Leading Edge Review Cell 140, January 8, 2010 ��2010 Elsevier Inc. 33 Introduction A basic property of living systems is the ability to respond to extracellular signals by evoking an internal response, which often leads to changes in gene expression and phenotypic alterations. Synthetically modifying an organism to respond differently to a signal, or to respond to an artificial signal, is accomplished through rewiring existing pathways or adding new modules that in some cases may be a complete path- way (for instance, the transplantation of a pathway to another cell type or species). The two main objectives of engineering signaling pathways are to understand how natural networks function and to build synthetic networks with specific applica- tions or functionalities. Progress in this field may have a major impact on biomedical engineering, such as gene therapy or tissue engineering, and industrial biotechnology. Synthetic biology has naturally evolved as a field in bio- technology that has a broader engineering scope: to modify entire systems. This would not have been possible without the development of systems biology, which has benefited tremen- dously from high-throughput technologies (DNA microarrays, ultrasequencing, mass spectrometry, automated microscopy, and computation). Initially, synthetic biology focused on the engineering of genetic circuits, which were first designed to study negative and positive feedback loops, oscillations, noise, and robustness within a system (Becskei and Serrano, 2000 Becskei et al., 2001 Elowitz and Leibler, 2000 Gardner et al., 2000 Fung et al., 2005 Swinburne et al., 2008). Later, gene circuits were designed to couple gene expression with metabolism, to understand quorum-sensing pathways (Bulter et al., 2004 Balagadd�� et al., 2008), and to synthesize new chemical compounds (for example, the production of terpe- noids in engineered Escherichia coli see Martin et al., 2003). They have also been used to study memory (Friedland et al., 2009), spatial patterning (Basu et al., 2005), and network evolu- tion (Isalan et al., 2008). The design of synthetic circuits in eukaryotes began with the accessibility of a wide range of molecular tools (many coming from work in bacterial engineering, such as inducible expres- sion systems reviewed in Gossen and Bujard, 2002), which have since evolved tremendously in terms of their complexity. For example, the design of an inducible gene silencing mod- ule based on RNA interference and repressor proteins (Deans et al., 2007) as well as the first tunable synthetic mammalian oscillator in hamster ovary cells (CHO) and human embryonic kidney (HEK293) cells (Tigges et al., 2009) have recently been reported. Several recent reviews have covered the engineering of synthetic gene circuits (see Dueber et al., 2004 Andrianan- toandro et al., 2006 Drubin et al., 2007 Greber and Fusseneg- ger 2007 Serrano, 2007 Michalodimitrakis and Isalan, 2009). However, not covered in these reviews is a detailed analysis of the challenges and differences of engineering gene circuits as compared to signaling systems, and when using prokaryotic versus eukaryotic cells. The aim of this review is to address these two features of synthetic biology. The first part describes the conceptual dif- ferences between engineering signaling pathways as com- pared to genetic circuits. We highlight the different properties of the components and the general properties of the circuits. In the second part, we compare signaling pathway engineering in prokaryotes as opposed to eukaryotes, contrasting the dif- ferences in architecture and components of these two classes of systems and how these differences impose different design considerations. This is complemented by reviewing examples of pioneering and the latest engineering approaches. The review concludes with our perspective on how the field might evolve and what remaining challenges are left to be faced. Engineering of Gene Circuits versus Signaling Pathways What are the differences and what should be considered when engineering signal transduction pathways as opposed to gene circuits? There are two main points to consider, one being the general properties of the system and the other being its compo- nents (Figure 1). Regarding the general properties of the system: (1) Signaling pathways operate fast (milliseconds to minutes). In contrast, transcriptional responses can range from minutes (prokaryotes) to hours (eukaryotes) (see the recent review by Pryciak, 2009) (although expression changes might be faster if the system involves noncoding RNA, as it would not require Engineering Signal Transduction Pathways Christina Kiel,1 Eva Yus,1 and Luis Serrano1,2,* 1EMBL-CRG Systems Biology Unit, Design of Biological Systems, Centre de Regulaci�� Gen��mica, Dr. Aiguader 88, 08003 Barcelona, Spain 2Intituci�� Catalana de Recerca i Estudis Avan��ats (ICREA), 08010 Barcelona, Spain *Correspondence: luis.serrano@crg.es DOI 10.1016/j.cell.2009.12.028 Cells respond to their environment by sensing signals and translating them into changes in gene expression. In recent years, synthetic networks have been designed in both prokaryotic and eukaryotic systems to create new functionalities and for specific applications. In this review, we discuss the challenges associated with engineering signal transduction pathways. Furthermore, we address advantages and disadvantages of engineering signaling pathways in prokaryotic and eukaryotic cells, highlighting recent examples, and discuss how progress in synthetic biology might impact biotechnology and biomedicine.

34 Cell 140, January 8, 2010 ��2010 Elsevier Inc. nuclear export and translation. (2) Signal transduction pathways usually depend on subcellular localization, and therefore elicit spatially restricted, context-specific responses. (3) Operations at the level of protein activity, unlike protein levels, allow for a larger degree of control and tuning capability. Thus, engineering signal transduction pathways could allow for more versatility and design options, but the predictability of the engineered pathway, at least in eukaryotes, could be low because of the complexity of signal propagation and the greater number of different molecules involved. (4) Genetic circuits tend to be noisy because mRNA and protein synthesis occur in bursts and are the major source of biological noise (Pedraza and Paulsson, 2008), whereas sig- naling pathways usually involve larger number of molecules and thus tend to be less stochastic. (5) Signaling systems employing amplification cascades need to avoid spontaneous activation, which is usually achieved by negative feedback regulation (such as in epidermal growth factor signaling see Amit et al., 2007) and also in many cases by the requirement of a double trigger- ing signal (as in B cell signaling reviewed in Kurosaki, 2002). Regarding the components of the two systems, there are some fundamental differences (Figure 1): (1) Gene circuit engineering is done on the level of DNA and DNA binding proteins. DNA is easy to modify, given that it has a modular structure and nucleotides can be exchanged without hav- ing a large impact on DNA structure (Benner and Sismour, 2005). Therefore, placement of DNA sequences where they can be recognized by DNA binding proteins in regulatory regions of a gene is, in principle, relatively easy in prokary- otes or, when plasmids are used, in eukaryotes. However, when inserting a construct in eukaryotic chromosomes, one needs to consider context effects (such as heterochromatin versus euchromatin, and epigenetic regulation). On the other hand, there have been some advances for DNA binding pro- teins, in rationally modifying their DNA binding specificities (Ashworth et al., 2006 Redondo et al., 2008), but this is not yet a well-established methodology. However, engineering DNA binding can be greatly facilitated by taking advantage of the extensive work on modular zinc-finger proteins to cre- ate transcription factors with new DNA binding specificities (Townsend et al., 2009). (2) Engineering of signal transduc- tion networks requires the modification of proteins alone, or protein-protein interactions, either by mutagenesis, inser- tion of unstructured recognition sequences, or alteration of domain composition. Although in some cases polypeptides (linear motifs) can be exchanged as easily as DNA modules, introduction of mutations inside domains or globular proteins is more complicated given that the conformation of an amino acid side chain greatly depends on the conformation of its neighboring residues and vice versa. As a consequence, their replacement could cause structural changes that can lead to protein misfolding and aggregation (L��pez De La Paz et al., 2002 Chiti and Dobson, 2006). Thus, manipulating DNA can often be done without compromising its structural compo- sition and the function of the various components, whereas manipulating proteins requires the use of protein design tools (Dahiyat, 2006), or a combination of directed evolution with selection (Looger et al., 2003). In summary, the three main differences between engineering signal transduction as opposed to gene circuits are as follows: (1) signaling systems operate fast, and thus designing inter- connections and feedbacks requires accurate prediction of the system behavior, (2) subcellular localization plays an important role in signaling and must be considered, and (3) engineering of proteins is more difficult than DNA because protein structure and folding is less understood and less predictable. Hence, the main challenges in engineering genetic systems are (1) coping with the inherent stochasticity of transcription- translation (in prokaryotes this can be diminished by engi- neering negative feedback of the transcription factors on their own promoters), (2) generating enough variants of selected DNA binding proteins to ensure recognition of almost all DNA sequences, (3) finding more transcription factors that can be modulated by chemical compounds (for example, the TET repressor), or postranslational modifications, to ensure a high dynamic range of regulation and the possibility of combining more than one transcription factors, (4) getting a better grasp of the regulatory role of chromatin structure in eukaryotes and possibly in prokaryotes, (5) incorporating other regulatory mol- ecules (such as riboswitches and small RNAs), and (6) taking advantage of the possibility of playing with the histone code. Figure 1. The Engineering of Signal Transduction Pathways and Gene Networks When engineering signal transduction pathways as opposed to gene circuits, there are two main points to consider: the general properties of the system and its components. Regarding the components, nucleotides can usually be exchanged without having a large impact on neighbor nucleotides and DNA structure (���independence approximation���). In contrast, amino acid substitu- tions in proteins can lead to structural changes and misfolding. With respect to the circuit properties, gene circuits are modular, they usually respond slow- ly, and the responses can be stochastic. In contrast, signaling pathways are highy modular, respond fast, often involve signal amplication, and make use of spatial localization.

Cell 140, January 8, 2010 ��2010 Elsevier Inc. 35 Tools for Signaling Pathway Engineering Signal transduction relies on a series of mechanisms, which to some degree are conserved in both prokaryotes and eukaryotes (Table 1). Essentially, they include all or some of the following mechanisms: localization, complex assembly, competition, activation-deactivation, diffusion and/or active transport, modularity, degradation, negative and positive feedbacks, specificity, and crosstalk (reviewed in Teruel and Meyer, 2000 Jordan et al., 2000 Aravind et al., 2003 Galp- erin, 2004 Galperin and Gomelsky, 2005). Thus, in order to design or modify signal transduction pathways, one could tackle any of the characteristics above. Depending on which segment of the pathway one would like to target, different tools can be employed (Figure 2). Below, we will describe both the design tools that have been used already (some examples are highlighted in the next section) and those that are available but have not yet been tested in synthetic approaches. In every case, we will point out the peculiarities of prokaryotes and eukaryotes that could constrain the use of particular tools. Localization As signal transduction pathways in eukaryotes are often spa- tially restricted, modifying the location of proteins is a power- ful tool for rewiring a pathway. A protein can be retargeted by using scaffold proteins that recognize a protein domain, adaptor proteins, or lipids (Harris et al., 2001 Park et al., 2003 Bashor et al., 2008), as well as by introducing post- translational modifications recognized by other proteins or by the membrane (for instance, lipid anchoring see Kamalak- kannan et al., 2004). Another tool for rewiring signal trans- duction routes by localization in single cells is the spatially restricted expression of proteins using localized transfection, Table 1. Comparing Signal Transduction in Prokaryotes and Eukaryotes Prokaryotes Eukaryotes Mechanism of Signal Transduction Phosphorrelay One-component systems yes no Two-component systems yes yes (only in yeast and plants) Kinases and phosphatases Histidine-Aspartic acid phosphotransfer yes (mainly) yes (rare) Serine/threonine kinases yes (rare) yes (mainly) Tyrosine kinases yes (rare) yes (not in plants, rare in yeast) Other posttranslational modification (such as methylation, acetylation) yes yes Second messengers and alarmones Nucleotide derivatives yes yes Lipids no yes Allosteric regulation yes yes Modularity Multidomains fewer majority Autoinhibition yes yes Anchoring yes yes Complex formation Oligomerization yes yes Receptor clustering yes yes Proteolysis and degradation yes yes (complex regulation) Network Properties Active transport yes (simple) yes (complex) Pathway length short long cascades Regulation by localization Subcellular localization yes yes Scaffolds poorly studied yes Gradients unknown important Regulation by scaffolds yes (but not well established) yes Noise resistance/robustness yes, often required for function yes Negative and positive feedback regulation yes (mainly transcriptional) yes, important (mainly at protein level) Crosstalk between pathways yes (rare, and physiological relevance often unknown) yes (very important)