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Directed Evolution: New Parts and Optimized Function Michael J Dougherty1 and Frances H Arnold1,2

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Directed Evolution: New Parts and Optimized Function Michael J Dougherty1 and Frances H Arnold1,2 COBIOT-658; NO OF PAGES 6 Available online at www.sciencedirect.com Directed evolution: new parts and optimized function Michael J Dougherty1 and Frances H Arnold1,2 Constructing novel biological systems that function in a robust Genome sequencing efforts have provided an enormous and predictable manner requires better methods for number of sequences that encode new biological parts. discovering new functional molecules and for optimizing their Unfortunately, a sequence only provides a hint as to its assembly in novel biological contexts. By enabling functional detailed biological properties, and the vast majority of diversification and optimization in the absence of detailed these parts remain uncharacterized, much less standar- mechanistic understanding, directed evolution is a powerful dized. Furthermore, these parts are all from the large but complement to ‘rational’ engineering approaches. Aided by nonetheless limited set of biologically relevant ones. clever selection schemes, directed evolution has generated Many synthetic biology applications will require parts new parts for genetic circuits, cell–cell communication that are not biologically relevant, parts that solve human systems, and non-natural metabolic pathways in bacteria. problems and not necessarily problems for the organisms Addresses that make them. An alternative to culling parts from 1 Division of Chemistry & Chemical Engineering, California Institute of natural sources is to construct them in the laboratory. Technology, Pasadena, CA 91125, USA In many cases it may be more efficient to start from a well- 2 Division of Biochemistry & Molecular Biophysics, California Institute of characterized part, for example, an enzyme or a transcrip- Technology, Pasadena, CA 91125, USA tion factor, and engineer it to exhibit new properties than Corresponding author: Arnold, Frances H ([email protected]) to try to find the exact desired sequence in Nature. By tuning one property, for example ligand-binding speci- ficity, without dramatically changing others (e.g. ability to Current Opinion in Biotechnology 2009, 20:1–6 be expressed in a particular host organism), this approach This review comes from a themed issue on can in principle generate families of parts that differ along Systems and synthetic biology one desired dimension but whose behaviors along others Edited by Sven Panke and Ron Weiss are at least similar and are therefore more likely to be predictable. Ellis et al. demonstrated this very nicely by generating a library of promoters and using them to 0958-1669/$ – see front matter construct feed-forward networks with different, predict- Published by Elsevier Ltd. able input–output characteristics [3]. The diverse DOI 10.1016/j.copbio.2009.08.005 families of biological molecules that exist in Nature were all generated by evolution. Not surprisingly, directed evolution — iterations of mutagenesis and artificial selec- tion or screening — can be an efficient way to generate Introduction new biological parts in the laboratory [4,5]. The emerging field of synthetic biology promises useful fuels and chemicals, sensors, pharmaceuticals, therapies, The next step is assembly into circuits or pathways. and materials as well as increased understanding of Unfortunately, novel arrangements or contexts can sig- natural biological systems. To rationalize the time-con- nificantly affect how these molecules perform, and these suming, ad hoc construction of new biological systems [1], effects are very difficult to predict. In addition to its role some researchers advocate efforts to ‘standardize’ bio- in creating new functional molecules, evolution also plays logical parts in such a way that their behavior in novel the role of Chief Editor in nature, turning disjoint para- assemblies or environments becomes more predictable graphs into great literature. In synthetic biology, however, [2]. The notorious complexity and context-dependency natural evolution is tremendously destructive, relent- of the behavior of biological parts and their assemblies, lessly chipping away at painstakingly engineered net- however, make such standardization extremely challen- works and allowing the organism to escape control. ging. It is unlikely, in fact, that biological parts can ever Nonetheless, evolution can be very helpful when tamed. be fully standardized, and engineering methods that Thus directed evolution can be used to discover enable rapid optimization of synthetic biological systems mutations that fine-tune circuits and pathways and opti- will be very useful. Evolution is Nature’s optimization mize their performance, all without requiring a detailed algorithm; it is also the mechanism by which all the understanding of the mechanisms by which those natural biological parts were generated. Here we will improvements are achieved. discuss how evolution in the laboratory — directed evol- ution — supports synthetic biology efforts, both in the This review covers recent applications of directed evol- generation of new parts and in the optimization of their ution in synthetic biology. The discussion is limited to assemblies. bacterial systems, where directed evolution experiments www.sciencedirect.com Current Opinion in Biotechnology 2009, 20:1–6 Please cite this article in press as: Dougherty MJ, Arnold FH. Directed evolution: new parts and optimized function, Curr Opin Biotechnol (2009), doi:10.1016/j.copbio.2009.08.005 COBIOT-658; NO OF PAGES 6 2 Systems and synthetic biology are relatively easy to perform. Applications have communication circuits. These systems consist of a sig- included modifying protein transcription factors, con- nal synthase, which produces a diffusible signal, and a structing synthetic genetic regulatory and cell–cell com- receptor protein, which binds to the signal and activates munication circuits, and engineering enzymes for non- transcription at a specific promoter. The well-studied QS natural metabolic pathways. Some future challenges and transcription factor LuxR, from Vibrio fischeri,hasbeena opportunities for directed evolution in synthetic biology target for directed evolution. Random mutagenesis and arealsodiscussed. dual selection were used to switch the specificity of LuxR so that it responds to decanoyl-homoserine lactone rather Engineering protein transcription factors than the native 3-oxo-hexanoyl-homoserine lactone sig- A significant fraction of synthetic biology research has nal [12]. This work demonstrated the utility of dual focused on the construction of synthetic genetic circuits selection for identifying variants of transcription factors based on transcriptional regulators. The goal is to program that could respond to new signals and no longer to the cellular behavior in both time and space, which extends native ones. LuxR sensitivity to its native signal was also genetic engineering to a much richer set of behaviors and increased using directed evolution [13]. In addition, may provide a better understanding of how much more these authors demonstrated how artificial positive feed- complex natural circuits work. The construction of simple back loops can increase the effective sensitivity of LuxR- biological devices in bacteria, such as toggle switches, based circuits. The signal synthase component of QS pulse generators, and oscillators, as well as ‘minimal systems has also been engineered: the substrate speci- circuits’ that aim to reconstruct specific biological func- ficity of the RhlI synthase was modified to increase the tions, has relied largely on a very limited number of well- production of the non-native signal hexanoyl-homoser- studied transcription factors: LacI, TetR, AraC, LuxR, ine lactone [14]. and lCI [6]. The construction of more complex circuits will require a larger set of parts to choose from. Because These successes notwithstanding, efforts to direct the ‘wiring’ in biological circuits occurs through chemical evolution of protein transcription factors have all been binding specificity, these transcription factors will need limited to conferring the ability to respond to new sig- to respond to diverse signaling molecules and activate or naling molecules that are closely related to the native repress transcription at diverse DNA sequences. While signals. Large changes in a signal that elicits a regulatory there are a few reports of novel functions that have been response have not been achieved, at least yet. DNA- engineered rationally into transcription factors [7], binding specificity appears to be similarly difficult to designing a protein that can bind a chemical signal, modify in these proteins. No code for DNA binding transmit that signal into a conformational change that has been deduced. One recent study reported some alters DNA binding and regulates transcription is extre- success in using comparative genomic data to guide mely challenging. A very high degree of modularity has changes in DNA-binding specificity, but unanticipated significantly simplified the problem for eukaryotic sig- activation between noncognate protein–operator pairs naling pathways [8,9]; many bacterial transcription fac- was also frequently found [15]. In our experience, sig- tors, however, do not exhibit the same degree of nificant changes in ligand-binding and DNA-binding modularity and appear far more difficult to modify. functions are difficult to obtain, at least in the LuxR family of transcriptional activators. There may be The E. coli transcription factor AraC is widely used in inherent limitations to transcription factor engineering, combination with the
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