize this project we separated cardenolide biosynthetic pathway into three modules. Module 1 consists in the optimization of a recombinant sterol cleavage system according to Duport et al. (1998). Module 2 comprises the steps of the cardenolide biosynthesis where the coding genes for the envolved enzymes are available, namely the 3␤-hydroxysteroid dehydrogenase, the 3ketosteroid isomerase, the progesterome 5␤-reductase and the cytochrome P-450c21. In this module a 5 step conversion proceeds from pregnenolone to 5␤-pregnane-3␤,21-diol-20-one. The 3rd module contains the 14␤-hydroxylation step, the formation of the 21-O-malonyl hemiester and subsequent butenolide ring formation. The coding sequences for the enzymes involved in module 3 are so far unknown and candidate genes are searched for. We started with the reconstitution of module 2. We cloned the D.l.3␤HSD, C.t.3-KSI, A.th.P5␤R F343A F153A and the M.m.CYP21A1 into one yeast expression vector. To obtain an expression system yielding in high amounts of the final product, in vitro studies with in E.coli overexpressed enzymes were conducted to choose the most suitable enzymes. All intermediates of the reaction sequence were already tested as substrates in yeast in vivo which demonstrated that all genes were functionally expressed. Moreover, our results demonstrate the successful combination of the enzymatic reactions, as feeding early intermediates led to higher number of downstream products. http://dx.doi.org/10.1016/j.nbt.2016.06.1361

P29-8 Scaffolding for metabolic engineering endeavors Bernd Albrecht 1,∗ , Matthias Steiger 1 , Diethard Mattanovich 2 , Michael Sauer 2 1

ACIB GmbH, Austria BOKU – University of Natural Resources and Life Sciences Vienna, Austria 2

Production of valuable metabolites at high titers is often hindered by interference with other metabolic processes, toxicity of pathway intermediates and loss of substrates due to diffusion into the cell periphery and export into the medium. Spatial organization of enzymes has the potential to overcome these problems and facilitate metabolic engineering. This can be achieved for instance by docking enzymes onto co-expressed synthetic scaffolds. Here we suggest scaffolds made out of defined RNA modules. One RNA module itself contains a docking domain for protein binding and a self-assembly domain for the formation of large scaffold complexes. The docking domain harbors two or four stemloop structures called aptamers which function as specific peptide interaction sites. Respective peptides that bind to these aptamers are fused to proteins of interest. The efficiency of targeting onto docking domains is evaluated by using a bimolecular fluorescence complementation (BiFC) assay. Therefore, the yellow fluorescent protein (YFP) venus is split into a N-terminal (YN) and a C-terminal (YC) part. Both parts are fused to a certain targeting peptide spaced by a flexible linker. Co-expression of tagged split variants with a corresponding scaffold in E. coli brings YN and YC in close proximity and promotes refolding into functional YFP. Fluorescence signal output is measured using microplate reader, flow cytometry and fluorescence microscopy. We can show that our scaffold designs